THE ROLE OF CEP120 AND SPICE1 IN HUMAN CENTRIOLE ...

THE ROLE OF CEP120 AND SPICE1 IN HUMAN

CENTRIOLE DUPLICATION

By

David Comartin

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Department of Molecular Genetics

University of Toronto

© Copyright by David Comartin (2015)

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The Role of CEP120 and SPICE1 in Human Centriole Duplication

David Comartin

Doctorate of Philosophy

Department of Molecular Genetics

University of Toronto

2015

ABSTRACT

The centrosome is a collection of proteins scaffolded upon microtubule based structures called

centrioles. Centrosomes play critical roles as the primary microtubule organizing centers in

interphase and mitotic cells, and template the formation of cilia and flagella. A mitotic cell

contains two centrosomes, each with two centrioles, and following mitosis daughter cells inherit

one centrosome. Before the next mitotic division, centrioles are duplicated in a process where

one procentriole forms adjacent to each existing centriole. Mutations in proteins required for

centriole duplication and/or centrosome function cause developmental defects such as primordial

dwarfism, microcephaly and ciliopathies. Additionally many tumor cells contain excess

centrosomes, and extra centrosomes are a driver of cancer progression. Thus, the correct number

and function of centrosomes is critical for human health, and understanding the proteins involved

in centriole assembly will offer insights into important diseases. Here, I describe the functions of

CEP120 and SPICE1 as proteins required for centriole assembly. I show that CEP120 and

SPICE1 cooperate with CPAP in the assembly of centrioles. Within the centriole duplication

pathway, CEP120 and SPICE1 are dependent upon the presence of SASS6, STIL, CPAP and

CEP135, and are in turn required for CEP135, CP110, CEP97 and Centrin recruitment. Further,

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CEP120 interacts with CPAP, and they cooperate with SPICE1 in centriole microtubule

assembly and elongation. Following up on this work, I identify a group of proteins with

previously undescribed roles in centriole duplication, including the human nucleosome assembly

protein NAP1L1. These proteins were identified by screening for proteins in the proximity of

CEP120 and SPICE1, and several of them are important for centriole elongation, suggesting a

functional link to CEP120 and SPICE1 in centriole assembly. The results herein represent

important advances in our understanding of how CEP120 and SPICE1 function, and in our

understanding of centriole assembly and elongation.

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ACKNOWLEDGEMENTS

I would like to first thank my supervisor Dr. Laurence Pelletier, for his mentorship, scientific

direction, valuable advice, and the resources provided throughout the course of my Ph.D. I have

greatly benefitted from the many lessons learned and opportunities I was given while in the

Pelletier Lab. Thank you for the great experience!

To my committee members, Dr. Frank Sicheri, Dr. David Bazett-Jones and Dr. Ian Scott: Thank

you all for your support, advice, guidance, and the time investment each of you made in

attending meetings and reading reports. I am very fortunate to have had input from such a great

group of scientists.

Many thanks to the groups I have had collaborations with throughout my Ph.D.: Dr. James

Hutchins and Dr. Jan-Michael Peters, Dr. Marco Archinti and Dr. Jens Luders, Dr. Eden Fussner

and Dr. David Bazett-Jones, Doug Holmyard, Dr. Etienne Coyaud and Dr. Brian Raught, and

finally Stine Morthorst and Dr. Lotte Pedersen. Your contributions, and the opportunity to work

with each of you, have been amazing.

To the members of the Pelletier Lab, past and present who made this experience fun, thank you

all! Special thanks to Deborah Pinchev, Sally Cheung, Dr. Steffen Lawo, Dr. Monica Hasegan

and Dr. Gagan Gupta for your work in contribution to the publication of a paper during my Ph.D.

Thank you also to Christina Yeh, Dr. Nicole St.-Denis, Dr. Joao Goncalves, Bahareh Adhami

Mojarad, Dr. Ladan Gheiratmand, Dr. Johnny Tkach, Christine Holly, Dr. Mariana Gomez-

Ferreria, Dr. Mikhail Bashkurov, Andrea Tagliaferro, Rachel Ford, Dr. Yi Luo, Yi Liu, and

Qiazhu-Wu for being great colleagues. Thank you to Dr. Suzanna Prosser, Dr. Monica Hasegan,

Dr. Johnny Tkach, Dr. Gagan Gupta, and soon-to-be-Dr. Christina Yeh for editing this thesis.

Lastly, very special thanks to Dr. Gagan Gupta, Dr. Nicole St.-Denis, and Dr. Steffen Lawo for

frequent advice and guidance on many scientific problems, and for your general wisdom.

Most importantly, a special thank you to my mother, father and brother for encouraging me to

undertake this project, and supporting me throughout the long process.

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Table of Contents

ABSTRACT....................................................................................................................................i

ACKNOWLEDGEMENTS.........................................................................................................iv

LIST OF TABLES.....................................................................................................................viii

LIST OF FIGURES.....................................................................................................................ix

LIST OF ABBREVIATIONS.....................................................................................................xi

LIST OF PROTEINS DISCUSSED..........................................................................................xii

1. Chapter I: Introduction ........................................................................................................ 1 1.1. Functions of the Centrosome............................................................................................ 2

1.1.1. The Functions of Centrosomes in Interphase ...................................................................................... 2 1.1.2. The Functions of Centrosomes in Mitosis ........................................................................................... 3 1.1.3. The Functions of Centrosomes in Cilia Formation .............................................................................. 5

1.2. The Centrosome in Disease .............................................................................................. 8 1.2.1. Cancer.................................................................................................................................................. 8 1.2.2. Developmental Diseases .................................................................................................................... 10

1.3. The Structure of the Centrosome ................................................................................... 11 1.3.1. Centrioles .......................................................................................................................................... 11 1.3.2. The PCM ........................................................................................................................................... 15

1.4. The Centrosome Cycle ................................................................................................... 17 1.4.1. Disengagement .................................................................................................................................. 17 1.4.2. Centrosome Separation ...................................................................................................................... 18 1.4.3. Centrosome Maturation ..................................................................................................................... 19

1.5. Centriole Duplication ..................................................................................................... 19 1.5.1. Procentriole Formation ...................................................................................................................... 20 1.5.2. Procentriole Elongation and Length Regulation ................................................................................ 25 1.5.3. The Regulation of Centriole Duplication .......................................................................................... 30

1.6. CEP120 and SPICE1 ...................................................................................................... 34

1.7. Rationale of this Thesis .................................................................................................. 35 1.8. Figures ............................................................................................................................ 36

2. Chapter II: CEP120 and SPICE1 Cooperate with CPAP in Centriole Elongation ..... 44 2.1. Statement of Contributions............................................................................................. 45 2.2. Summary ........................................................................................................................ 47

2.3. Introduction .................................................................................................................... 48 2.3.1. Combining Super-Resolution Imaging with PLK4 Induced Centriole Overduplication to Study

Procentriole Assembly ....................................................................................................................................... 48 2.3.2. The Use of Chemical Manipulation of Microtubules to Study Centriole Elongation ........................ 49

2.4. Results. ........................................................................................................................... 50 2.4.1. CEP120 and SPICE1 Interact and are Required for Centriole Duplication in Cycling Cells. ........... 50 2.4.2. CEP120 and SPICE1 are Required for PLK4 Induced Centriole Overduplication ........................... 51 2.4.3. Using 3D SIM to Study Centriole Assembly. ................................................................................... 53 2.4.4. Defining the Roles of CEP120 and SPICE1 in Procentriole Assembly ............................................ 54 2.4.5. Investigating the Role of CEP120 and SPICE1 in Procentriole Structure ......................................... 56 2.4.6. Comparison of the Effects of CEP120 or SPICE1 Depletion versus Microtubule Depolymerization

on PLK4 Induced Centriole Overduplication ..................................................................................................... 58 2.4.7. CEP120 and SPICE1 Cooperate with CPAP in Centriole Elongation .............................................. 60

2.5. Discussion ...................................................................................................................... 62 2.5.1. Placing CEP120 and SPICE1 in the Centriole Assembly Pathway ................................................... 62 2.5.2. CEP120 and SPICE1 Cooperate with CPAP in Centriole Elongation .............................................. 63 2.5.3. CEP120 and SPICE1 are Important for Microtubule Formation during Procentriole Assembly ...... 65 2.5.4. CEP135 Localization Requires CEP120 and SPICE1, and is a Microtubule Dependent Event

During Centriole Duplication ............................................................................................................................. 66

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2.5.5. CEP120 and SPICE1 are Required for Tubulin Incorporation into Procentrioles ............................. 67 2.6. Figures ............................................................................................................................ 70 2.7. Materials and Methods ................................................................................................. 103

2.7.1. Cell lines and Tissue Culture ........................................................................................................... 103 2.7.2. RNA Interference ............................................................................................................................ 103 2.7.3. Cloning of CEP120 siRNA Resistant Construct ............................................................................. 104 2.7.4. PLK4 Induced Centriole Overduplication Assays ........................................................................... 104 2.7.5. Taxol Induced Centriole Elongation ................................................................................................ 105 2.7.6. CPAP or CEP120 Induced Centriole Elongation ............................................................................ 105 2.7.7. Immunofluorescence Microscopy (IFM) and 3D-SIM .................................................................... 105 2.7.8. Cloning ............................................................................................................................................ 107 2.7.9. Statistical Methods .......................................................................................................................... 107 2.7.10. Western Blots .................................................................................................................................. 107 2.7.11. Electron Microscopy ....................................................................................................................... 108

3. Chapter III: Identification of CEP120 and SPICE1 Associated Proteins Required for

Centriole Duplication and Elongation .................................................................................... 109 3.1. Statement of Contribution, Rights and Permissions..................................................... 110

3.2. Summary ...................................................................................................................... 111 3.3. Introduction .................................................................................................................. 112

3.3.1. BioID ............................................................................................................................................... 112 3.3.2. Human Nucleosome Assembly Proteins ......................................................................................... 113 3.3.3. The Role of tubulin Glutamylation in Microtubule and Centriole Stability .................................... 114

3.4. Results .......................................................................................................................... 116 3.4.1. Identification of Potential Functional Interactors of CEP120 and SPICE1 by BioID ..................... 116 3.4.2. Characterization of CEP120/SPICE1 Associated Proteins .............................................................. 117 3.4.3. NAP1L1 is a CEP120 and SPICE1 Associated Protein Required for Centriole Duplication .......... 120

3.5. Discussion .................................................................................................................... 124 3.5.1. Identification of CEP120 and SPICE1 Associated Proteins Critical for PLK4 Induced Centriole

Duplication ....................................................................................................................................................... 124 3.5.2. Multiple CEP120 and SPICE1 Associated Proteins Are Required for CEP120 or SPICE1

localization ....................................................................................................................................................... 124 3.5.3. Depletion of CEP120 and SPICE1 Associated Proteins Affects the Microtubule Cytoskeleton ..... 129 3.5.4. NAP1L1 is Required for Centriole Duplication .............................................................................. 131

3.6. Figures .......................................................................................................................... 135 3.7. Materials and Methods ................................................................................................. 180

3.7.1. Cell Culture, RNAi, PLK4 Assay, and Microscopy Sample Preparation and Imaging ................... 180 3.7.2. BioID Analysis ................................................................................................................................ 180 3.7.3. PLK4 Screen ................................................................................................................................... 181 3.7.4. Analyses of CEP120 and SPICE1 localization, Microtubule Glutamylation and Centriole

Elongation following Depletion of CEP120 and SPICE1 Associated Proteins in U-2 OS .............................. 181 3.7.5. Bioinformatic Analysis of NAP1L-family members and Isoforms ................................................. 181 3.7.6. Cloning of Rescue Constructs ......................................................................................................... 182 3.7.7. Quantitative PCR to Detect NAP1L1 and NAP1L4 Transcripts Following treatment with

siRNAs Against NAP1L1 ................................................................................................................................ 182 4. Chapter IV: Conclusion and Future Directions ............................................................. 190

4.1. The Functions of CEP120 and SPICE1 in Procentriole Assembly and Elongation .... 191 4.2. Identification of CEP120 and SPICE1 Associated Proteins with Uncharacterized Roles

in Centriole Assembly ............................................................................................................ 193 4.3. A Role for NAP1L1 in Centriole Duplication ............................................................. 195

5. LITERATURE CITED ..................................................................................................... 197

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LIST OF TABLES

Table 3.1 CEP120 Associated Proteins Detected by BioID 137

Table 3.2 SPICE1 Associated Proteins Detected by BioID 140

Table 3.3 Summary of the Phenotypes of CEP120 and SPICE1 Associated Proteins in

Secondary Assays

158

Table 3.4 Summary of the NAP1L1 siRNAs and Their Associated Phenotypes 179

Table 3.5 Primary Antibodies Used in this Work 184

Table 3.6 Secondary Antibodies Used for Microscopy in this Work 185

Table 3.7 Sequences of Small Interfering RNA Sequences Used in this Work 186

Table 3.8 Primers for esiRNA Used in this Study 188

Table 3.9 Plasmids Used in this Study 189

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LIST OF FIGURES

Figure 1.1 ...................................................................................................................................... 36

Figure 1.2 ...................................................................................................................................... 38

Figure 1.3 ...................................................................................................................................... 40

Figure 1.4 ...................................................................................................................................... 42

Figure 2.1.......................................................................................................................................70

Figure 2.2 ...................................................................................................................................... 71

Figure 2.3.. .................................................................................................................................... 73

Figure 2.4 ...................................................................................................................................... 74

Figure 2.5 ...................................................................................................................................... 76

Figure 2.6 ...................................................................................................................................... 78

Figure 2.7 ...................................................................................................................................... 80

Figure 2.8 ...................................................................................................................................... 82

Figure 2.9 ...................................................................................................................................... 84

Figure 2.10 .................................................................................................................................... 86

Figure 2.11 .................................................................................................................................... 88

Figure 2.12. ................................................................................................................................... 90

Figure 2.13 .................................................................................................................................... 92

Figure 2.14 .................................................................................................................................... 94

Figure 2.15 .................................................................................................................................... 96

Figure 2.16 .................................................................................................................................... 98

Figure 2.17 .................................................................................................................................... 99

Figure 2.18 .................................................................................................................................. 101

Figure 3.1 .................................................................................................................................... 135

Figure 3.2 .................................................................................................................................... 144

Figure 3.3 .................................................................................................................................... 145

Figure 3.4 .................................................................................................................................... 146

Figure 3.5 .................................................................................................................................... 148

Figure 3.6 .................................................................................................................................... 150

Figure 3.7 .................................................................................................................................... 152

Figure 3.8 .................................................................................................................................... 154

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Figure 3.9 .................................................................................................................................... 156

Figure 3.10 .................................................................................................................................. 159

Figure 3.11 .................................................................................................................................. 160

Figure 3.12 .................................................................................................................................. 162

Figure 3.13 .................................................................................................................................. 164

Figure 3.14 .................................................................................................................................. 166

Figure 3.15 .................................................................................................................................. 168

Figure 3.16 .................................................................................................................................. 170

Figure 3.17 .................................................................................................................................. 172

Figure 3.18 .................................................................................................................................. 174

Figure 3.19 .................................................................................................................................. 175

Figure 3.20 .................................................................................................................................. 176

Figure 3.21 .................................................................................................................................. 178

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LIST OF ABBREVIATIONS

3D-SIM Three-dimensional structured illumination microscopy

APC Anaphase promoting complex

AP-MS Affinity purification followed by mass spectrometry

Co-IP Co-immunoprecipitation

CDK Cyclin-Dependent-kinase

CDKI Cyclin-Dependent-kinase Inhibitor

CTD Carboxy-terminal domain of a protein

DAPI 4', 6-diamidino-2-phenylindole

DNA Deoxyribonucleic acid

EM Electron microscopy

esiRNA Endoribonuclease-prepared siRNA

FSG Fish skin gelatin

G1 Gap 1 phase of the cell cycle

G2 Gap 2 phase of the cell cycle

HU Hydroxyurea

IP Immunoprecipitation

kDa kilo-Daltons

M Mitosis/mitotic-phase of the cell cycle

MAP Microtubule-associated protein

Min Minute

MS Mass spectrometry

MT Microtubule

MTOC Microtubule-organizing center

NA Numerical Aperture

NTD Amino-terminal domain of a protein

PAGE Polyacrylamide gel electrophoresis

PALM Photoactivated Localization Microscopy

PBS Phosphate buffered saline

PCM Pericentriolar material

RNA Ribonucleic acid

RNAi RNA interference

SCF Skp1, Cullin, F-box E3 ubiquitin ligase complex

SDS Sodium dodecyl sulfate

SEM Standard error of the mean

siRNA Small interfering RNA

S-phase Synthesis phase of the cell cycle

STED Stimulated Emission Depletion Microscopy

STORM Stochastic Optical Reconstruction Microscopy

TBST Tris buffered saline containing Tween-20

TEM Transmission electron microscopy

.tif Tagged image file (.tif)

γ-TuRC Gamma-tubulin Ring Complex

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LIST OF PROTEINS DISCUSSED

HUGO Gene Symbol/Protein Name SYNONYM USED

ANK2/Ankyrin2 ANK2

AURKA/Aurora-kinase A Aurora-A

BirA Biotin Ligase BirA

BTF3/Basic Transcription Factor 3 BTF

BTRCP/-transducin repeat containing E3 ub. Prot. ligase TRCP

CEP350/Centrosomal protein 350 kDa CAP350

CCDC77/coiled-coil domain containing 77 CCDC77

CCDC138/coiled-coil domain containing 138 CCDC138

CCNA2/cyclin A2 Cyclin-A

CCNE1/cyclin E1 Cyclin-E

CCNF/cyclin-F, FBXO1 FBXO1

CDC20/cell division cycle 20 CDC20

CDK1/Cyclin-Depedent-Kinase-1 CDK1

CDK2/Cyclin-Depedent-Kinase-2 CDK2

CDK5RAP2/CDK5 regulatory subunit assoc. prot. 2 CDK5RAP2

CDKN1A/cyclin-dependent-kinase-inhibitor 1A, p21,cip1 p21

CENPJ/Centromere protein J CPAP

CEP83/CCDC41 CEP83

CEP89/CCDC123 CEP89

CEP97/Centrosomal protein 97 kDa CEP97

CEP120/ Centrosomal protein 120 kDa CEP120

CEP131/Azi1 CEP131






CEP250/Centrosomal protein 250 kDa C-NAP1

CETN2/Centrin 2 CETN2/Centrin

CDH3/chromodomain helicase DNA binding protein 3 CDH3

CNTROB/Centrosomal BRCA2 interacting protein Centrobin

CP110/centriolar coiled-coil protein 110kDa CP110

CROCC/cilia rootlet coiled-coil Rootletin

CSPP1/Centrosome and spindle pole assoc. protein 1 CSPP1

CUL1/Cullin 1 CUL1

DCTN2/dynactin-2, p50 Dynactin Dynactin

DCTN4/dynactin-4, p62 dynactin DCTN4

ESPL1/extra spindle pole bodies homolog 1 Separase

FBF1/Fas binding factor 1 FBF1

FBXW5/F-box and WD repeat domain containing 5 FBXW5

FOP/ FGFR1 oncogene partner FOP

FZR1/fizzy/cell division cycle 20 related 1 Cdh1

HIST3H2BB/ histone cluster 3, H2bb HIST3H2BB

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HUGO GENE SYMBOL/NAME SYNONYM USED H2AFY/H2A histone family, member Y H2AFY

IPO(4,5,7,8)/Importin(4,5,7,8) IPO4, IPO5, IPO7, IPO8

KIAA0586 KIAA0586

KIAA1731 KIAA1731

KIAA0753 KIAA0753

KIF24/kinesin-family-member 24 KIF24

KIF11/kinesin-family-member 11, Eg5 Eg5

LRRC45/leucine rich repeat containing 45 LRRC45

MAP7/microtubule assoc. protein 7 MAP7

MAP9/microtubule assoc. protein 9 MAP9

MAP7D3/MAP7 domain containing 3 MAP7D3

MIB1/Mindbomb E3 ubiquitin protein ligase 1 MIB1

MTOR/Mechanistic Target of Rapamycin mTOR

MTUS1/microtubule assoc. tumour suppressor prot. 1 MTUS1

NAP1L1/nucleosome assembly prot.1-like 1 NAP1L1





NEDD1/neural precursor cell expressed, developmentally

down-regulated 1

NEDD1

NEK2/Nima-related kinase 2 NEK2

NIN/Ninein Ninein

NPM1/Nucleophosmin Nucleophosmin

OFD1/orofacial-digital syndrome 1 OFD1

OFD14/orofacial-digital syndrome 14 OFD14, C2cd3

PCNT/Pericentrin Pericentrin

PLK1/Polo-like kinase 1 PLK1



POC1A/POC centriolar protein A POC1 or POC1A

POC1B/POC centriolar protein B POC1 or POC1B

hPOC5/POC centriolar protein 5 hPOC5

POLH/Polymerase (DNA Directed) eta POLH

PP1CA/prot. phosphatase 1, catalytic subunit, isozyme PP1

RAD21/SCC1, Sister Chromatid Cohesion 1 SCC1

SASS6/spindle assembly protein 6 homolog (C.elegans) SASS6

SAV1/Salvador Homolog 1 hSAV1

SCLT1/sodium channel and clathrin linker 1 SCLT1

SPICE1/spindle and centrosome assoc. Prot.1 SPICE1

STIL/SCL/TAL1 interrupting locus STIL

STK3/MST2, serine/threonine kinase 3 MST2

TOP2(A,B)Topoisomerase-2(A,B) TOP2A,TOP2B

TP53/tumour protein p53 p53

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HUGO GENE SYMBOL/NAME SYNONYM USED TP54BP2/tumour protein p53 binding protein 2 p53BP2

TTLL/ tubulin tyrosine ligase-like family member (1-13) TTLL1, TTLL2, etc.

TUB1A/tubulin, 1a -tubulin

TUBB/tubulin, polypeptide -tubulin

TUBG1/tubulin, polypeptide tubulin

TUBGPC (2, 6)/tubulin, complex assoc. prot. (2,6) GCP2, GCP6

USP33/ ubiquitin specific peptidase 33 USP33

USP37/ ubiquitin specific peptidase 37 USP37

ZC2HC1A/Zinc-finger, C2HC-type containing 1A ZC2HC1A

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1. Chapter I: Introduction

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1.1. Functions of the Centrosome

My thesis describes the function of two proteins critical for centriole, and accordingly

centrosome, duplication. To understand why centrosomes and centrioles are worth studying, I

briefly discuss their cellular functions in the first part of this introduction. In the second part of

the introduction, I describe some of the diseases that can arise from, or be driven by, defects in

centrosome function and centriole assembly. Together, these sections of the introduction

highlight the importance of research aimed at furthering our understanding of these organelles.

1.1.1. The Functions of Centrosomes in Interphase

The human centrosome is a combination of tubulin based structures called centrioles and a

surrounding matrix of proteins called the pericentriolar material (PCM). The centrosome has

over a hundred associated proteins that perform a variety of cellular functions (Andersen et al.,

2003; Jakobsen et al., 2011). In interphase, the PCM that surrounds centrioles nucleates and

anchors the majority of the cellular microtubules, leading to the term ‘microtubule organizing

center’ (MTOC) (Gould and Borisy, 1977; Nigg and Raff, 2009; Woodruff et al., 2014). By

anchoring the majority of cellular microtubules in interphase, centrosomes play an important role

in cellular organization, shape, motility and intracellular signaling (Arquint et al., 2014; Fu et al.,

2015). For example, the presence of an intact microtubule network organized by the centrosome

is required for the correct positioning of the Golgi apparatus around the centrosome (Rios, 2014).

Golgi apparatus positioning around the centrosome is important in polarized cells. During cell

migration, for example into a wound in a wound-healing assay, centrosomes and the Golgi

become localized between the nucleus and the edge of the cell facing the wound in preparation

for directed cell migration (Yadav and Linstedt, 2011; Yadav et al., 2009). Disruption of Golgi

apparatus structure, or of the connection between the Golgi and the centrosome, prevents this

polarization of the cell and directed migration (Hurtado et al., 2011; Yadav et al., 2009).

Polarization of the centrosome and Golgi apparatus is also important for determining the position

of neuronal axon formation, and in T-cells is important for immunological synapse formation

and directed vesicular secretion (de Anda et al., 2005; Stinchcombe and Griffiths, 2014;

Stinchcombe et al., 2006; Yadav and Linstedt, 2011).

3

Progression through the cell cycle also requires a functional centrosome. In G1 there is a

centrosome-dependent checkpoint that arrests cells if centrosomes are not functioning properly

(Mikule et al., 2007; Pihan, 2013). The checkpoint can be activated by depletion of multiple

centrosomal proteins, before or during the G1 phase of the cell cycle, and is independent of

mitotic defects (Mikule et al., 2007; Pihan, 2013). In this case p53 is activated by p38

phosphorylation, which occurs at the centrosome, and p53 accumulates in the nucleus and drives

transcription of the CDK2 inhibitor p21, causing a block of cell cycle progression into S-phase

(Besson et al., 2008; El-Deiry, 1993; Kishi et al., 2001; Mikule et al., 2007; Sherr and Roberts,

1999). A distinct p53 induced G1 arrest occurs when centrosomes are lost due to prolonged

chemical inhibition of centriole duplication in human cell lines with functional p53 (Wong et al.,

2015). The same inhibitor, when applied to cancer cell lines lacking normal p53, does not stop

them from progressing through the cell cycle (Wong et al., 2015). Upon washout of the

chemical inhibitor, cancer cell lines initiate de novo assembly of centrioles, whereas G1 arrested

cells with functional p53 are not able to enter the cell cycle or assemble centrioles (Wong et al.,

2015). Importantly, the p53-dependent G1 arrest upon chemically induced loss of centrioles

does not involve p38 activation, and is thus different from that observed when centrosomal

proteins are disrupted (Mikule et al., 2007; Wong et al., 2015). These results are consistent with

two distinct p53-dependent checkpoints monitoring cells for the presence and function of

centrosomes as a requisite for progress through the cell cycle. p53 activation in response DNA

damage occurs through phosphorylation by the ATM kinase, but ATM is not involved in either

of the centrosome related G1 arrests (Ciciarello, 2001; Mikule et al., 2007; Shiloh, 2001;

Tritarelli et al., 2004; Wong et al., 2015). Finally, prolonged mitosis is also a known trigger for

p53 activation, through another pathway as discussed in the next section (Chavali et al., 2014;

Insolera et al., 2014; Mikule et al., 2007; Mogensen et al., 2000; Pihan, 2013; Uetake and Sluder,

2010).

1.1.2. The Functions of Centrosomes in Mitosis

In mitosis, each centrosome contains two tightly connected centrioles surrounded by PCM that

nucleates and anchors microtubules to segregate DNA (Gould and Borisy, 1977; Kuriyama and

Borisy, 1981). Centrosomes are localized to the mitotic spindle pole, to which the mitotic

spindle microtubules are anchored. Despite their normal localization at the spindle poles, mitosis

4

has been observed in the absence of centrioles. When centrioles are disassembled in HeLa cells,

mitosis proceeds with spindle poles lacking centrioles (Bobinnec et al., 1998a; Debec et al.,

2010). Similarly, in monkey fibroblast cells, laser-ablation of one or both centrosomes does not

prevent bipolar spindle assembly (Khodjakov et al., 2000). In the context of whole organisms,

results have been more variable. For example, in Drosophila that lack centrioles, asymmetric

cell division is abnormal and spindle formation is slowed during development (Basto et al., 2006;

Debec et al., 2010). These flies, however, die due to failure of cilia formation rather than mitotic

defects (Basto et al., 2006; Debec et al., 2010). Recently, analogous mouse models have been

generated that lack a protein essential for centriole assembly (CPAP/SAS-4), and unlike

Drosophila the mouse embryos die at a stage earlier than mouse embryos that cannot make cilia

(Bazzi and Anderson, 2014). In these acentriolar embryos, mitosis is delayed by the lack of

centrosomes, and widespread p53-dependent apoptosis occurs leading to lethality (Bazzi and

Anderson, 2014). Similar effects were seen when microtubule poisons were used to prolong

mitosis in cultured embryonic cells, indicating that in the context of mouse embryos one or more

checkpoints activate p53-mediated programmed cell death in cases where the mitotic spindle is

slow to form (Bazzi and Anderson, 2014; Fridman and Lowe, 2003). Mammalian female

meiotic cell divisions occur without centrosomes, and spindle assembly in this case is achieved

by a distinct pathway (Manandhar, 2005; Ohkura, 2015). Therefore, in some contexts

centrosomes ensure timely mitotic divisions that are essential during development, but in

cultured somatic cells where centrosomes are ablated, spindle formation is still achieved.

Asymmetric cell divisions produce two distinct daughter cells, for example a progenitor (stem)

cell and a differentiated cell (Morrison and Kimble, 2006). In both Drosophila and mouse

development, a requirement has been demonstrated for centrosomes in regulating the

maintenance of progenitor pools through ensuring asymmetric cell division (Wang et al., 2009;

Yamashita et al., 2007). This is achieved in some contexts through centrosome-based control of

mitotic spindle orientation to ensure retention of only one daughter cell within a stem-cell niche

(Yamashita and Fuller, 2008). For example, in Drosophila male germline stem cells, the older

centrosome maintains close contact with the apical cortex throughout the cell cycle, setting up

spindle orientation to ensure division that leads to a daughter cell being excluded from the niche

(Yamashita and Fuller, 2008; Yamashita et al., 2003, 2007). In the case of Drosophila

neuroblast stem cells, a similar microtubule-based anchoring of one centrosome to the cortex

5

near the niche is also observed, and in this context the younger (daughter) centrosome is retained

(Conduit and Raff, 2010; Januschke et al., 2013). In Drosophila embryos engineered to lose

centrioles early in development, asymmetric cell division still succeeds 70% of the time in larval

neuroblasts, so evidently mechanisms exist that can achieve asymmetric divisions in the absence

of centrioles (Basto et al., 2006). In embryonic mice, the basal epidermal cells undergo

asymmetric cell division with specified spindle orientation, where one daughter cell is retained in

the original niche and the other daughter cell divides perpendicular to the basal layer and

becomes differentiated (Lechler and Fuchs, 2005). Centrosomes may also carry cell-fate

determinants. For example, in the brains of developing mouse embryos, radial glia stem cells

inherit the older centrosome in each asymmetric division (Wang et al., 2009). When the

expression of the mother-centriole subdistal appendage protein Ninein is knocked down,

centrosome inheritance becomes random with subsequent depletion of radial glia progenitor

cells, and this depletion of progenitors is independent of spindle orientation (Bouckson-Castaing

et al., 1996; Mogensen et al., 2000; Wang et al., 2009). Thus centrosome inheritance,

presumably through differential retention of proteins to the mother centrosome, can influence

cell differentiation (Wang et al., 2009). Overall there is good evidence that centrosomes are

required for both timely cell division during development and the maintenance of stem cell

niches through asymmetric cell divisions.

1.1.3. The Functions of Centrosomes in Cilia Formation

Outside of mitotic cell division, the centrosome has an important role in many tissues as a basal

body, the structure from which cilia and flagella are built (Bettencourt-Dias et al., 2011;

Ishikawa and Marshall, 2011; Nigg and Raff, 2009). Cilia and flagella are structures that are

formed by the fusion of the mother centriole with the cell membrane, followed by the elongation

of the distal portion of the mother centriole to form a ciliary axoneme (Bettencourt-Dias et al.,

2011; Goetz and Anderson, 2010; Ishikawa and Marshall, 2011; Nigg and Raff, 2009). The

ciliary axoneme is isolated from the cytoplasm by a barrier (transition zone) but remains

surrounded by the cell membrane along its length (Bettencourt-Dias et al., 2011; Goetz and

Anderson, 2010; Ishikawa and Marshall, 2011; Nigg and Raff, 2009). Cilia can be motile or

non-motile, and cells can contain one (primary) cilium or many (motile) cilia (Bettencourt-Dias

et al., 2011; Ishikawa and Marshall, 2011; Nigg and Raff, 2009). Primary (non-motile) cilia

6

allow cells to receive and internalize extracellular signals and activate critical signaling pathways

(Goetz and Anderson, 2010). Motile cilia and flagella rely on the sliding of microtubule motors

between microtubule doublets to allow them to ‘beat’, i.e. bend and deform in a coordinated

effort to generate fluid movement in the extracellular space, or cell propulsion in the case of

flagella (Lindemann and Lesich, 2010; Roberts et al., 2013). The importance of these organelles

are made clear by the variety of developmental defects that can occur as a result of failure of cilia

formation, collectively called the ‘ciliopathies’ (Goetz and Anderson, 2010; Nigg and Raff,

2009; Sharma et al., 2008; Tobin and Beales, 2009; Waters and Beales, 2011). These include

patterning defects such as polydactyly or situs inversus/heterotaxy, orofacialdigital syndrome

and Jeune asphyxiating thoracic dystrophy, as well as defects in kidneys, retina and brain

development (Pennekamp et al., 2015; Sharma et al., 2008; Waters and Beales, 2011). Both

motile cilia and primary cilia have been shown to be important for proper development (Sharma

et al., 2008; Tobin and Beales, 2009). Mouse embryos that lack cilia and centrioles due to the

absence of the centriole assembly protein STIL show developmental failures consistent with loss

of Sonic Hedgehog (Shh) signaling (David et al., 2014; Huangfu et al., 2003; Izraeli et al., 1999).

On the other hand, induction of excess centriole formation in cultured mouse embryonic

fibroblasts leads to the formation of multiple primary-cilia per cell upon serum starvation

(Mahjoub and Stearns, 2012). The formation of excess cilia diffuses the localization of

Smoothened (Smo) to the ciliary membrane and leads to a reduction of downstream

transcriptional activation following addition of Shh protein to the cells (Mahjoub and Stearns,

2012). Hence control of centriole number is important for ensuring formation of a single

primary cilium capable of activating cellular responses to external signals (Mahjoub and Stearns,

2012).

The breaking of left-right (LR) symmetry during development requires the function of cilia

within a structure called the ‘node’ (Blum et al., 2014; Lee and Anderson, 2008). The nodal cilia

exhibit a distinct rotational movement that establishes a directed flow in the extracellular fluid

towards the left side of the embryo (called leftward-flow), and this flow is both necessary and

sufficient to break the symmetry of the developing embryo (Blum et al., 2014; Lee and

Anderson, 2008; Nonaka et al., 1998, 2002). The fluid current is in turn detected by sensory cilia

on cells to the left of the node, which then initiate a signaling cascade that is unique to one side

of the embryo (Blum and Vick, 2015; Blum et al., 2014; Lee and Anderson, 2008). Thus cilia

7

also play a central role in the breaking of embryonic symmetry and correct establishment of the

vertebrate body plan. The many important roles of cilia during development all depend on

properly functioning centrosomes capable of acting as basal bodies to facilitate their assembly.

The presence of centrioles correlates strongly with the ability to form cilia or flagella throughout

various organisms, and both centrioles and cilia/flagella are thought to have been present in the

last eukaryotic common ancestor (LECA) (Azimzadeh, 2014; Carvalho-Santos et al., 2011;

Debec et al., 2010). Mitotic spindle pole formation and accurate DNA segregation had been

widely thought to be the important role of centrioles, however as discussed, spindle formation

can be achieved without centrioles (Debec et al., 2010; Dumont and Desai, 2012). In organisms

that do not make cilia or flagella, such as yeasts or certain amoebas (Dictyostelium discoidum),

their microtubule organizing centers (called spindle pole bodies and nuclear associated bodies,

respectively) do not contain centrioles and are structurally distinct from centrosomes

(Azimzadeh, 2014; Debec et al., 2010). Further, in planarians only their multiciliated cells

assemble centrioles, and planarians lack the genes that encode certain conserved PCM proteins

required to generate functional centrosomes (Azimzadeh, 2014; Azimzadeh et al., 2012).

Accordingly, it has been proposed that the essential role of a centriole that results in its

maintenance through evolution may be the ability to template cilia/flagella, and that centrioles

exist as centrosomes at mitotic spindle poles primarily to ensure daughter cells inherit the

capacity to make cilia/flagella in some species (Azimzadeh, 2014; Carvalho-Santos et al., 2011;

Debec et al., 2010; Friedländer and Wahrman, 1970). Indeed, in human cells, when de novo

assembled centrioles are not tethered to parental centrioles within PCM-nucleating centrosomes,

they are randomly segregated between daughter cells (Wang et al., 2011). Overall, in some

eukaryotes centrioles have been lost along with cilia/flagella and other microtubule organizing

centers evolved, whereas in others the formation of cilia and flagella is achieved through an on-

demand de novo assembly of centrioles, and in species such as humans centrioles act as

centrosomes in mitosis at least partly to ensure the centrioles are passed on equally to every cell.

Aside from the ancestral role of centrosomes in centriole distribution, additional essential

functions of centrosomes have since evolved in complex multicellular organisms (Debec et al.,

2010). For example, as discussed in the previous sections, there are essential roles for

centrosomes in mitosis and interphase. Further, centrioles are absent from female oocytes that

retain PCM proteins, and in some species including humans the sperm donates centrioles without

8

PCM during fertilization to activate embryogenesis (Debec et al., 2010; Dumont and Desai,

2012; Schatten and Sun, 2010). Thus centrioles may have been co-opted to help regulate the

onset of embryonic development in some sexually reproducing species (Debec et al., 2010;

Dumont and Desai, 2012; Schatten and Sun, 2010).

1.2. The Centrosome in Disease

1.2.1. Cancer

In 1914 Theodore Boveri published a book describing the centrosome as a duplicating cellular

structure required for cell division, including the observation that abnormal numbers of

centrosomes gave abnormal cell divisions that gave rise to aneuploidy and the hypothesis that

aneuploidy could lead to cancer (recently translated in Boveri, 2008; Hansford and Huntsman,

2014; Scheer, 2014). Indeed, aneuploidy and extra centrosomes are both now known as

hallmarks of cancer (Chan, 2011; Godinho and Pellman, 2014). In mice lacking the tumour

suppressor gene p53, centrosome amplification is observed and mice develop tumors

(Donehower et al., 1992; Fukasawa, 2005, 2008; Fukasawa et al., 1997). There is debate as to

whether centrosome numerical abnormalities initiate cancer, although they are thought to drive it

(Nigg, 2002; Nigg and Raff, 2009). Extra centrosomes are known to lead to multipolar cell

divisions which are observed in cancers (Godinho and Pellman, 2014; Nigg, 2002). Experiments

using cancer cell lines have revealed that multipolar cell divisions normally result in cells that

eventually die, suggesting that this is not a common initial cause of cancer (Ganem et al., 2009).

Cancer cells with excess centrosomes can form bipolar spindles through a process called

centrosome clustering, to avoid potentially fatal multipolar divisions (Ganem et al., 2009;

Quintyne et al., 2005). These bipolar spindles form after a transient multipolar state during

mitosis, and during this temporary multi-polar state misaligned centrosomes can form

attachments to either kinetochore through spindle microtubules (Ganem et al., 2009). Later in

mitosis, centrosomes from the ‘extra’ poles are pulled together into a bipolar formation, and this

results in kinetochores with attachments to both spindle poles (merotelic) (Ganem et al., 2009;

Godinho and Pellman, 2014; Gregan et al., 2011; Quintyne et al., 2005; Ring et al., 1982). As a

result of merotelic attachment, chromosomes are incorrectly segregated leading to aneuploidy, or

9

lag behind during pole-ward movement in anaphase, resulting in DNA damage (Ganem et al.,

2009; Janssen et al., 2011). Recently, experiments have tested the impact of extra centrosomes

on developing organisms. When embryonic mice are engineered to have extra centrosomes

within their neural cells, multipolar mitosis leads to cell death and microcephaly, rather than

over-proliferation (Marthiens et al., 2013). In Drosophila, neural stem cells generated to have

excess centrosomes initiate tumour formation upon transplantation to an adult Drosophila (Basto

et al., 2008). While this suggests tumorigenic potential in cells with too many centrosomes, the

caveat here is that the flies that have excess centrosomes are not themselves prone to genomic

instability or other obvious defects (Basto et al., 2008). Thus in many models tested so far,

centrosome amplification alone appears unlikely to initiate cancer directly (Godinho and

Pellman, 2014; Nigg, 2002). In a fly model where asymmetric stem-cell division is disrupted by

targeting cell polarity regulators, these stem-cells generate tumors upon transplantation to an

adult fly (Caussinus and Gonzalez, 2005). Despite initially being genetically normal with two

centrosomes, a fraction of the tumors that later developed from these cells obtained both extra

centrosomes and aneuploidy (Caussinus and Gonzalez, 2005). Based on this finding, coupled

with the common occurrence of superfluous centrosomes in cancers, the hypothesis that extra

centrosomes drive aneuploidy in cancer but don’t commonly initiate it, has been proposed

(Godinho and Pellman, 2014; Nigg, 2002).

The role of centrosome amplification in tumour aggressiveness has been linked to an effect of

interphase centrosomes on cellular migration. PLK4 is a kinase required for centriole

duplication, and when overexpressed PLK4 causes the formation of multiple centrioles around a

parental centriole (Bettencourt-Dias et al., 2005; Habedanck et al., 2005; Kleylein-Sohn et al.,

2007). In 3D MCF10A mammary epithelia cell cultures cells that have extra centrioles due to

PLK4 overexpression have increased migration, decreased cell-cell adhesion, and form dynamic

cellular protrusions that promote invasiveness in culture (Godinho et al., 2014). The extra

centrosomes in these cells cluster in interphase, and accordingly nucleate greater numbers of

microtubules, leading to Rac1 activation and RhoA deactivation (Godinho et al., 2014). Rac1

activation leads to lamellipodia and cell migration through stimulation of actin filament

formation at the leading edge of cells (Burridge and Wennerberg, 2004; Lawson and Burridge,

2014; Sadok and Marshall, 2014). The invasive phenotype, and formation of cellular protrusions

were reversed by depletion of CEP192, a protein required for centriole duplication and

10

microtubule nucleation within the PCM (Godinho et al., 2014; Gomez-Ferreria et al., 2007;

Joukov et al., 2010, 2014; Zhu et al., 2008). CEP192 depletion reduced the activation of Rac1,

presumably through reducing-tubulin and microtubule nucleation at centrosomes (Godinho et

al., 2014; Zhu et al., 2008). Engineered aneuploidy in MCF10A cells in the absence of

centrosome amplification did not lead to the formation of invasive cellular protrusions (Godinho

et al., 2014). Thus the amplification of centrosomes can likely drive metastasis through

changing the interphase microtubule landscape, providing a mechanism for centrosome

involvement in aggressive cancers beyond aneuploidy (Godinho et al., 2014).

1.2.2. Developmental Diseases

Aside from ciliopathies, several developmental diseases have been linked to proteins of the

centrosome. Notably, these diseases share the characteristic of decreasing the size of patient

tissues, as in the case of primordial dwarfism or microcephaly wherein the brain is under-sized

(Barbelanne and Tsang, 2014; Chavali et al., 2014; Faheem et al., 2015; Nigg et al., 2014).

Autosomal recessive primary microcephaly (MCPH) has been linked to 13 genes to date

(MCPH1-13) and many of these are key centriole duplication proteins (Barbelanne and Tsang,

2014; Chavali et al., 2014; Faheem et al., 2015; Nigg et al., 2014). MCPH6, 7, 8, 9 and 13 have

been identified as the essential centriole duplication proteins CPAP, STIL, CEP135, CEP152 and

CEP63 respectively (Barbelanne and Tsang, 2014; Nigg et al., 2014). The other MCPH genes

can be broadly described as genes that affect mitosis or proliferation (Barbelanne and Tsang,

2014; Chavali et al., 2014; Faheem et al., 2015; Nigg et al., 2014).

How do defects in proteins affecting centriole duplication or centrosome function lead to these

developmental effects? There are two major models for how centrosome defects contribute to

microcephaly; one is disruption of the asymmetric versus symmetric cell division balance, and

the other is a reduction in cell viability as a result of mitotic defects (Chavali et al., 2014).

Mouse models have shown that loss of mother-centrosome functionality leads to stem cell

depletion presumably by disrupting asymmetric cell division (Wang et al., 2009; Yamashita et

al., 2007). Conversely, in mouse models that lack centrosomes, delays in mitosis cause cell

death and microcephaly (Bazzi and Anderson, 2014; Insolera et al., 2014). When p53 is also

removed in such mice to block apoptosis, microcephaly is suppressed without restoration of

correct spindle orientation (Chavali et al., 2014; Insolera et al., 2014). This suggests that in

11

developing mammalian brains, the predominant effect of loss of centrosome function may be

apoptosis driven by inefficient mitosis (Insolera et al., 2014). Consistent with this idea,

depletion of an MCPH protein (WDR62/MCPH2) causes prolonged mitosis with fragmentation

of the PCM after bipolar spindle formation (Barbelanne and Tsang, 2014; Bogoyevitch et al.,

2012). Mice engineered to have reduced WDR62 expression display dwarfism and

microcephaly, with widespread mitotic spindle checkpoint arrest and programmed cell death

leading to a reduction in the progenitor pools in their brains (Chen et al., 2014). Extra centrioles

may also cause developmental microcephaly. Mice which constitutively over-express PLK4

form extra centrioles, and are born smaller than normal (dwarfism) and with microcephaly

(Marthiens et al., 2013). This result is attributed to programmed cell death that occurs in

response to aneuploidy stemming from multipolar mitoses (Marthiens et al., 2013). Consistent

with this idea, several of the MCPH patient mutations to centrosomal genes can cause centriole

amplification (Nigg et al., 2014). Overall, abnormalities of centriole number or centrosome

function are causative in human developmental diseases.

1.3. The Structure of the Centrosome

To understand the results described in the data chapters of this thesis, it is important to have an

understanding of the anatomy of the centrioles and centrosomes. This section of the introduction

will describe the general anatomy of a centriole, and make clear the distinction between a

centriole and a centrosome. The structures of both the centrosome and centriole(s) change

through the cell cycle, and these changes are the topic of the subsequent section of the

introduction.

1.3.1. Centrioles

The structure of the centrosome is maintained by, and organized around the centrioles (Abal et

al., 2005; Bobinnec et al., 1998a; Bornens et al., 1987; Kuriyama and Borisy, 1981). The human

centriole is a barrel-shaped structure with a nine-fold symmetrically organized array of

microtubule structures individually referred to as ‘microtubule triplets’ (Bernhard and De

Harven, 1956; Winey and O’Toole, 2014). and tubulin hetero-dimers can form polymers

called ‘protofilaments’, and microtubules are comprised of a number of protofilaments arranged

12

side-by-side to form a tube (Amos and Klug, 1974; Meurer-Grob et al., 2001; Nogales et al.,

1999). The exact number of protofilaments in a microtubule can vary between cell types both

within and between organisms, but the most often observed microtubules in eukaryotes are made

of 13 protofilaments, having a diameter of ~25 nm, and with the tubulin dimers having a

periodicity of 8 nm along the long-axis (Amos and Klug, 1974; Choi et al., 2009; Meurer-Grob

et al., 2001; Nogales et al., 1999). The structure of the centriolar microtubule triplets from the

model organisms Chlamydomonas and Trichonympha have been studied in sub-nanometer

resolution using cryo-electron tomography (Guichard et al., 2013; Li et al., 2012; Winey and

O’Toole, 2014). The centriole microtubule triplets in these organisms have a single complete

microtubule with 13 protofilaments, connected to two partial microtubules with 10

protofilaments each (Guichard et al., 2013; Li et al., 2012; Winey and O’Toole, 2014). These

microtubules are called the “A”, “B” and “C” microtubules, respectively (Guichard et al., 2013;

Li et al., 2012; Winey and O’Toole, 2014). In mature centrioles the C microtubule extends only

partway up the centriole, and the distal portion is a doublet made up of only the A and B

microtubules (Bornens et al., 1987; Ibrahim et al., 2009; Paintrand et al., 1992).

In most organisms that have centrioles, the nine microtubule triplets of the centriole are initially

organized around a central structure referred to as the ‘cartwheel’ based on its structure having a

central ‘hub’ (ring) from which emanates 9 ‘spokes’ at 40° angles that make contact with the

triplets (Figure 1.1) (van Breugel et al., 2011; Kitagawa et al., 2011; Winey and O’Toole, 2014).

In Caenorhabditis elegans, centrioles differ from the triplet and cartwheel organization of human

centrioles, as their centrioles contain nine single microtubules arranged around a central tube

(Pelletier et al., 2006; Sharp et al., 1999; Winey and O’Toole, 2014). The diameter of the human

centriole is approximately 250 nm, and this diameter is flexible (Ibrahim et al., 2009; Paintrand

et al., 1992; Winey and O’Toole, 2014). Each human centriole cartwheel is composed of 9

dimers of SASS6, where the carboxy-terminal domains (CTDs) of SASS6 point outwards as the

‘spokes’ of the cartwheel towards the microtubule triplets, and facilitate SASS6 dimerization

(van Breugel et al., 2011; Kitagawa et al., 2011). The amino-terminal domains (NTDs) of

SASS6 comprise the ‘central hub’, and their interactions facilitate formation of the 9-dimer hub

(van Breugel et al., 2011; Kitagawa et al., 2011). In other model organisms, including

Drosophila and Chlamydomonas, SASS6 homologs likewise are critical for correct 9-fold

symmetry and formation of the central cartwheel (Nakazawa et al., 2007; Rodrigues-Martins et

13

al., 2007). Similar to the human cartwheel, the central tube in C.elegans requires SAS-6 and

SAS-5, the homologs of SASS6 and STIL respectively (Arquint et al., 2012; Leidel et al., 2005;

Pelletier et al., 2006). Thus despite the variations in cartwheel structure, many centriole

assembly proteins are conserved across species, as is the nine-fold symmetry of centrioles. In

some cases, non-nine-fold centrioles are observed, as in male germ line cells of the gnat Sciara

where massive centrioles are observed with over 20 centriolar microtubules (Gönczy, 2012;

Phillips, 1967). The lumen of these giant centrioles does not appear to contain a cartwheel

(Phillips, 1967). How these centrioles are assembled has not been studied.

The proximal end of a newly formed centriole (procentriole) contains multiple cartwheels.

Recently, the application of cryo-electron tomography in the model organism Trichonympha has

revealed the structure of the cartwheel stacks and their connections to the microtubule triplets in

basal bodies (Figure 1.1) (Guichard et al., 2012, 2013). Trichonympha was used in this work

because its basal bodies have substantially longer centrioles with more cartwheels than humans

or other model organisms (Guichard et al., 2012, 2013; Hirono, 2014). Trichonympha

cartwheels are stacked as pairs where the cartwheel spoke tips of each pair of cartwheels make

contact at a structured dubbed the ‘spoke junction’ (SP-J), which is joined to the spoke-tip (SP-

T) which in turn connects to the A-tubule of the triplet through a structure called the ‘pinhead’

(Figure 1.1) (Guichard et al., 2012, 2013; Hirono, 2014). These observations have yet to be

repeated outside Trichonmypha. Similar spacing of cartwheels is observed within the centriole

lumen in Chlamydomonas, so inter-cartwheel organization may be a conserved feature among

organisms where centrioles contain cartwheels (Hirono, 2014).

There are discs of unknown composition and function within the distal lumen of human

centrioles, making connections with the triplet and doublet microtubules (Ibrahim et al., 2009;

Winey and O’Toole, 2014). These disks are tilted with respect to the longitudinal axis of the

centriole, unlike the cartwheels, and their composition and function is unknown (Ibrahim et al.,

2009; Winey and O’Toole, 2014). The distal lumen of the human centriole contains proteins that

include Centrin and an interacting partner protein hPOC5 (Azimzadeh et al., 2009; Kleylein-

Sohn et al., 2007; Paoletti et al., 1996). The structure and function of the distal lumen of human

centrioles is much less studied than the cartwheel structures of the proximal lumen.

14

At full maturity, the distal portion of a human centriole is decorated with a set of nine distal and

nine sub-distal appendages (Bornens et al., 1987; Brito et al., 2012; Jana et al., 2014; Paintrand et

al., 1992; Winey and O’Toole, 2014). There are several proteins associated with subdistal

appendages. Ninein is a subdistal appendage protein, and it is required for anchoring of

microtubules to the centrosome (Brito et al., 2012; Delgehyr, 2005; Mogensen et al., 2000;

Shinohara et al., 2013; Winey and O’Toole, 2014). Likewise, EB1 is another protein of the

subdistal appendages that plays a role in microtubule anchoring (Askham et al., 2002; Brito et

al., 2012; Louie, 2004). EB1 recruitment to centrosomes requires an interacting protein called

FOP, and FOP in turn requires an interacting protein called CAP350 for its centrosome

localization (Brito et al., 2012; Yan et al., 2006). Depletion of any of these three proteins leads

to loss of microtubule organization by the interphase centrosomes, and FOP and CAP350 are

dispensable for microtubule nucleation but required for microtubule anchoring at the centrosome

following nocodazole washout (Louie, 2004; Yan et al., 2006). CEP170 is another subdistal

appendage protein that localizes to microtubules when overexpressed, and its depletion alters the

microtubule cytoskeleton (Brito et al., 2012; Guarguaglini et al., 2005). The subdistal appendage

protein Centriolin/CEP110 is required along with ODF2 to recruit several proteins important for

recycling endosome function to the mother centriole, and Centriolin is required to target SNARE

and exocyst complexes to the midbody in a process essential for abscission during cytokinesis

(Gromley, 2003; Gromley et al., 2005; Hehnly et al., 2012). Lastly, the -tubulin protein is

localized to subdistal appendages, and its depletion blocks the formation of microtubule asters

and centriole duplication following sperm centrosome addition in Xenopus egg extracts (Chang

et al., 2003). In summary, the functions of subdistal appendages include anchoring microtubules

to the centrosome, and the localization of proteins important for recycling endosome formation.

Mature centrioles are also the templates from which cilia and flagella are assembled. The distal

centriole appendages are required for ciliogenesis, as evident by the fact that disruption of distal

appendage proteins inhibits this process. ODF2 is required for cilia formation, and assembly of

both distal and subdistal appendages (Ishikawa et al., 2005). The function of ODF2 in distal

appendage assembly and cilia formation requires a specific isoform of ODF2, now called

Cenexin1 (Chang et al., 2013). C2CD3/OFD14 is critical for the localization of five distal

appendage proteins; CEP164, SCLT1, CCDC41/CEP83, CCDC123/CEP89, and FBF1 (Ye et al.,

15

2014). CEP164, SCLT1, CEP83, CEP89, FBF1 are all proteins required for cilia formation

(Graser et al., 2007; Tanos et al., 2013). OFD14 is also required for the assembly of the distal

portion of centrioles, so the failed distal appendage formation is likely an indirect effect rather

than a direct recruitment of the five proteins by OFD14 (Thauvin-Robinet et al., 2014; Ye et al.,

2014). OFD1, an interacting protein of OFD14, is also required for distal appendage assembly

and cilia formation (Singla et al., 2010). This is probably also due to a role in regulation of

centriole length, as depletion of OFD1 leads to elongation of the distal portion of the centriole

and failure of distal appendages to form (Singla et al., 2010). These two proteins highlight an

important role for regulators of centriole length in proper cilia formation (Thauvin-Robinet et al.,

2014). CEP83 is required for recruitment of the CEP164, SCLT1, CEP89 and FBF1, while

CEP164 and FBF1 were also dependent on SCLT1 for localization (Tanos et al., 2013).

Disruption of the distal appendage proteins by CEP83 depletion inhibits membrane docking of

the mother centriole during ciliogenesis, consistent with distal appendages playing a critical role

in that step of ciliogenesis (Tanos et al., 2013).

1.3.2. The PCM

The pericentriolar material is organized by centrioles and plays a critical role in nucleating

microtubules at the centrosome (Gould and Borisy, 1977). This function of the PCM is achieved

through localization and regulation of -tubulin ring complexes (-TuRC) (Kollman et al., 2010,

2011). The -TuRC is a conserved multi-subunit complex including -tubulin that acts as a

template to seed the assembly of microtubules (Kollman et al., 2010, 2011). The PCM itself was

until recently viewed as a disorganized mass of proteins, due to limitations in both the resolution

of immunofluorescence microscopy and the capabilities of immuno-electron microscopy to

differentiate between multiple antigens in the same sample (Mennella et al., 2014). Innovations

in fluorescence microscopy have provided the additional resolution required to for examination

of the PCM structure, and microscopy in human and Drosophila cells shows that individual

proteins of the PCM occupy distinct regions surrounding the centrioles (Fu and Glover, 2012;

Lawo et al., 2012; Mennella et al., 2012, 2014; Sonnen et al., 2012). Pericentrin and

CDK5RAP2 are two large scaffold proteins of the PCM, and Pericentrin can be co-

immunoprecipitated with CDK5RAP2 (Kraemer et al., 2011; Wang et al., 2010). Whereas most

PCM proteins studied to date are organized within distinct regions around the centrioles,

16

Pericentrin and CDK5RAP2 appear to span larger regions of the PCM and are polarized with

regard to the centriole, having their CTDs closer to the centrioles (Lawo et al., 2012).

Overexpression of Pericentrin or CDK5RAP2 in interphase results in an excess of PCM around

centrioles, reminiscent of the PCM expansion normally observed in mitosis, with both proteins

and -tubulin recruited to the enlarged PCM (Lawo et al., 2012). CDK5RAP2 is critical for

interphase -TuRC recruitment to the centrosomes and depletion of CDK5RAP2 reduces -

tubulin localization and microtubule organization (Fong et al., 2008). In Drosophila, there is

evidence that the homolog of CDK5RAP2 (CNN) controls the size of the PCM by regulating the

rate of its incorporation. In flies lacking CNN expression, the amount of GFP-CNN expressed is

proportional to how fast GFP-CNN becomes incorporated into the PCM around centrioles, and to

the total amount of CNN that gets incorporated (Conduit et al., 2010). The incorporation of

CNN into the PCM occurs first with CNN appearing at the wall of the centriole, then migrating

outwards into the peripheral PCM (Conduit et al., 2010). In C. elegans, SAS-4 depletion leads to

shorter centrioles, and along with shorter centrioles a reduction in the amount of PCM observed

at the centrosomes (Kirkham et al., 2003; Schmidt et al., 2009; Tang et al., 2009a). When

centrioles are abnormally elongated, as in the case of SAS-4 overexpression, they accumulate

PCM along their lengths and this additional PCM is capable of nucleating microtubules

(Kohlmaier et al., 2009). In human cells, destruction of centrioles by antibody injection leads to

fragmentation of the PCM (Bobinnec et al., 1998a). Only mature centrioles are able to nucleate

PCM, and only after they have passed through mitosis successfully (Wang et al., 2011). When

de novo centriole assembly is induced, newly formed procentrioles are unable to nucleate

microtubule regrowth in interphase, and in mitosis are randomly segregated between daughter

cells when they lack attachment to a mature centriole (Wang et al., 2011). When procentriole

assembly is inhibited, the amount of PCM organized at each mitotic spindle pole is comparable

to that of a spindle pole with a centriole pair (Wang et al., 2011). Newly formed centrioles gain

the ability to nucleate PCM only after passage through mitosis and entry to G1 in the cell cycle,

and this requires the activity of PLK1 around the G2/M transition, but is independent of centriole

engagement (Wang et al., 2011). In sum, mature centrioles are critical to the organization of

PCM, and their length is also proportional to how much PCM they can recruit and ultimately

how they function as centrosomes.

17

1.4. The Centrosome Cycle

The duplication of centrioles and centrosomes is regulated throughout the cell cycle, with distinct

events predictably occurring in specific phases (Figure 1.2) (Vorobjev and YuS, 1982; Winey

and O’Toole, 2014). Following mitosis, a cell contains a pair of loosely connected (disengaged)

centrioles, capable of acting as sites for the formation of new centrioles (procentrioles) (Chrétien

et al., 1997; Firat-Karalar and Stearns, 2014). In S-phase, the procentrioles are formed and begin

to elongate (Chrétien et al., 1997; Kuriyama and Borisy, 1981). By G2, the cell has two parental

centrioles, each with a single tightly associated procentriole. These centrioles must separate to

become distinct centrosomes, and accumulate additional PCM, in preparation for the next mitotic

division. The steps of the centrosome cycle are described below.

1.4.1. Disengagement

When procentrioles assemble, they form in close association with the wall of the parental

centriole, and remain tightly anchored there throughout S phase and G2. Late in mitosis, each

parental centriole loses tight association with its procentriole, in a process known as

disengagement. Disengagement is regulated by PLK1 and the protease Separase (Firat-Karalar

and Stearns, 2014; Tsou and Stearns, 2006; Tsou et al., 2009). Separase is known to target the

Scc1 subunit of the Cohesin protein complex, and the cleavage of Cohesin by Separase is a key

step in mitosis that allows separation of sister chromatids (Firat-Karalar and Stearns, 2014; Hauf

et al., 2001). Separase cleavage of the cohesin subunit SCC1 is critical for centriole

disengagement in vitro using purified centrosomes, however, in vivo expression of a non-

cleavable SCC1 does not block centriole disengagement (Schöckel et al., 2011; Tsou et al.,

2009). Further, in the absence of Separase, disengagement can eventually occur during the

subsequent interphase (Tsou et al., 2009). Chemical inhibition of PLK1 in G2, but not later in

mitosis, inhibits centriole disengagement (Tsou et al., 2009). A combination of Separase

depletion and PLK1 inhibition in G2/early-mitosis strongly inhibited centriole disengagement

throughout S-phase of the following cell cycle, demonstrating that a combination of PLK1

activity early in mitosis with Separase activity are important for disengagement (Tsou et al.,

2009). The critical substrate of Separase in disengagement may in fact be Pericentrin, cleavage

of which by Separase is essential for disengagement (Lee and Rhee, 2012; Matsuo et al., 2012).

Pericentrin cleavage may facilitate removal of the PCM protein CDK5RAP2, as evident from the

18

fact that depletion of CDK5RAP2 allows disengagement in the presence of a non-cleavable

Pericentrin construct (Lee and Rhee, 2012; Matsuo et al., 2012; Pagan et al., 2014). Consistent

with this, loss of CDK5RAP2 function leads to premature disengagement and re-duplication of

centrioles in mouse embryonic fibroblast cells (MEFs) (Barrera et al., 2010). Thus centriole

engagement is maintained the PCM components Pericentrin and CDK5RAP2, and

disengagement requires Separase cleavage of Pericentrin along with the activity of PLK1 in early

mitosis.

1.4.2. Centrosome Separation

During S and G2 phase, the two parental centrioles remain connected by a linker that formed in

the preceding G1. In order for bipolar mitosis to occur, the connected parental centrioles must

separate to form two distinct centrosomes. Rootletin and LRRC45 form the linker and are

connected through their interacting protein C-Nap1 to CEP135 at the centrioles (Agircan et al.,

2014; Bahe, 2005; Fry et al., 1998; He et al., 2013; Kim et al., 2008; Mayor et al., 2000). This

linker is dissolved at the onset of mitosis to allow centrosome separation (or disjunction), after

which the two centrosomes can participate in bipolar spindle formation (Agircan et al., 2014).

The dissolution of the linker is regulated by NEK2 kinase, which phosphorylates the linker

proteins C-Nap1, as well as Rootletin and LRRC45, leading to their loss from the centrosomes

and centrosome separation (Agircan et al., 2014; Bahe, 2005; Fry et al., 1998; He et al., 2013).

The activity of NEK2 is regulated by Protein Phosphatase 1 (PP1). The PP1subunit interacts

with NEK2, and PP1 can de-phosphorylate NEK2 reducing its activity (Helps et al., 2000;

Meraldi and Nigg, 2001). NEK2 undergoes an activating auto-phosphorylation, and also

regulates PP1 through a deactivating phosphorylation (Helps et al., 2000; Meraldi and Nigg,

2001). PP1 can also reverse phosphorylation of C-Nap1 by NEK2, indicating that the complex

of NEK2 and PP1 works to regulate C-Nap1 phosphorylation (Helps et al., 2000; Meraldi and

Nigg, 2001). NEK2 localization to centrosomes and subsequent centrosome separation also

requires hSav1 and the kinase MST2, which phosphorylates NEK2 promoting its centrosomal

localization (Agircan et al., 2014; Mardin et al., 2010). The activity of PLK1 also regulates

NEK2 activity, where phosphorylation of MST2 by PLK1 prevents association of PP1 with the

complex of NEK2 and MST2, allowing NEK2 to promote centrosome separation at the onset of

mitosis (Agircan et al., 2014; Mardin et al., 2010, 2011). The physical separation of centrosomes

19

once the linker is dissolved is orchestrated by microtubule motor proteins, including Eg5 and

dynein (Agircan et al., 2014).

1.4.3. Centrosome Maturation

In preparation for mitosis, the PCM undergoes an increase in size, with the additional

recruitment of key PCM proteins important for microtubule nucleation and/or anchoring (Piehl et

al., 2004; Woodruff et al., 2014). The most important regulator of PCM maturation is the kinase

PLK1, which phosphorylates multiple PCM proteins and is required for PCM maturation (Haren

et al., 2009; Lane and Nigg, 1996; Woodruff et al., 2014). One of the key targets of PLK1 for

this process is Pericentrin, as its phosphorylation by PLK1 is critical for recruitment of PCM

proteins involved in microtubule nucleation including -tubulin, Nedd1, CEP192, Aurora-kinase-

A and PLK1 itself (Haren et al., 2009; Joukov et al., 2014; Lee and Rhee, 2011). In mitosis,

Pericentrin acts as a scaffold to which CEP192 is localized, and CEP192 in turn is a scaffold for

PLK1 and Aurora-A that facilitates PLK1 phosphorylation and activation by Aurora-A (Joukov

et al., 2014; Zhu et al., 2008). CEP192 is a substrate of PLK1 in this pathway, and its ability to

bind -tubulin and function in microtubule organization requires phosphorylation by PLK1

(Joukov et al., 2014). CEP192 is a critical component of the mitotic PCM, and is required for

Nedd1 and -tubulin recruitment and bipolar spindle formation (Gomez-Ferreria et al., 2007;

Joukov et al., 2014; Zhu et al., 2008). Nedd1 interacts with the -TuRC subunit GCP2 and -

tubulin, and is likewise required for mitotic spindle formation and centrosomal microtubule

nucleation (Haren, 2006; Lüders et al., 2006). Centrosome maturation is reversed by a reduction

of PCM protein concentrations at the centrosomes after metaphase, in a process thought to

involve phosphatases and microtubule motor driven fragmentation (Woodruff et al., 2014).

1.5. Centriole Duplication

Proteins required for the process of centriole duplication are the focus of this thesis. The

following section provides an in-depth view of the current understanding of the process of

centriole assembly and the conserved proteins involved. How centriole length is regulated is

also a central theme of this thesis, and thus positive and negative regulators of centriole length

20

are discussed. Finally, the regulatory mechanisms that control centriole duplication are covered

to provide the reader with a complete picture of the process of centriole assembly from which to

understand the results described in this thesis.

1.5.1. Procentriole Formation

The process of centriole assembly requires a core set of conserved proteins found in a variety of

organisms that have centrioles (Carvalho-Santos et al., 2010). In the model organism C. elegans,

a set of 5 core proteins was described as being critical for this process: Spd-2, Zyg-1, SAS-4,

SAS-5, SAS-6 (Delattre et al., 2004, 2006; Kemp et al., 2004; Kirkham et al., 2003; Leidel and

Gönczy, 2003; Leidel et al., 2005; O’Connell et al., 2001; Pelletier et al., 2006). The structural

details of this assembly pathway were described using electron microscopy following

fertilization in C. elegans oocytes (Pelletier et al., 2006). Procentriole assembly begins with the

recruitment of Spd-2 (Cep192 homolog) and Zyg-1 kinase to the sperm-donated centrioles

(Pelletier et al., 2006). Spd-2 localization is required for (but not dependent upon) recruitment of

Zyg-1, and both Spd-2 and Zyg-1 are required for recruitment of the SAS proteins (Pelletier et

al., 2006). The assembly of the procentrioles begins with the formation of a central tube, which

then elongates and increases in diameter before gaining “hooks” at sites where single

microtubules eventually assembled (Pelletier et al., 2006). Central tube formation was blocked

by depletion of SAS-5 or SAS-6 in this assay, whereas SAS-4 depletion allowed central tube

formation and elongation, but did not permit central-tube widening or the formation of hooks or

single microtubules around the central tube (Pelletier et al., 2006). Despite the unique structure

of C. elegans centrioles, the centriole assembly factors described in C. elegans have functional

homologs conserved in higher organisms (Carvalho-Santos et al., 2012).

The Zyg-1 kinase in C. elegans is essential for initiation of centriole duplication, though the

critical substrate is not known (Lettman et al., 2013; O’Connell et al., 2001; Pearson and Winey,

2010). The functional homolog of Zyg-1 in other species is the kinase SAK/PLK4 (Bettencourt-

Dias et al., 2005; Habedanck et al., 2005; Pearson and Winey, 2010). PLK4 localization to

centrioles requires CEP192 and another protein CEP152 (Cizmecioglu et al., 2010; Hatch et al.,

2010; Sonnen et al., 2013). Both CEP192 and CEP152 interact with PLK4 and are together

important for its recruitment to the mother centriole (Cizmecioglu et al., 2010; Hatch et al., 2010;

Sonnen et al., 2013). Human CEP152 and the Drosophila homolog Asterless (ASL) have both

21

been shown to interact with PLK4/SAK and CPAP/SAS-4, and to be critical for centriole

duplication (Blachon et al., 2008; Cizmecioglu et al., 2010; Dzhindzhev et al., 2010; Hatch et al.,

2010; Sonnen et al., 2013). CEP152 in turn depends on CEP192 and another protein required for

centriole duplication, CEP63, for its localization to centrioles (Brown et al., 2013; Kim et al.,

2013; Sonnen et al., 2013). CEP192, like Spd-2, is essential for centriole duplication (Delattre et

al., 2006; Kemp et al., 2004; Pelletier et al., 2004; Zhu et al., 2008). Both CEP192 and CEP152

interact with the cryptic polo-box-domain of PLK4 through a conserved sequence in their NTDs,

and the binding appears to be competitive and mutually exclusive (Cizmecioglu et al., 2010;

Hatch et al., 2010; Kim et al., 2013; Sonnen et al., 2013). The nature of the proposed

cooperation of CEP192 and CEP152 was initially obscure, as CEP152 depletion alone increased

PLK4 recruitment to mother centrioles, whereas CEP192 depletion decreased PLK4 recruitment,

but simultaneous depletion of both CEP152 and CEP192 was more effective than CEP192

depletion alone at preventing PLK4 recruitment (Kim et al., 2013; Sonnen et al., 2013). An

understanding of the regulation of PLK4 localization was only recently achieved through a

combination of crystallography and three-dimensional structured illumination microscopy (3D-

SIM) to study sub-resolution centrosomal domains and localizations (Park et al., 2014). PLK4 is

initially recruited to CEP192 in early G1 and forms a ring of similar diameter to CEP192 around

the centrioles, but when CEP152 is recruited later in G1/S, the PLK4 ring expands in diameter

(Park et al., 2014). When CEP152 is absent, the PLK4 ring stays smaller, indicating this

transition requires CEP152 (Park et al., 2014). Indeed, co-crystal-structures of the binding

domains of CEP192 and CEP152 with PLK4 show that CEP192-PLK4 and CEP152-PLK4

interactions are mutually exclusive, and CEP152-PLK4 binding is several-fold tighter than

CEP192-PLK4 binding (Park et al., 2014). Overall, CEP192 recruits and sequesters PLK4

before CEP152 arrives at the centriole and ‘steals’ PLK4 as a pre-requisite for initiation of

centriole duplication (Park et al., 2014).

PLK4 has multiple known substrates important for centriole duplication. STIL, the human

homolog of Ana2 and SAS-5, is a critical substrate of PLK4 in centriole duplication (Ohta et al.,

2014; Stevens et al., 2010; Vulprecht et al., 2012). As with SAS-5 and SAS-6 in C. elegans,

STIL and SASS6 are co-dependent for targeting to procentriole assembly sites, and STIL is

essential for centriole duplication in human cells (Ohta et al., 2014; Tang et al., 2011; Vulprecht

et al., 2012). STIL interacts with SASS6 through its STAN motif, and a PLK4 phosphorylation

22

appears to be critical for this interaction (Ohta et al., 2014). When the residue is substituted to

alanine, SASS6 is not recruited to procentriole assembly sites, whereas STIL harboring a

phosphorylation-mimicking residue at S1061 can interact in vitro with SASS6 and facilitate its

delivery to procentriole assembly sites in vivo (Ohta et al., 2014). Endogenous PLK4 initially

appears as a ring around centrioles in G1, but later (G1/S) becomes focused at a single spot

where a procentriole will assemble (Ohta et al., 2014; Park et al., 2014). When STIL or SASS6

are depleted, or the proteasome is inhibited by MG132, PLK4 remains ring-like and fails to focus

to a single spot (Ohta et al., 2014). Overexpression of STIL, or STIL that lacks the SASS6-

interaction domain, can also protect PLK4 rings around the mother centriole, leading to the

model that STIL can prevent PLK4 degradation (Ohta et al., 2014). Consistent with this,

overexpression of STIL inhibits PLK4 ubiquitination in vivo (Ohta et al., 2014). Similarly, in

Drosophila, SAK phosphorylation of Ana2 is required for SAS-6 localization, the SAS-6/Ana2

interaction, and centriole duplication (Dzhindzhev et al., 2014). Thus one conserved critical

substrate for PLK4 in centriole duplication is STIL, and a PLK4-phosphorylation dependent

interaction of STIL with SASS6 plays a critical role in limiting PLK4 localization and centriole

duplication to a single procentriole assembly site (Dzhindzhev et al., 2014; Ohta et al., 2014). In

C. elegans, the interaction of SAS-6 and SAS-5 has been studied in vitro, and the association of

SAS-5 prevents abnormal tetramers of SAS-6 from forming through its coiled-coil domains

(Qiao et al., 2012). Further, in vitro SAS-5 and SAS-6 complexes form partial rings that are of a

diameter consistent with the C. elegans central tube, consistent with SAS-5 and SAS-6

cooperating to form the tube (Pelletier et al., 2006; Qiao et al., 2012).

PLK4 also phosphorylates the-TuRC component GCP6, and the -TuRC is required for

centriole duplication (Bahtz et al., 2012; Kleylein-Sohn et al., 2007). When HeLa cells are

depleted of GCP6 the PCM has reduced amounts of -tubulin, and -TuRC structure, mitotic

spindle formation and centriole duplication are impaired (Bahtz et al., 2012). PLK4 can be used

to co-IP GCP6, and PLK4 interacts with and phosphorylates GCP6 in vitro (Bahtz et al., 2012).

Twenty residues were identified in GCP6 that were phosphorylated by PLK4 in vitro, and

mutants of GCP6 that have all 20 serine residues substituted for alanines are unable to rescue

centriole duplication defects (Bahtz et al., 2012). The 20-alanine mutant GCP6 localizes to the

centrosome and rescues the localization of -tubulin to the centrosome, so PLK4 phosphorylation

23

of GCP6 specifically regulates -TuRC function, but not -TuRC assembly (Bahtz et al., 2012).

Therefore -TuRC regulation is another important aspect of how PLK4 regulates centriole

duplication.

The conserved protein SASS6 is the structural component of the cartwheel hub of centrioles,

conferring nine-fold symmetry to centrioles through formation of a nonamer of dimers (van

Breugel et al., 2011; Kitagawa et al., 2011). Disengagement of two centrioles in late mitosis or

early G1 is a requisite for centriole duplication in the subsequent S-phase (Kuriyama and Borisy,

1981; Tsou and Stearns, 2006). In early S-phase, there is a small but detectable population of

SASS6 initially recruited to the proximal lumen of the existing disengaged centrioles (Fong et

al., 2014). In super-resolution microscopy experiments, this population of SASS6 appears to be

ring-like and polarized from N to C termini, suggesting the strong likelihood of SASS6

cartwheels forming anew in the lumen of the parental centrioles in S-phase (Fong et al., 2014).

Critically, this population of SASS6 is lost in S-phase, when SASS6 accumulates outside the

mother centrioles where the procentrioles are assembling (Fong et al., 2014). The transition of

SASS6 from the parental lumen to the procentriole site requires the presence of both PLK4 and

STIL, and when either are absent the SASS6 population within the parental lumen persists

throughout S-phase (Fong et al., 2014). The de novo re-formation of cartwheels of SASS6

within the lumen of mother centrioles is possible only when centrioles are disengaged, and this is

hypothesized to be part of the reason engagement of centrioles inhibits re-duplication during the

cell cycle (Fong et al., 2014). Taken together with the evidence that PLK4 initially forms a ring

around parental centrioles in G1 before a STIL-and-SASS6 dependent transition to a single focus

at the site of procentriole assembly in S-phase, this work provides an appealing model for how a

single procentriole is formed adjacent to the mother centriole per cell cycle (Fong et al., 2014;

Ohta et al., 2014). New questions arise, such as whether the SASS6 cartwheel is directly moved

from the lumen to the side wall without disassembly, and how can such a transition be

experimentally captured in real-time (Fong et al., 2014)? How many cartwheels are assembled

within the parental centriole lumen, and what is the ‘trigger’ mechanism for their release from

the parental centrioles (Fong et al., 2014)? These questions are challenging but important to

address in pursuit of a complete understanding of centriole duplication.

24

Human CEP135 is a largely conserved protein required for human centriole duplication

downstream of SASS6, although there is no homolog in C. elegans (Carvalho-Santos et al.,

2010; Kleylein-Sohn et al., 2007; Lin et al., 2013a). When CEP135 is depleted, microtubule

organization by interphase centrosomes is disrupted, and monopolar or abnormal mitotic spindles

are observed (Ohta, 2002). In Chlamydomonas and Tetrahymena thermophila, the CEP135

homolog Bld10 forms part of the connection between the cartwheel spokes and the microtubule

triplets (Bayless et al., 2012; Hiraki et al., 2007; Matsuura, 2004). In Chlamydomonas, when

truncated Bld10 constructs are expressed, shorter cartwheel spokes and loss of connection

between spokes and microtubule triplets are observed (Hiraki et al., 2007). Likewise,

Drosophila Bld10 localizes to the cartwheel spokes, and mutant flies lacking CEP135/Bld10

have defective cartwheel attachments to microtubule triplets, although centrioles can form

(Roque et al., 2012). In humans, CEP135 interacts with SASS6 and CPAP/SAS-4 and has a

microtubule binding domain (Lin et al., 2013a). CEP135 localizes along the centriolar

microtubules, and within the proximal and distal portions of the lumen of centrioles and

procentrioles (Kleylein-Sohn et al., 2007; Lin et al., 2013a). The localization of CEP135 is thus

consistent with the hypothesis that it forms part of the cartwheel spoke tip, but may have

additional function in the stability of the centriole.

Subsequent to central tube formation, the assembly of microtubules in C. elegans procentrioles

requires SAS-4 (Pelletier et al., 2006). Likewise, the SAS-4 homolog CPAP is also required for

procentriole formation in humans (Kleylein-Sohn et al., 2007). CPAP interacts directly with

STIL in vivo and in vitro, and the interaction involves the CTD of CPAP (residues 895-1338)

and the NTD of STIL (residues 281-781) (Tang et al., 2011). Both SASS6 and STIL are

required for CPAP localization to procentrioles during S-phase, but CPAP is not required for

SASS6 or STIL recruitment (Cottee et al., 2013; Kleylein-Sohn et al., 2007; Tang et al., 2011).

CPAP also directly binds CEP135, and depletion of CEP135 appears to prevent the localization

of CPAP when CPAP is overexpressed (Lin et al., 2013a). The same region of CPAP appears to

be required for the CEP135 and STIL interactions (residues 895-1338), and the NTD of CEP135

(residues 50-460) is important for its interaction with CPAP (Lin et al., 2013a). CPAP

localization to both mature centrioles and procentrioles also requires Centrobin, another protein

essential to human centriole duplication (Gudi et al., 2014; Zou et al., 2005). Thus, CPAP

recruitment during centriole duplication is downstream of CEP152, PLK4 SASS6, STIL, and

25

Centrobin, while its localization to centrosomes when overexpressed also appears to depend on

CEP135 (Cizmecioglu et al., 2010; Hatch et al., 2010; Kleylein-Sohn et al., 2007; Lin et al.,

2013a). The interactions of CPAP suggest that along with CEP135 and STIL it occupies a

region connecting SASS6 cartwheel spokes to the microtubule triplets (Lin et al., 2013a). This

idea is consistent with the recent evidence that SASS6 recruitment to the parental centriole

lumen in G1 requires CPAP (Fong et al., 2014).

Lastly, CP110 was identified as a CDK2 substrate required for centriole duplication, and further

characterized as one of the proteins required for procentriole formation following PLK4

overexpression in human cells (Chen et al., 2002; Kleylein-Sohn et al., 2007). In the human

centriole duplication pathway, CP110 is not recruited when CPAP or CEP135 are absent, placing

it downstream in terms of recruitment dependencies (Kleylein-Sohn et al., 2007). CP110 is

localized to the distal portion of centrioles and procentrioles, and it acts like a “cap” on the

microtubules under which procentriole microtubules elongate (Kleylein-Sohn et al., 2007). The

process of procentriole assembly is summarized in Figure 1.3. There are numerous other

proteins required for centriole duplication, and the discovery and characterization of novel

proteins required for this process is an area of intensive research.

1.5.2. Procentriole Elongation and Length Regulation

Following formation in S-phase, procentrioles continue to elongate during in G2, and only reach

full length in mitosis (Azimzadeh and Marshall, 2010; Brito et al., 2012; Chrétien et al., 1997).

The human centriole is typically described as reaching a length of ~500 nm at maturity, with an

outer diameter of ~250 nm (Gönczy, 2012; Jana et al., 2014). In reality, “~500 nm” represents

an average of a range of lengths, as mature single centrioles isolated from KE37 human

lymphoblasts measure 512 ± 53 nm in length (n=165) (Chrétien et al., 1997). Overall though,

this shows a remarkable conservation of length. Several proteins are known to be required for

centriole elongation, and these are counter-balanced by other proteins that restrict the length of

the centriole.

CPAP plays a pivotal role in promoting centriole elongation, and excess CPAP leads to centriole

elongation beyond the normal length (Jana et al., 2014; Kohlmaier et al., 2009; Schmidt et al.,

2009; Tang et al., 2009a). This activity is partly regulated by PLK2 phosphorylation at two

26

specific serine residues, as neither overexpression of a mutated CPAP where these residues are

replaced with alanine residues nor depletion of PLK2 followed by overexpression of CPAP can

drive centriole elongation (Chang et al., 2010). Centrobin is also required for CPAP driven

centriole elongation (Gudi et al., 2011). Similar to CPAP, Centrobin can drive abnormal

centriole elongation when overexpressed (Gudi et al., 2015). Given that Centrobin is required

for CPAP centriolar-localization, and when overexpressed raises both overall CPAP levels and

the concentration of CPAP at centrosomes, it is probable that Centrobin affects centriole length

partly through regulating CPAP levels (Gudi et al., 2014, 2015). Alternatively, Centrobin has a

tubulin-dimer binding domain and could play a direct role in centriole stabilization and/or

tubulin incorporation during elongation (Gudi et al., 2011). The relative importance of

Centrobin tubulin binding activity versus CPAP regulation in centriole elongation is not yet

clear. CPAP also has two tubulin binding domains, called PN2-3 and A5N (Hsu et al., 2008).

The A5N domain binds to polymerized microtubules, whereas the PN2-3 domain binds tubulin

dimers and microtubule plus-ends, prevents microtubule polymerization, and can destabilizing

pre-formed taxol-stabilized microtubules in vitro (Hsu et al., 2008; Hung et al., 2004). High-

levels of full length CPAP overexpression can sequester tubulin leading to disruption of the

microtubule skeleton (Hsu et al., 2008). CPAP carrying a mutation in the PN2-3 that inhibits

tubulin binding does not drive abnormal centriole elongation, and is not functional in centriole

duplication (Tang et al., 2009a). Thus the PN2-3 domain may not be acting as a microtubule

destabilizing domain in the context of centriole elongation, or its destabilizing effects may be

required for elongation through some unknown mechanism. In Drosophila, SAS-4 has been

shown to bind PCM components and help incorporate them into centrosomes, in a process

requiring the PN2-3 domain and regulated by tubulin binding (Gopalakrishnan et al., 2011,

2012). In C. elegans, SAS-4 was initially described as regulating PCM size at centrosomes, but

this observation was probably the result of shorter centrioles following SAS-4 depletion

(Kirkham et al., 2003). The exact mechanisms by which CPAP and Centrobin cooperate with

each other and utilize their respective tubulin interactions to promote centriole elongation

remains unclear (Gönczy, 2012). Whether CPAP is involved in PCM recruitment or PCM

anchoring to centrioles in organisms other than Drosophila has not been investigated, though it

has been reported that a population of CPAP exists within the outer-regions of the PCM of

27

human centrosomes in addition to the population within the centriole lumen (Sonnen et al.,

2012).

In addition to cartwheel-microtubule interactions, CEP135 plays a role in microtubule

stabilization. The microtubule binding domain of Drosophila Bld10, which is within the NTD of

Bld10 as it is in Homo sapiens CEP135, can stabilize microtubules when expressed alone as a

truncated peptide (Carvalho-Santos et al., 2012; Lin et al., 2013a). Conversely, loss of Bld10

disrupts spindle formation and results in loss of flagella formation with abnormal basal bodies in

Chlamydomonas (Bayless et al., 2012; Matsuura, 2004). CEP135 localizes to both the proximal

and distal portions of the lumen in centrioles and T. thermophila basal bodies (Bayless et al.,

2012; Kleylein-Sohn et al., 2007). Drosophila mutants lacking Bld10 have shorter centrioles and

spermatid basal bodies, and are incapable of forming the ‘central pair’ of microtubules normally

found in motile cilia and flagella (Carvalho-Santos et al., 2012; Mottier-Pavie and Megraw,

2009). Consistent with a role in microtubule stability, depletion of Bld10 in T. thermophila leads

to loss of already-assembled basal bodies over time due to the mechanical stress of ciliary

beating (Bayless et al., 2012). Depletion of CEP135 appears to prevent the localization of CPAP

and centriole elongation when CPAP is overexpressed (Lin et al., 2013a). In summary,

Bld10/CEP135 is required for cilia/flagella and centriole microtubule stability, and plays a role

in centriole elongation (Bayless et al., 2012; Carvalho-Santos et al., 2012; Hiraki et al., 2007;

Kleylein-Sohn et al., 2007; Matsuura, 2004; Mottier-Pavie and Megraw, 2009; Roque et al.,

2012).

Poc1 is essential for centriole duplication, and its overexpression drives centriole elongation in

S-phase, in contrast to CPAP which is G2 specific (Keller et al., 2009). Two lines of evidence

suggest that the role for Poc1 in centriole length may relate to stabilizing of centriolar or ciliary

microtubules. First, it has been demonstrated that depletion of Poc1 in T. thermophila leads to

formation of unstable basal bodies (Pearson et al., 2009). Second, depletion of Poc1 in human

cells leads to immature procentrioles that lack acetylation (Venoux et al., 2013). There are two

Poc1 paralogs, called Poc1A and Poc1B (Keller et al., 2009). Each of these proteins is

independently capable of centrosome localization and functioning in centriole assembly, and

only co-depletion of both blocks this process (Venoux et al., 2013).

28

hPOC5 is one of two proteins that appear to drive centriole elongation specifically within distal

portion of the procentriole, and it is recruited to nascent centrioles in G2 (Azimzadeh et al.,

2009). hPOC5 interacts with Centrin, and co-localizes with Centrin within the distal portion of

the centriole (Azimzadeh et al., 2009). In the absence of hPOC5, cells do form procentrioles that

lack hPOC5 but contain Centrin and recruit PCM (Azimzadeh et al., 2009). In HeLa cells

depletion of hPOC5 leads to an S-phase arrest, but the procentrioles formed are shorter than

procentrioles in otherwise S-phase arrested controls and are made up of only doublet

microtubules (Azimzadeh et al., 2009). The authors offer two explanations for how hPOC5

affects procentrioles in S-phase despite not being detected on S-phase procentrioles; it is possible

that hPOC5 is present in S-phase at undetectable levels, or hPOC5 participates in some extra-

centriolar regulation of procentriole assembly related to its essential role in cell-cycle

progression (Azimzadeh et al., 2009). Overall, hPOC5 is an essential protein that is required for

procentriole microtubule assembly beginning in S-phase, and it appears to be critical for the

formation of the C-microtubule and the distal part of the procentriole (Azimzadeh et al., 2009).

OFD14 (formerly C2CD3) was identified as mutated in oral-facial-digital/orofacial-digital

syndrome patients, and is the other protein known to drive abnormal elongation of the distal

portion of the centrioles when overexpressed (Thauvin-Robinet et al., 2014). OFD14 is critical

for cilia formation, being required for both distal and subdistal appendage formation on mature

centrioles (Hoover et al., 2008; Thauvin-Robinet et al., 2014). OFD14 depletion results in loss

of Centrin and OFD1 from centrioles, and the centrioles remain shorter (~300 nm) (Thauvin-

Robinet et al., 2014). Consistent with a role in distal centriole assembly, OFD14 localizes in the

region near the distal appendage marker CEP164, where it interacts with OFD1 (Thauvin-

Robinet et al., 2014).

Prolonged chemical inhibition of proteolysis within a cell has been shown to lead to abnormal

centriole elongation, and this discovery was leveraged in an assay to look for proteins required

for centriole elongation (Korzeniewski et al., 2010). This approach identified CAP350, FOP,

CEP170, C-Nap1 and SASS6 as being required for this to occur (Korzeniewski et al., 2010).

While the presence of CEP170, C-Nap1, Ninein and SASS6 as hits in this screen suggest that

some of the proteins found are only indirectly required for centriole elongation, CAP350 has

since been shown to be important for centriole microtubule stability (Le Clech, 2008;

29

Korzeniewski et al., 2010). It seems likely that other proteins will be identified that are required

for centriole elongation, and these will be important for furthering our understanding of the

intricate regulation of centriole elongation that produces consistent centriole lengths in each

organism.

CP110, and its interacting partner CEP97, are critical for restricting the length of centrioles

(Spektor et al., 2007). CP110 and CEP97 are co-dependent for centriole localization, and

depletion of either leads to elongation of the centrioles (Spektor et al., 2007). CP110 decorates

the distal tips of centrioles, and even elongated centrioles when CPAP is overexpressed

(Kleylein-Sohn et al., 2007; Schmidt et al., 2009). Initially it was reported that cilia formation

occurred upon depletion of CP110 or CEP97, however, the elongated structures observed upon

CP110 depletion are actually abnormal centrioles (Schmidt et al., 2009). The abnormal

elongation of centrioles upon CEP97 depletion can be inhibited by simultaneous overexpression

of KIF24, a microtubule de-polymerizing kinesin that acts specifically on centriolar microtubules

(Kobayashi et al., 2011). Consistent with their role in restricting centriole length, it is clear that

CP110 and CEP97 are absent from basal bodies, and when localized there by overexpression

they will suppress cilia formation (Spektor et al., 2007). How exactly CP110 and CEP97

cooperate to regulate tubulin incorporation, or how the cell ‘measures’ the length of a centriole to

ensure uniformity, remain important open questions.

Like CP110/CEP97, depletion of OFD1 gives abnormal centriole elongation in mouse embryonic

stem cells (Singla et al., 2010). OFD1 localizes to both mature centrioles and procentrioles, in

the distal portion of each, near CP110 and hPOC5 (Singla et al., 2010). Depletion of OFD1 only

affects the lengths of the parental centrioles (not procentrioles), leading to elongation of the

distal portion of the centrioles in G2 specifically, as indicated by an expanded region of hPOC5

localization (Singla et al., 2010). The distal structure of the mother centriole is also affected in

that subdistal appendage proteins localize along an unusually long portion of the centriole, while

distal appendages do not form at all (Singla et al., 2010). Remarkably, the distal centriole

elongation observed upon overexpression of OFD14 is reversible by simultaneous

overexpression of OFD1, suggesting that these two proteins are antagonistically striking a

balance to establish the correct distal centriole length and facilitate appendage formation

(Thauvin-Robinet et al., 2014). The loss of OFD1 correlates with a loss of centriolar

30

microtubule glutamylation, which is unexpected because glutamylation is associated with

microtubule/centriole stability (Bobinnec et al., 1998a; Janke and Bulinski, 2011). The fact that

centrioles without OFD1 elongate abnormally in their distal regions but are not glutamylated

may be explained by a lag between elongation and stabilization, or a requirement for OFD1 in

recruiting glutamylation enzymes. Resolving this contradiction will be important for furthering

our understanding of centriole length regulation and stabilization by glutamylation.

In summary, a variety of proteins positively and negatively regulate centriole elongation (Figure

1.3), and likely more remain to be discovered. These proteins can act on distinct domains within

the centriole (proximal, distal), and imbalances between them can have dramatic effects on

centriole length, structure, mitotic function and capability in cilia formation. Several of these

proteins, such as CPAP (MCPH6), OFD1 and OFD14 are affected in diseases (Barbelanne and

Tsang, 2014; Gul et al., 2006; Singla et al., 2010; Thauvin-Robinet et al., 2014). Thus,

understanding how key proteins function in centriole length regulation may provide important

insights into human developmental diseases.

1.5.3. The Regulation of Centriole Duplication

The levels of a number of centriole duplication proteins have been shown to be cell cycle

regulated. This is achieved largely through proteolysis ensuring their presence at appropriate

levels at during the proper cell cycle stage. Careful regulation of their levels is critical to

ensuring formation of one and only one procentriole per parental centriole in a cell cycle. The

degradation of these proteins is mediated by ubiquitin-dependent proteolysis. Ubiquitin ligases

catalyze the transfer of ubiquitin molecules to specific substrate proteins within the cell, and

ubiquitination can make these proteins substrates for degradation by the 26S proteasome (Finley,

2009; Voges et al., 1999). The anaphase promoting (APC) and Skp, Cullin,-F-box (SCF)

complexes are ubiquitin ligase complexes that regulate a variety of key cellular proteins, and

play critical roles in the cell cycle (Cardozo and Pagano, 2004; Silverman et al., 2012). The SCF

E3 ubiquitin ligase complexes are formed on a scaffold of CUL1, which brings RBX1 and an E2

(ubiquitin conjugating enzyme) together with Skp1 and an adaptor protein of the F-box family

(Cardozo and Pagano, 2004; Silverman et al., 2012). F-box proteins act as the substrate

specificity factors, and so each one has specific protein targets, including key regulators of the

cell cycle and centriole duplication (Cardozo and Pagano, 2004; Silverman et al., 2012; Skaar et

31

al., 2013). The APC consists of 19 subunits at its core, which bring together an E2 with one of

two adaptor molecules, CDC20 or Cdh1 (Peters, 2006; Sivakumar and Gorbsky, 2015). CDC20

and Cdh1 have both been implicated in recognition and regulation of centriole duplication

proteins (Arquint and Nigg, 2014; Arquint et al., 2012). Both the APC and SCF E3 ubiquitin

ligase complexes can regulate centriole duplication through controlling the stability of core

centriole assembly proteins.

SASS6 is normally degraded during late-mitosis/early-G1 following ubiquitination by the APC-

Cdh1 E3 ubiquitin ligase complex, and is undetectable in early G1 at centrosomes (Arquint and

Nigg, 2014; Puklowski et al., 2011; Strnad et al., 2007). A SASS6 mutant construct lacking its

KEN domain, the target for Cdh1, drives excess centriole formation when overexpressed (Strnad

et al., 2007). Conversely, SASS6 becomes ubiquitinated when Cdh1 is overexpressed

(Puklowski et al., 2011; Strnad et al., 2007). Levels of STIL also decrease in late-mitosis and

G1, and this is likewise achieved by the activity of the APC and critical for limiting centriole

duplication (Arquint and Nigg, 2014; Arquint et al., 2012). SASS6 is also a substrate of the SCF

E3 ubiquitin ligase complex through its interaction with the SCF adaptor FBXW5 (Puklowski et

al., 2011). Overexpression of FBXW5 causes increased ubiquitination of SASS6, leading to

degradation of SASS6 and inhibition of centriole duplication (Puklowski et al., 2011). FBXW5

itself is regulated by APC dependent ubiquitination (Puklowski et al., 2011). To ensure FBXW5

levels are low during G1, the APC-Cdh1 and APC-CDC20 complexes ubiquitinates FBXW5 to

promote its degradation (Puklowski et al., 2011).

PLK4 induces centriole duplication partly by negatively regulating FBXW5 (Puklowski et al.,

2011). FBXW5 is kept at low levels in mitosis and G1 through APC regulation, but at G1/S it

appears PLK4 phosphorylation may become critical for inhibiting FBXW5 to stabilize SASS6

for centriole duplication (Puklowski et al., 2011). When co-overexpressed with FBXW5, PLK4

protects SASS6 from degradation, and the kinase activity of PLK4 is required for this effect

(Puklowski et al., 2011). FBXW5 is phosphorylated by PLK4 in vitro, and when the target

serine (151) of FBXW5 is mutated to alanine, the resulting FBXW5 ubiquitinates SASS6

regardless of PLK4 activity (Puklowski et al., 2011). Finally, the loss of centriole duplication

upon PLK4 RNAi is partially rescued by co-depletion of FBXW5, indicating that PLK4

32

regulation of SASS6 stability through FBXW5 de-activation is a critical aspect of centriole

duplication (Puklowski et al., 2011).

Work in Drosophila and later in human cells showed that SAK (Drosophila PLK4) and PLK4

were both negatively regulated by the SCF-complex with the F-box protein Slimb (Drosophila)/

TrCP (Homo Sapiens) (Cunha-Ferreira et al., 2009; Guderian et al., 2010; Rogers et al., 2009).

Accordingly, depletion of Slimb/TrCP drives centriole amplification in both systems, with

elevated SAK/PLK4 protein levels and an increase in centrosome localized SAK/PLK4 (Cunha-

Ferreira et al., 2009; Guderian et al., 2010; Rogers et al., 2009). The targeted degradation of

PLK4 by SCF-Slimb/-TrCP depends on the phosphorylation of each SAK/PLK4 molecule by a

second molecule of SAK/PLK4 in trans (Cunha-Ferreira et al., 2013; Guderian et al., 2010). In

both Drosophila and Homo sapiens, SAK/PLK4 has a conserved phosphodegron with a pair of

residues (S293, T297 in Drosophila,S285 and T289 in Homo sapiens) that along with

phosphorylation sites outside the degron are targets of trans auto-phosphorylation and required

for the interaction with Slimb/-TrCP (Cunha-Ferreira et al., 2013; Guderian et al., 2010; Klebba

et al., 2013). Mutation of the two critical residues in the phosphodegron to alanines prevents the

Slimb/-TrCP interaction, drives centriole overduplication and leads to elevated SAK/PLK4

levels (Cunha-Ferreira et al., 2013; Guderian et al., 2010). In Drosophila and C. elegans, the

phosphorylation of PLK4 is further regulated by protein-phosphatase-2A, which stabilizes PLK4

during mitosis by de-phosphorylating the phosphodegron, allowing PLK4 to be present on

mitotic centrosomes at the same time as SCF-Slimb without being degraded (Brownlee et al.,

2011; Rogers et al., 2009; Song et al., 2011).

The ubiquitin ligase MIB1 is also a regulator of PLK4 stability and function (Cajanek et al.,

2015). MIB1 is localized to the centrosome during S-phase, and enriched there following PLK4

overexpression (Cajanek et al., 2015). When overexpressed, MIB1 drives ubiquitination of

PLK4, and this can occur regardless of -TrCP expression (Cajanek et al., 2015). Depletion of

MIB1 does not cause centriole overduplication in U-2 OS cells the way TrCP depletion does,

but MIB1 overexpression does prevent PLK4 overexpression-induced centriole overduplication

and reduces PLK4 levels in this context (Cajanek et al., 2015). Further, PLK4 ubiquitination by

MIB1 reduced the binding of PLK4 to both CEP192 and CEP152, indicating that in addition to

regulation by degradation, MIB1 ubiquitination of PLK4 can regulate its interactions during

33

centriole duplication (Cajanek et al., 2015). Thus MIB1 along with -TrCP helps regulate PLK4

levels and function in centriole duplication (Cajanek et al., 2015).

PLK4 can positively regulate its own activity in centriole duplication through another

phosphorylation at serine 305 (Sillibourne et al., 2010). A PLK4 peptide where serine 305 is

substituted with glutamic acid to mimic phosphorylation promotes centriole overduplication

more efficiently than wild-type PLK4 when overexpressed (Sillibourne et al., 2010). PLK4

constructs that mimic (S305E) or inhibit (S305A) phosphorylation do not differ in their stability

from wild-type PLK4 when overexpressed, indicating that this phosphorylation is not regulating

PLK4 levels (Sillibourne et al., 2010). When a non-degradable PLK4 construct is overexpressed

it partitions both the soluble and insoluble (centrosome containing) fractions of cell lysates, but

S305-phosphorylated PLK4 is only detectable in the insoluble fractions, suggesting PLK4

phosphorylated at S305 is sequestered to the centrosome (Sillibourne et al., 2010). Therefore

PLK4 can also regulate its own activity through phosphorylation, independent of promoting

degradation.

CPAP is a cell cycle regulated protein, with reduced levels in late mitosis and G1 (Tang et al.,

2009a). The APC co-factor Cdh1 interacts with CPAP, which is ubiquitinated in vivo, and

proteasome inhibition by MG132 stabilizes CPAP throughout the cell cycle (Tang et al., 2009a).

CP110 is likewise regulated during the cell cycle by the SCF-FBXO1/Cyclin-F ubiquitin ligase

complex, so that CP110 levels are decreased beginning in G2 (D’Angiolella et al., 2010). When

a CP110 mutant is expressed that is not targeted by the SCF complex, cells are observed with

extra (more than 4) centrioles (D’Angiolella et al., 2010). The ubiquitination of CP110 by SCF-

FBXO1 is counter-acted by the activity of a deubiquitinating (DUB) enzyme called USP33,

which interacts with CP110, and is present on centrosomes during S and G2 (Li et al., 2013).

When FBXO1 is overexpressed, CP110 becomes ubiquitinated, and this is reversed by

simultaneous overexpression of USP33 (Li et al., 2013). USP33 can also de-ubiquitinate CP110

in vitro (Li et al., 2013). Lastly, when FBXO1 is depleted CP110 accumulates at centrosomes

and centriole overduplication is observed (D’Angiolella et al., 2010; Li et al., 2013). When

USP33 is simultaneously depleted, CP110 levels are reduced at centrosomes and centriole

overduplication is prevented, an affect attributed to residual FBXO1 in the cells following

34

depletion (Li et al., 2013). Therefore, in addition to ubiquitin ligases, DUBS can play a role in

regulating centriole duplication factors.

1.6. CEP120 and SPICE1

Shortly after joining the lab, I was given the opportunity to collaborate on a study of CEP120 as

a potential centriole duplication protein (Hutchins et al., 2010). Prior to this, one function of

CEP120 had been described (Xie et al., 2007). CEP120 was shown to be a centrosomal protein

highly expressed in the brains of mouse embyros (Xie et al., 2007). Further, CEP120 was

important for a process called interkinetic nuclear migration (INM), a microtubule dependent

nuclear migration between opposite ends of a polarized cell during the cell cycle, and loss of

CEP120 led to depletion of the neural progenitor cells (Xie et al., 2007). A yeast-2-hybrid

screen for interactors of CEP120 led to a validated interaction between CEP120 and the

transforming acidic coiled-coil proteins (TACC-2B, TACC3) which are also important for INM

(Xie et al., 2007). CEP120 or TACC3 overexpression increased the size of the microtubule

asters from centrosomes, whereas depletion of CEP120 or TACC3 reduced the size of

microtubule bundles linking the centrosomes to the nuclei in cultured neurons and increased the

distances between the nuclei and the centrosomes in mouse embryonic neural stem cells (Xie et

al., 2007). These results were taken as supportive of a role for CEP120 in centrosome and

microtubule function (Xie et al., 2007). I demonstrated that CEP120 was required for centriole

duplication, and my collaborators demonstrated that CEP120 had an interaction with a protein

called CCDC52, now known as SPICE1(Hutchins et al., 2010). Otherwise, at the beginning of

my Ph.D. nothing was known about how either protein functioned in centriole duplication.

During my time in the lab, a paper came out describing CEP120 function in greater detail

(Mahjoub et al., 2010). This paper supported my conclusion that CEP120 was required for

centriole duplication, and added the insights that CEP120 was preferentially localized to

daughter centrioles in G1, was found on the microtubule walls of centrioles, and was dependent

on its coiled-coil domain for centriole localization (Mahjoub et al., 2010). Subsequent to the

CEP120 paper, another paper from our collaborators in the Luders lab showed that CCDC52,

which they renamed SPICE1, was a centriole and spindle associated protein with separable roles

in centriole duplication and mitotic spindle assembly (Archinti et al., 2010). SPICE1 contains

35

two coiled coil domains, the importance of which was investigated. The N-terminal portion of

SPICE1 (1-450), containing one of two coiled-coil domains, was nuclear in interphase, and

spindle and centriole localized in mitosis (Archinti et al., 2010). The central fragment (350-750)

having both coiled-coil domains of SPICE1, and localized to the centrosome but not the spindle

(Archinti et al., 2010). The C-terminal portion, containing only the second coiled-coil domain

(444-856), did not localize properly to either centrioles or the spindle (Archinti et al., 2010).

Overall, these papers advanced our understanding of how CEP120 and SPICE1 localized, and

validated their roles in centriole duplication.

1.7. Rationale of this Thesis

Answering the question of how CEP120 and SPICE1 participated in centriole duplication was

the first aim of my research. Were they cooperating in the process? Where were they acting

within the centriole assembly pathway? What aspect of centriole structure/assembly were they

important for? What were their interacting partners (if any) in this process? These important

questions formed the rationale for the first data chapter of this thesis. As part of our publication

of a paper addressing these questions (Comartin et al., 2013), we used a screen to identify

proteins that were in the proximity of CEP120 in vivo. This screen yielded a surprising variety

of proteins, some with no or poorly characterized functions. Since then, we have expanded our

search to include proteins in the proximity of SPICE1. The rationale of my second data chapter

was that by investigating proteins identified in the proximity of CEP120 and SPICE1, I would

find proteins required for centriole duplication. Further, within these I might uncover proteins

that functionally cooperate with CEP120/SPICE1 in centriole duplication, centriole elongation,

or mitosis. The overall goal of the thesis presented here is to advance the understanding of

centriole assembly through characterization of proteins critical for this process.

36

1.8. Figures

Figure 1.1

37

Figure 1.1. Structure of the Human Centriole.

(A) A mature mother centriole surrounded by PCM (blue) with distal and subdistal appendages is

shown with an associated procentriole. Note the procentriole does not accumulate PCM proteins

until after passage through mitosis(Wang et al., 2011). The known distal and subdistal

appendage proteins are listed (see text for discussion). (B) (Centre) Schematic of the cross-

sectional view of the proximal end of a procentriole containing a central cartwheel structure and

nine microtubule triplets. (Left) The central hub of the cartwheel and its spokes are made from a

set of nine dimers of SAS-6 (van Breugel et al., 2011; Hirono, 2014; Kitagawa et al., 2011;

Winey and O’Toole, 2014). The cartwheel dimensons, SASS6 dimer dimension, and model are

based on the report of the crystal structure of Chlamydomonas reinhardtii SAS-6 (Kitagawa et

al., 2011). (Right) Two microtubule triplets are shown, with A, B and C microtubules, and the

A-C linker shown (red)., based on cryotomography of Trichonympha centrioles (Diagram based

on Figure 5, Guichard et al., 2013). Each circle of the microtubule represents a protofilament, of

which the A tubule has 13, and the B and C tubules each contain 10 (Guichard et al., 2013;

Winey and O’Toole, 2014). The A-C linker connects protofilament A8 to C9 between each

triplet (Guichard et al., 2013; Winey and O’Toole, 2014). (C) Cross-sectional view of cartwheel

stacks, with physical measurements, as seen in cryotomography based 3D-models of

Trichonympha centrioles (Diagram based on Figures 2, 3 and 6 of Guichard et al., 2013). The

central cartwheel (blue), spoke junction (green), spoke tips (red), pin-heads and pin-feet (blue)

are shown with connection to a microtubule (Guichard et al., 2013; Hirono, 2014; Winey and

O’Toole, 2014).

38

Figure 1.2

39

Figure 1.2 The Centrosome Throughout the Cell Cycle.

The general centrosome duplication cycle is outlined through various stages of the cell cycle.

(From top) In late mitosis, the newly assembled procentrioles from the previous cell cycle lose

their tight connection to the parental centrioles in a process called disengagement, and gain the

ability to recruit PCM proteins. Both of these events are critical for the centriole to participate in

the subsequent round of centriole duplication. After each mitosis, daughter cells enter G1 with a

single centrosome containing two disengaged centrioles connected by a fibrous linker (see text).

In S-phase, a procentriole forms adjacent to each of these centrioles, and these procentrioles

continue to grow and elongate through S and G2. In late G2, the amount of PCM recruited by

the two centrosomes increases (centrosome maturation). Also in late G2, the two parental

centrioles lose their fibrous linker to separate from each other (centrosome separation) in

preparation for bipolar spindle formation in mitosis. During mitosis, the younger of the two

parental centrioles will also complete its maturation with the acquisition of distal and subdistal

appendages.

40

Figure 1.3

41

Figure 1.3. Contemporary Model of Centriole Assembly.

(A) (Top) The process of procentriole assembly on a single parental centriole is shown (see text

for references). (From top-left) In G1 PLK4 is first recruited as a ring to CEP192 on the parental

centriole, then migrates farther away from the centriole barrel by interacting with CEP152 which

is newly recruited in G1. At the start of S-phase, the formation of a SASS6 cartwheel occurs

within the lumen of each disengaged parental centriole. The recruitment of SASS6 into the

parental lumen is mediated through binding to CPAP therein. Subsequently, dependent upon

PLK4 and STIL, SASS6 is released from the parental centrioles and localizes to the proximal

outer wall of the centrioles along with a single focus of PLK4 and STIL. Thereafter, assembly of

a procentriole is initiated requiring the combined activities of -tubulin, /-tubulin, CPAP,

CEP135, Centrobin and CP110. In S-phase, the elongation of the procentrioles is driven by

CPAP, Centrobin and Poc1, whereas in G2 elongation is further promoted by CPAP, Centrobin,

hPOC5 and OFD14. The figure legend is shown below the pathway. (B) A summary of the

proteins known to promote or restrict centriole elongation is shown, with the affected portion of

the centriole indicated. *OFD1 restricts the length of the parental centriole specifically.

(Bottom) Legend is shown.

42

Figure 1.4

43

Figure 1.4. Summary of key Centriole Duplication Proteins and their Localizations.

(Top) A model of a centriole in S-phase, showing an early procentriole (right) adjacent to the

mature centriole (left). Approximate localizations of proteins involved in centriole duplication

are shown in purple, with their localization during duplication shown in green (the procentriole

assembly site). (Bottom) A brief summary of the interactions and functions of select proteins

with critical roles in centriole duplication.

44

2. Chapter II:

CEP120 and SPICE1 Cooperate with CPAP in

Centriole Elongation

45

2.1. Statement of Contributions

Almost all the data in this chapter has been published as a report in the journal Current Biology:

David Comartin*1,2, Gagan D. Gupta1, Eden Fussner3,4, Etienne Coyaud5,6, Monica Hasegan1,

Marco Archinti7, Sally W.T. Cheung1, Deborah Pinchev1, Steffen Lawo1,2, Brian Raught5,6,

David P. Bazett-Jones3,4, Jens Luders8, and Laurence Pelletier1,2. CEP120 and SPICE1 Cooperate

with CPAP in Centriole Elongation, Current Biology.2013 July 22. 23(14): 1360-1366.

DOI: 10.1016/j.cub.2013.06.002.

www.sciencedirect.com.myaccess.library.utoronto.ca/science/article/pii/S0960982213006933

This publication was a collaboration involving researchers from:

(1) The Samuel Lunenfeld Research Institute at Mt. Sinai Hospital, Toronto, Ontario, Canada.

(2) The Department of Molecular Genetics at The University of Toronto, Toronto Ontario

Canada.

(3) The Department of Biochemistry at the University of Toronto, Toronto, Ontario Canada.

(4) The Hospital For Sick Children, Toronto, Ontario, Canada.

(5) The Ontario Cancer Institute, Toronto, Ontario, Canada.

(6) The Department of Medical Biophysics at the University of Toronto, Toronto, Ontario

Canada.

(7) ERA Biotec, Barcelona, Spain.

(8) Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain.

Dr. Gagan D. Gupta (Pelletier Laboratory) carried out image analysis and quantification of

various centrosome signals using scripts written in MATLAB. He also generated box and

whisker plots shown in this chapter (Figures 2.8 and 2.13).

Sally Cheung (Pelletier Laboratory) carried out multiple Western blots and co-

immunoprecipitation experiments (Figures 2.5d and 2.16).

Dr. Marco Archinti (Luders Laboratory) made and generously provided SPICE1 DNA in

plasmid form encoding full-length SPICE1 as well as fragments of SPICE1. He further

performed the co-IP experiments shown in Figure 2.3 in their entirety.

Dr. Eden Fussner (Bazett-Jones Laboratory) operated the electron microscope for the acquisition

of all tomography data sets discussed within this thesis. Further, she carried out tomogram

reconstruction and analysis of microtubule length contributing to Figure 2.11.

Doug Holmyard (SickKids Hospital and Mt .Sinai Hospital Toronto Joint Advanced Bioimaging

Centre) fixed and prepared all samples for electron microscopy, and provided training and

assistance on the electron microscope.

Deborah Pinchev (Pelletier Laboratory) performed the rescue experiment for CEP120 siRNA

shown in Figure 2.2D, E. She also generated the cell line used in that experiment.

46

Figure 2.1 of this chapter has been published in part in a paper in the journal Science:

James R. A. Hutchins1*, Yusuke Toyoda2*, Björn Hegemann1*, †Ina Poser2*, Jean-Karim

Hériché 3, 4, Martina M. Sykora1, Martina Augsburg2, Otto Hudecz1, Bettina A. Buschhorn1,

Jutta Bulkescher4, Christian Conrad4, David Comartin5,6, Alexander Schleiffer1, Mihail Sarov2,

Andrei Pozniakovsky2, Mikolaj Michal Slabicki2, Siegfried Schloissnig2,7, Ines Steinmacher1

Marit Leuschner2, Andrea Ssykor2, Steffen Lawo 5,6, Laurence Pelletier5,6, Holger Stark8, Kim

Nasmyth1, ‡Jan Ellenberg4, Richard Durbin3, Frank Buchholz2, Karl Mechtler1, Anthony A.

Hyman2 and Jan-Michael Peters1 Systematic Analysis of Human Protein Complexes Identifies

Chromosome Segregation Proteins. Science, 2010 April 30. 328, 593-599.

Reprinted with permission from AAAJ.

(1) Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.

(2) Max Planck Institute (MPI) for Molecular Cell Biology and Genetics, Pfotenhauerstrasse

108, D-01307 Dresden, Germany.

(3) Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge

CB10 1HH, UK.

(4) Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse

1, D-69117 Heidelberg, Germany.

(5) Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue,

Toronto, Ontario, Canada.

(6) Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8,

Canada.

(7) German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg,

Germany.

(8) Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen,

Germany.

47

2.2. Summary

Centrosomes play an important role in maintaining genome stability, and along with the

genome must be duplicated during the cell cycle. Centrioles are the barrel-shaped

microtubule structures that organize the PCM at the centrosome. Procentriole assembly is

achieved by a group of proteins, and centriole lengths are remarkably consistent throughout

human cells. The length of procentrioles is thought to be regulated at least partly through the

balance of the pro-elongation factors such as CPAP and hPOC5 with negative regulators of

centriole length such as CP110 and CEP97 (Azimzadeh et al., 2009; Schmidt et al., 2009;

Spektor et al., 2007; Tang et al., 2009a). How procentriole assembly and growth to a specific

length are achieved is the subject of continuing investigation.

Here, we show that CEP120 and SPICE1 are required for centriole duplication, specifically

impacting the recruitment of CEP135, Centrin, CP110, CEP97 and each other during

assembly. CEP120 and SPICE1 recruitment is dependent upon SASS6, STIL and CPAP

during centriole overduplication. We show that CEP120 complexes with SPICE1 and CPAP

in vivo, and depletion of each of these proteins resulted in procentrioles either lacking

microtubules or with fewer, shorter microtubules. Overexpression of CEP120 or CPAP

drove abnormal centriole elongation in G2 specifically, and CPAP, CEP120 and SPICE1 had

to be expressed for this to occur in either case. Using colchicine to depolymerize

microtubules, I show that CEP135 recruitment during procentriole assembly requires

microtubules whereas SASS6, SPICE1, CEP120, CPAP, and CP110 all localized to the

region of procentriole assembly without microtubules present. Lastly, I demonstrated that

artificially stabilizing microtubules in G2 arrested cells led to elongated centrioles and

procentrioles, but this was not observed when CPAP, CEP120, or SPICE1 were depleted.

Based on these results, I conclude that CEP120 and SPICE1 play a role in procentriole

assembly and that together with CPAP they are critical for delivery or incorporation of

tubulin into centrioles. Overall our findings identify a shared function for CEP120, SPICE1

and CPAP in procentriole assembly and elongation.

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2.3. Introduction 2.3.1. Combining Super-Resolution Imaging with PLK4 Induced Centriole

Overduplication to Study Procentriole Assembly

The centriole duplication pathway involves several conserved proteins, and the human pathway

was partly illuminated using a system where PLK4, the initiating kinase, was overexpressed

upon addition of tetracycline to cells (Kleylein-Sohn et al., 2007). This system effectively

amplifies centriole duplication from one procentriole per parent to multiple procentrioles per

parent. Much of the data presented in this chapter was obtained using the same PLK4 over-

expression system. There are several advantages to this approach: First, detection of failure of

one procentriole to form is more challenging than detection of 6-8 failing to form, making

defects more obvious experimentally (Kleylein-Sohn et al., 2007). Second, and perhaps more

importantly, the system amplifies the amount of protein localized during duplication, making it

much easier and more reliable to assess the dependencies of recruitment within the pathway

(Kleylein-Sohn et al., 2007). Third, when the parental centriole is oriented such that the observer

is looking down the length of the barrel, PLK4 appears ring-like surrounding the parental

centriole. This provides an important and reliable cue for determining where procentrioles

should form and the orientation of the centrioles and procentrioles being imaged.

In combination with the PLK4 induced centriole overduplication system, I show data within this

chapter that were obtained using a super-resolution microscopy method known as three

dimensional structured illumination microscopy (3D-SIM). The resolution of wide-field

fluorescence microscopy is fundamentally limited by the fact that light diffracts, and thus is often

referred to as ‘diffraction limited’ (Lakadamyali and Cosma, 2015; Oddone et al., 2014;

Yamanaka et al., 2014). The lateral resolution achievable in a quality wide-field microscope is

approximately half the wavelength of the fluorescent light being imaged, on the order of ~200

nm (Lakadamyali and Cosma, 2015; Oddone et al., 2014; Yamanaka et al., 2014). This means

that two coplanar fluorescent point-sources within 200 nm of each other will appear as one spot

in an image (Lakadamyali and Cosma, 2015; Oddone et al., 2014; Yamanaka et al., 2014). With

diffraction limited microscopy, resolving certain structural features of centrosomes, for example

the organization of the PCM proteins, is not possible (Lawo et al., 2012). Several super-

resolution microscopy methods allow resolution beyond the limits of conventional fluorescence

49

(Huang et al., 2009, 2010; Lakadamyali and Cosma, 2015; Oddone et al., 2014; Schermelleh et

al., 2010; Yamanaka et al., 2014). These methods include stimulated-emission-depletion-

microscopy (STED), stochastic optical reconstruction microscopy (STORM) photoactivated

localization microscopy (PALM), and 3D-SIM (Huang et al., 2009; Schermelleh et al., 2010;

Yamanaka et al., 2014). 3D-SIM involves taking multiple images using patterned illumination,

with the pattern shifted or rotated for each image. The combination of the sample’s fluorescence

with the known illumination pattern in multiple positions provides additional information that

can be used to computationally reconstruct an image of higher resolution (Gustafsson, 2000;

Gustafsson et al., 2008; Huang et al., 2009; Schermelleh et al., 2008). 3D-SIM has the advantage

of allowing rapid 3D imaging with multiple fluorophores and achieves double the lateral and

axial resolution of conventional microscopy (Gustafsson, 2000; Huang et al., 2009, 2010;

Schermelleh et al., 2008; Yamanaka et al., 2014). Several studies have used this approach to

visualize features of the centrosomes and centrioles otherwise unresolvable using diffraction

limited approaches (Kim et al., 2013; Lawo et al., 2012; Mennella et al., 2012; Park et al., 2014;

Sonnen et al., 2012). Combining the resolution of 3D-SIM with the benefits of the PLK4 assay

can provide important insights into the process of procentriole assembly.

2.3.2. The Use of Chemical Manipulation of Microtubules to Study Centriole

Elongation

Centrioles are nine-fold symmetrical microtubule based structures, and once assembled these

structures are resilient to chemical destabilization of microtubules, for example by the colchicine

derivative colcemid (Kuriyama, 1982). Normally centrioles reach a consistent length, and

procentrioles elongate through the cell cycle, but when the microtubule stabilizing chemical

taxol is added centrioles undergo abnormal elongation (Kuriyama et al., 1986). Conversely,

microtubule destabilizing chemicals can be used to prevent centriole duplication or limit

procentriole elongation reversibly, depending on concentration used (Kuriyama, 1982). The use

of chemicals to augment or inhibit centriole elongation is in some cases preferable to other

methods that involve protein depletion or overexpression, for example CPAP overexpression or

CP110 depletion (Schmidt et al., 2009). The use of chemicals in this context allows the

relationship between a protein of interest and the process of centriole microtubule nucleation or

elongation to be compared directly, without perturbing that protein’s cofactors. In other cases,

50

such as inferring cooperation or co-dependence for elongation or nucleation, altering the levels

of proteins involved in centriole length regulation would be important. Overall, these tools have

the potential to provide powerful insights into the mechanisms of centriole length regulation.

2.4. Results. 2.4.1. CEP120 and SPICE1 Interact and are Required for Centriole

Duplication in Cycling Cells.

As part of a large scale study of proteins important for mitosis in humans, CEP120 and CCDC52

(now SPICE1) were expressed at near endogenous levels from bacterial artificial chromosomes

(BACs) as fusions to localization-affinity purification tags (LAP-tags) and purified by modified

tandem-affinity-purification for the purpose of identifying interacting proteins (Cheeseman and

Desai, 2005; Hutchins et al., 2010; Poser et al., 2008). Endogenous CEP120 co-purified with

LAP tagged CCDC52, and endogenous CCDC52 co-purified with LAP tagged CEP120

(Hutchins et al., 2010). In addition, both proteins were found by fluorescence microscopy to

localize to the centrosome throughout the cell cycle in HeLa cells (Hutchins et al., 2010). To test

the possibility that CEP120 might play a role in centriole duplication, I used RNAi to deplete

CEP120 for 72 hours in cycling HeLa cells, and quantified the number of centrioles in mitotic

cells (Hutchins et al., 2010) (Figure 2.1A). I observed that CEP120 depletion led to an increase

in the number of cells with less than 4 centrioles in mitosis, indicating that CEP120 is likely

required for centriole duplication (Hutchins et al., 2010). To validate the specificity of the RNAi

silencing reagent, I demonstrated that the number of centrioles in mitosis was restored by

expression of an RNAi-resistant LAP-tagged mouse-CEP120 (Figure 2.1B). To further validate

the role of CEP120 in centriole duplication, I combined RNAi with an established technique for

inducing centriole overduplication by arresting U-2 OS cells in S-phase with hydroxyurea (HU)

(Balczon et al., 1995; Zhu et al., 2008). When CEP120 was depleted in this assay, the frequency

of centriole overduplication was significantly decreased (Hutchins et al., 2010) (Figure 2.1C).

These results indicate that CEP120 is required for centriole duplication in cycling cells, and

centriole overduplication in S-phase arrested cells.

51

Subsequent to our study (Hutchins et al., 2010) it was published that SPICE1 was required for

centriole duplication in cycling HeLa cells and for centriole overduplication in S-phase arrested

U-2 OS cells (Archinti et al., 2010) and elsewhere that CEP120 was required for centriole

duplication (Mahjoub et al., 2010). We established a collaboration with the lab of Dr. Jens

Luders, and I verified that SPICE1 depletion in cycling HeLa cells using their published siRNA,

and a second siRNA, inhibited centriole duplication (Figure 2.2A, B). The same experiment was

performed in HeLa cells to test two different siRNAs against CEP120, and treatment with either

siRNA had a similar effect (Figure 2.2B). To validate the specificity of our unpublished CEP120

siRNA (siRNA-1), Deborah Pinchev generated a HeLa T-REx inducible HeLa cell line that

expressed a FLAG-tagged CEP120 construct resistant to CEP120 siRNA-1. She then

demonstrated that when this siRNA-resistant FLAG-CEP120 was expressed, treatment of cells

with siRNA-1 against CEP120 did not lead to a defect in centriole duplication (Figure 2.2D, E).

Therefore the CEP120 siRNA-1 affects centriole duplication specifically by reducing CEP120

levels. Accordingly, unless otherwise specified the rescued siRNA targeting CEP120 (si-1) and

the published siRNA targeting SPICE1 were used for the remaining work described in this

thesis.

Dr. Marco Archinti from the Luders lab validated the interaction of CEP120 and SPICE1 by

reciprocal co-IP experiments, and mapped the domains of CEP120 and SPICE1 involved in this

interaction (Figure 2.3). A CEP120 fragment was expressed that corresponded to the amino-

terminal portion of the protein (residues 1-700) and a second fragment was expressed that

contained only the carboxy-terminal region including the coiled-coil domain (residues 700 to

986). Unlike the full length CEP120, neither fragment of CEP120 was able to co-IP SPICE1

(Figure 2.3A). SPICE1 fragments containing one or both of its coiled-coil domains were able to

co-IP CEP120, though the fragment containing both coiled-coil domains interacted more

strongly (Figure 2.3B). In summary, CEP120 and SPICE1 interact and are required for centriole

duplication in multiple human cancer cell lines.

2.4.2. CEP120 and SPICE1 are Required for PLK4 Induced Centriole

Overduplication

The creators of the PLK4 induced centriole overduplication assay generously provided us with

their cell line, which when treated with tetracycline will over-express PLK4 fused to a Myc-

52

epitope tag (Kleylein-Sohn et al., 2007). Initially, I sought to confirm that CEP120 and SPICE1

were important for PLK4 induced centriole overduplication, and to place CEP120 and SPICE1

within the centriole duplication pathway beginning with an examination of SASS6 localization to

procentrioles (Figure 2.4). SASS6 is a very early protein in the centriole duplication pathway,

and it forms the cartwheels of centrioles (Pelletier et al. 2006; Kitagawa et al. 2011; Leidel et al.

2005; Kleylein-Sohn et al. 2007). Two phenotypes were observed in each condition: One

population of parental centrioles showed no SASS6 recruitment at all, and a second population

of parental centrioles had SASS6 surrounding them but did not form procentrioles (Figure 2.4A,

B). The two phenotypes (SASS6 recruited, SASS6 not recruited) were also observed in positive

controls where CPAP and CEP135 were depleted in the assay (Figure 2.4A, B). The fact that

centriole duplication was inhibited in cells regardless of SASS6 recruitment to mother centrioles,

and that Western-blots showed CEP120, SPICE1, CPAP and CEP135 depletion, indicated that

the variable phenotypes were not a result of differences in RNAi efficiency (Figure 2.4C). In

trying to understand the variable SASS6 localization, I considered the possibility that differences

in the structure of the parental centrioles might have played a role. The U-2 OS cells were

asynchronous at the time of RNAi, and cycled for 48 hours prior to S-phase arrest. During the

~2 cell cycles of depletion prior to S-phase arrest, a fraction of the parental centrioles that would

later template overduplication were being assembled. If the centrioles assembled during the 48

hours were structurally defective, they might fail in recruiting SASS6, while (older) structurally

sound parental centrioles would retain the ability to recruit SASS6. This could explain the

variable phenotypes of SASS6 localization. To test this possibility I modified the PLK4 induced

centriole overduplication assay to reduce the number of cell cycles following RNAi treatment.

In the modified assay, cells were depleted by RNAi for 24 hours, then arrested in S-phase and

treated with tetracycline for another 24 hours before being released from S-phase by washout and

finally arrested in G2 for 16 hours (hereafter ‘the G2 PLK4 assay’, see Materials and Methods).

This G2 assay was intended to ensure a nearly uniform population of parental centrioles, but also

to allow sufficient time for depletion of target proteins before centriole overduplication.

Western-blots performed by Sally Cheung on whole-cell lysates I collected from the same

experiment showed that each protein was strongly depleted despite the reduced number of cell

cycles in the G2 PLK4 assay (Figure 2.5D). When CEP120, SPICE1, CPAP or CEP135 were

depleted in the G2 PLK4 assay, centriole duplication was still significantly inhibited, based on

53

Centrin recruitment (Figure 2.5A, B). Consistent with my hypothesis, when any of the target

proteins were depleted in the G2 PLK4 assay, SASS6 was localized to the region surrounding the

mother centrioles in the majority of cells where centriole overduplication failed (Figure 2.5A, B).

In summary, my results lead me to the conclusion that CEP120 and SPICE1 act downstream of

SASS6 recruitment during procentriole assembly, as do CPAP and CEP135 as previously

reported (Kleylein-Sohn et al., 2007).

2.4.3. Using 3D SIM to Study Centriole Assembly.

In the G2 PLK4 assay, I observed a subtle difference in SASS6 localization in control cells

compared to cells depleted of centriole assembly proteins. Namely, in controls SASS6

localization was often to clearly resolvable procentrioles, whereas in knock-downs of CEP120,

SPICE1, CPAP or CEP135 SASS6 localization appeared ring-like around the mother centriole

(Figure 2.5A). The frequency of the SASS6 localizations where individual procentrioles could

not be identified (i.e. rings of SASS6) was quantified following CEP120, SPICE1 and CPAP

depletion in the G2 PLK4 assay (Figure 2.5C). I found that depletion of these proteins led to an

increase in the frequency of smooth SASS6 rings surrounding mother centrioles (Figure 2.5C).

Thus, while dispensable for SASS6 recruitment to the mother centrioles, CEP120, SPICE1 and

CPAP depletion each affect the structure of SASS6 surrounding mother centrioles in the G2

PLK4 assay. To gain a better understanding of the ‘rings’ of SASS6, I used 3D SIM to examine

centrosomes following Myc-PLK4 overexpression in asynchronous U-2 OS cells. I imaged

centrosomes labeled with antibodies against SASS6 and the distal centriole markers Centrin and

CP110. Importantly, using PLK4-induced centriole overduplication in combination with 3D-

SIM, it is easy to determine the mother centriole based on the characteristic ‘flower’ shape of the

mother centriole surrounded by procentrioles, when the mother is oriented with its centriole

barrel along the z-axis (Figure 2.6A). Using centrosomes with this orientation, I was able to

compare the lengths of the procentrioles, as evident from both the elongation of SASS6 in some

procentrioles and the distances between the Centrin of the mother centriole and the Centrin in the

distal portion of procentrioles (Figure 2.6B). Centrosomes with procentrioles of varying lengths

were detectable, and representative images are shown in Figure 2.6B. Even using super-

resolution imaging, I found centrosomes where a ring of SASS6 was present, and these

centrosomes had the shortest procentrioles based on CP110 and Centrin localization (Figure

54

2.6B). From this observation, I conclude that SASS6 first localizes in a uniform distribution

around the mother centriole, and then subsequently becomes organized or resolvable into

individual procentrioles (Figure 2.6B). Consistent with this idea, a fraction of control

centrosomes were observed to have SASS6 rings in the G2 PLK4 assay (Figure 2.5C). I next

used 3D-SIM along with PLK4 induced centriole overduplication to examine the localization of

CEP120 and SPICE1 to mother centrioles and procentrioles. Both CEP120 and SPICE1

appeared to label the walls of the procentrioles (Figure 2.6C). This is consistent with the

immuno-gold labelling of CEP120 in EM experiments reported elsewhere during the course of

my research (Mahjoub et al., 2010). I observed a gap between the CEP120/SPICE1 labeling on

the mother centriole and the procentrioles (Figure 2.6C). The gap corresponded to the

localization of Myc-PLK4 around the mother, indicating that 3D-SIM makes it possible to

differentiate between localization of a protein to the mother centriole and localization to a

procentriole when Myc-PLK4 is labeled (Figure 2.6C). In summary, 3D-SIM in combination

with PLK4 induced centriole overduplication provides important insights not possible using

deconvolution fluorescence microscopy. Using this approach I found that SASS6 first localized

as a ring around the mother centriole before becoming resolvable within individual procentrioles

during PLK4 induced centriole overduplication, and that CEP120 and SPICE1 were localized to

the barrels of the procentrioles.

2.4.4. Defining the Roles of CEP120 and SPICE1 in Procentriole Assembly

Having established that CEP120 and SPICE1 are dispensable for SASS6 localization to

procentrioles, I next sought to place CEP120 and SPICE1 within the pathway of centriole

assembly. I repeated the G2 PLK4 assay depleting either CEP120 or SPICE1, and labeled

centrosomes for Myc-PLK4, Centrin, and one of a set of proteins known to be required for

centriole duplication. Using 3D-SIM, I observed that CEP120 or SPICE1 depletion resulted in

SASS6, STIL and CPAP localizing as rings partially overlapping the Myc-PLK4 signal (Figure

2.7). These rings were similar to SASS6 structures seen in the early stages of PLK4 induced

centriole overduplication (compare Figure 2.6B versus Figure 2.7). CEP152 localized as a ring

partly overlapping Myc-PLK4 in control conditions in this assay, but was not observed in the

region outside the Myc-PLK4 ring, consistent with its early role in PLK4 localization (Figure

2.7) (Cizmecioglu et al., 2010; Hatch et al., 2010; Sonnen et al., 2013). CEP120 and SPICE1

55

both appeared to be dispensable for the localization of CEP152 at the parental centriole in this

assay (Figure 2.7). Conversely, CEP120 and SPICE1 depletion resulted in centrosomes lacking

the distal centriole microtubule capping proteins CP110 and CEP97 (Figure 2.7). Lastly, the

localization of hPOC5 was not strongly affected by CEP120 or SPICE1 depletion in the G2

PLK4 assay (Figure 2.7).

When CEP120 or SPICE1 were depleted, I observed CEP135 localization was limited to the

parental centriole (Figure 2.8A). I repeated the G2 PLK4 assay with CEP135, CPAP, CEP120 or

SPICE1 depletion and immunolabeled the cells for Myc-PLK4, CEP135 and Centrin. For each

RNAi condition, I collected images of centrosomes using 3D-SIM under identical imaging and

image processing conditions. From these, I manually selected images where the parental

centriole was oriented along the z-axis such that Myc-PLK4 appeared as a distinct ring. I worked

with Dr. Gagan Gupta to develop an automated image analysis approach to quantify the

fluorescence intensity of CEP135 signal specifically in the region surrounding the parental

centriole outside of or overlapping the Myc-PLK4 signal. We termed this region the PCAR

(ProCentriole Assembly Region) (Figure 2.8B). The Myc-PLK4 signal was used to create a

‘mask’ in which the CEP135 signal that corresponded to the procentrioles was quantified. The

generation of signal masks, quantifications of signals, and generation of dot-plots were

performed by Dr. Gupta using MatLab scripts he authored for this assay. We observed that using

this experimental approach, there was a significant decrease in CEP135 signal intensity in the

PCAR under each of the depletion conditions compared to the control (Figure 2.8B). To validate

the importance of CEP120 and SPICE1 in CEP135 recruitment to procentrioles, I depleted

cycling HeLa cells stably expressing GFP-SASS6 of CEP120 or SPICE1 for 72 hours, then

imaged the S-phase centrosomes using 3D-SIM. Consistent with the results from the G2 PLK4

assay, when CEP120 or SPICE1 were depleted, I observed centrosomes where GFP-SASS6 was

evident adjacent to mother centrioles, but CEP135 was faint or not detectable (Figure 2.8C). To

quantify CEP135 levels at procentrioles in S-phase cells, I synchronized HeLa cells first in G2,

then released them and re-arrested them in S-phase following depletion of CEP120, SPICE1 or

CEP135. Centrosomes were then quantitatively imaged using 3-D SIM, and the frequency of

SASS6 positive procentrioles where CEP135 was not detectable was quantified for each

condition (Figure 2.8D, E). Further, the mean pixel intensity of CEP135 signal overlapping the

procentriole SASS6 signal was quantified for a number of centrosomes from each condition

56

(Figure 2.8D, F). I observed a frequent absence of CEP135 from the SASS6 positive

procentrioles, and a reduced overall average intensity of CEP135 signal concurrent with SASS6

signal, when CEP135, CEP120 or SPICE1 were depleted. Therefore CEP120, SPICE1, and

CPAP all appear to be important for CEP135 recruitment during both procentriole assembly.

Having determined that CEP120 and SPICE1 were important for CEP135 recruitment, I next

sought to establish which centriole assembly proteins were required for CEP120 and SPICE1

localization during centriole overduplication. To do this, I again employed RNAi in combination

with the G2 PLK4 induced centriole overduplication assay and 3D-SIM. I observed that when

CEP152, SASS6, STIL, CPAP or CEP135 were depleted, CEP120 and SPICE1 levels were

reduced in the regions surrounding parental centrioles in the majority of centrosomes (Figure

2.9A). In some cases, CEP120 or SPICE1 were retained on the mother centriole, most strongly

when CEP152/SASS6/STIL were depleted (Figure 2.9A). CEP135 and CPAP depletion reduced

CEP120 and SPICE1 recruitment to procentrioles and reduced their localization at the parental

centrioles (Figure 2.9B). When CEP120 or SPICE1 were depleted, both proteins were strongly

reduced in their localization to the procentriole assembly region (Figure 2.9B). Consistent with

published reports, CEP97 or CP110 depletion in the assay led to procentrioles that were

abnormally elongated (Schmidt et al., 2009; Spektor et al., 2007) (Figure 2.9C). Under CEP97

or CP110 depletion, CEP120 and SPICE1 were localized along the lengths of the abnormally

elongated procentrioles, indicating their localization was independent of CP110 and CEP97

(Figure 2.9C). When hPOC5 was depleted, although Centrin recruitment was affected, CEP120

localized along what appear to be procentrioles, and SPICE1 was also seen in the region of

procentriole assembly (Figure 2.9D). POC1 depletion did not affect CEP120 localization, but

did strongly impact SPICE1 recruitment (Figure 2.9D). Overall, from these results I conclude

that CEP120 and SPICE1 are dependent upon the functions of CEP152, SASS6, STIL, CPAP

and CEP135 for their recruitment to procentrioles, and that CEP120 and SPICE1 are important

for the recruitment of CEP135 and the distal procentriole assembly proteins CP110 and CEP97.

2.4.5. Investigating the Role of CEP120 and SPICE1 in Procentriole

Structure

The depletion of CEP120 or SPICE1 did not prevent accumulation of early procentriole

assembly proteins (SASS6, STIL, CPAP), however, even from super-resolution images it was

57

unclear whether there were de facto procentrioles or precursors being assembled under these

conditions. To understand more precisely the structures that exist in the PCAR when CEP120

and SPICE1 are absent, I pursued electron microscopy. Electron microscopy (EM) is the best

method for observing the structure of centrioles and procentrioles in fixed samples. I provided

samples from a G2 PLK4 induced centriole overduplication assay where CEP120, SPICE1, or

CPAP had been depleted to Doug Holmyard at the Mt. Sinai EM facility who prepared them for

transmission electron microscopy (TEM) (see Materials and Methods). CPAP was included as a

positive control because CPAP is known to be required for centriole elongation, and so depletion

in the G2 PLK4 assay would be expected to yield shorter procentrioles (Kohlmaier et al., 2009;

Schmidt et al., 2009; Tang et al., 2009a). Thin sectioning of EM samples (100 nm) results in

centrosomes that are often split into multiple sections, so to ensure proper detection of the full

length of each mother centriole and possible procentrioles, Doug Holmyard prepared serial

sections from each sample. In serial sections, the same sample block is cut and sequential slices

are mounted for viewing in order, allowing for the identification and examination of the same

centrosome in its entirety through a series of slices. Using this approach, I found multiple

centrosomes from each condition that could be examined in their entirety through multiple

sections. Such centrosomes are evident when the cell containing a centrosome of interest can be

identified in a serial section where the centrosome is no longer detectable. Figure 2.10 shows

representative centrosomes imaged through serial sections from each condition. In control

centrosomes, the procentrioles had well defined and resolvable microtubules (Figure 2.10). In

samples where CEP120, SPICE1 and CPAP had been depleted, regions of electron density were

visible adjacent to the parental centrioles (Figure 2.10). In some cases, these regions of electron

density included short but detectable microtubules, though overall no robust procentriole

structure was evident in most cases (Figure 2.10). These observations indicate that CEP120 and

SPICE1, like CPAP, act at an early stage in procentriole assembly, and that despite the presence

of SASS6 and STIL in the PCAR following CEP120 or SPICE1 depletion procentrioles are not

necessarily assembled. Regarding the organization of SASS6 in this region, I was unable to

detect cartwheel structures even in control procentrioles, and thus I cannot determine from these

results whether SASS6 forms cartwheels in the absence of CEP120 or SPICE1.

In the absence of CEP120, CPAP or SPICE1, procentriole precursors sometimes contained

detectable microtubules. I sought to compare the lengths of such microtubules to microtubules

58

in control procentrioles. To better resolve and effectively measure microtubules in three

dimensions, I collaborated with Dr. Eden Fussner from the lab of Dr. David Bazett-Jones to

perform electron tomography on samples from the G2 PLK4 assay. Briefly, electron tomography

involves imaging a thicker sample section at multiple angles by transmission electron

microscopy as it is tilted a number of times within the microscope. The samples are prepared

with gold particles present, and following acquisition of the ‘tilt-series’ of images, software is

used to reconstruct a three-dimensional image of the sample based on the experimental setup and

the positional information provided by the gold-particles. Dr. Fussner performed the image

acquisition for all tomograms, and carried out the processing to produce the final tomograms and

projections. I worked alongside Dr. Fussner selecting the appropriate centrosomes for each

condition. After useable tomograms were obtained for each condition, Dr. Fussner and I

measured detectable microtubules in control procentrioles and the procentriole precursors

observed under experimental conditions (see Materials and Methods). From 3D tomograms we

selected microtubules that we were able to measure along their entire lengths (Figure 2.11A).

We found that in control conditions, procentriole microtubules reached an average length of

>150 nm, whereas when CEP120, SPICE1 or CPAP were absent, the microtubules averaged less

than 100 nm in length (Figure 2.11B). In addition to the length of microtubules, we measured

the width of the procentrioles and precursor procentrioles from the outside of the detectable

microtubules, and found that this metric was similar across all samples (Figure 2.11C). Overall

the results of this experiment validated the observations from transmission electron microscopy

experiments, namely that CEP120, CPAP or SPICE1 depletion resulted in partially assembled

procentrioles that either lacked microtubules, or had short microtubules compared to control

procentrioles. Therefore, CEP120 and SPICE1 act like CPAP at an early stage of procentriole

assembly, and are required for proper formation of procentriole microtubules.

2.4.6. Comparison of the Effects of CEP120 or SPICE1 Depletion versus

Microtubule Depolymerization on PLK4 Induced Centriole Overduplication

Procentriole precursors assembled in the absence of CEP120 or SPICE1 have fewer

microtubules, and when present microtubules are shorter than in control conditions. One

possible explanation for this observation is that centriolar microtubules might not be properly

stabilized when CEP120 or SPICE1 are absent. To test the hypothesis that CEP120 or SPICE1

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depletion causes defects in centriole duplication by altering microtubule stability, I investigated

the impact of chemical inhibition of microtubule polymerization on procentriole assembly. First,

I repeated the G2 PLK4 assay in the presence or absence of 10 M colchicine, a chemical known

to inhibit microtubule polymerization and thus centriole assembly (Boisvieux-Ulrich et al., 1989;

Kuriyama, 1982). Initially, I verified that treatment with colchicine inhibited centriole

overduplication following PLK4 overexpression. I found that 10 M colchicine effectively

depolymerized cytoplasmic microtubules and prevented the formation of procentriole

microtubules (Figure 2.12A). I noted that CEP135 localization to the PCAR was reduced or

eliminated by the addition of colchicine during PLK4 induced centriole overduplication (Figure

2.12B). I imaged centrosomes using 3D-SIM in this assay, and provided images to Dr. Gupta for

automated quantification of protein signals at the centrosomes using MATLAB. Consistent with

my qualitative observations, there was a significant decrease in total CEP135 signal and total

glutamylated-tubulin signal at the centrosomes when colchicine was present (Figure 2.13A, B).

Conversely, the total levels of SASS6 at the centrosomes were not significantly affected by the

presence of colchicine, suggesting that SASS6 localization was independent of procentriole

microtubule polymerization (Figure 2.13B). Colchicine treatment caused SASS6 to localize in a

ring around the mother centriole, which when imaged using 3D-SIM appeared similar to the

localization of SASS6 following CEP120 or SPICE1 depletion (Figure 2.13A versus Figure 2.7).

I next examined how prevention of procentriole microtubule formation affected the localization

of other centriole assembly proteins following PLK4 overexpression. When colchicine was

present, I observed that CEP120 and SPICE1 could localize to the region of procentriole

assembly, indicating microtubule independent localization (Figure 2.13C). Similarly, CPAP and

CP110 showed robust localization as rings to the PCAR (Figure 2.13C). This was unexpected,

since CEP120 or SPICE1 depletion prevent CP110 localization to the PCAR in the G2 PLK4

assay, and since CP110 localizes to the distal tips of centriole microtubules (Schmidt et al.,

2009). In summary, the initial localization of CP110 is a microtubule and CEP135 independent

process, but requires CEP120 and SPICE1 (Figure 2.13C and Figure 2.7). On the other hand,

recruitment of CEP135 to the PCAR is reduced when microtubules are absent, or when CEP120

or SPICE1 are depleted, in the G2 PLK4 assay.

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2.4.7. CEP120 and SPICE1 Cooperate with CPAP in Centriole Elongation

The effects of CEP120 and SPICE1 depletion on procentriole assembly in the assays thus far

described are similar to those of CPAP depletion. CPAP is an early procentriole assembly

protein with a known role in procentriole elongation (Kleylein-Sohn et al., 2007; Tang et al.,

2009a). One of the phenotypes that was used to determine the function of CPAP was that when

overexpressed in G2 phase of the cell cycle, CPAP drove abnormal elongation of centrioles and

procentrioles (Tang et al., 2009a). I therefore sought to test whether CEP120 or SPICE1

overexpression gave a similar phenotype. When U-2 OS cells were transiently transfected with

plasmids encoding GFP-fusions to CEP120 or CPAP for 24 hours, a small but reproducible

fraction of cells had abnormally elongated centrioles based on acetylated tubulin and GFP

labeling (Figure 2.14A, B). Overexpression of a GFP-SPICE1 fusion in the same assay did not

result in an elongation of the centrioles, though SPICE1-GFP localized to acetylated cytoplasmic

microtubules (Figure 2.14A). CPAP induced centriole elongation is G2 specific (Kohlmaier et

al., 2009; Tang et al., 2009a). To see if CEP120 induced centriole elongation was cell cycle

specific, I generated a U-2 OS FLP-In TREx cell line that when treated with tetracycline

expressed FLAG-tagged CEP120. Similar to CPAP overexpression, CEP120 overexpression

drove the elongation of centrioles and procentrioles in G2 arrested cells, but not S-phase arrested

cells (Figure 2.14C, D). I next tested the possibility that CEP120 and CPAP were functionally

cooperating by probing whether each was required for centriole elongation following

overexpression of the other. I also tested whether SPICE1 was important for either CEP120 or

CPAP induced centriole elongation. When CPAP or SPICE1 were depleted prior to CEP120-

GFP overexpression, significantly fewer cells had elongated centrioles than in control transfected

cells or cells where SASS6 was pre-depleted as a control procentriole assembly protein not

involved in length regulation (Figure 2.15A, B). Similarly, centriole elongation following CPAP

overexpression required the presence of CEP120 and SPICE1, but was independent of SASS6

(Figure 2.15D, E). Therefore, CEP120 and SPICE1 appear to functionally interact with CPAP,

and to participate in centriole elongation and procentriole assembly along with CPAP.

To test whether CPAP was part of a complex with CEP120 and SPICE1, Sally Cheung

performed co-IP experiments to test whether CEP120 and CPAP interacted in vivo. HeLa cells

were transiently transfected with FLAG-CEP120 or FLAG-USP37 as a control and one of

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SPICE1-GFP, CPAP-GFP or GFP-USP37 (Figure 2.16). When FLAG-CEP120 was pulled

down, SPICE1-GFP and CPAP-GFP were both detected by Western blot, but neither GFP

construct was detected when FLAG-USP37 was pulled down. Therefore CEP120 functionally

and physically interacts with CPAP and SPICE1 in vivo to drive centriole elongation.

Finally, having shown that colchicine inhibition of microtubule polymerization mimicked some

of the characteristic phenotypes of CEP120 or SPICE1 depletion, I sought to determine whether

CEP120 and SPICE1 were required to elongate centrioles in a context where no protein was

being overexpressed. To achieve this, I used taxol as a chemical agent known to stabilize

microtubules and facilitate microtubule polymerization (Arnal and Wade, 1995; Kumar, 1981;

Schiff et al., 1979). It has been shown previously that taxol treatment leads to elongated

centrioles (Kuriyama et al., 1986). When U-2 OS cells were arrested in G2 for 48 hours in the

presence of taxol, I observed that mother centrioles (as detected by CEP164 staining), daughter

centrioles and procentrioles frequently became elongated (Control, Figure 2.17). Conversely,

depletion of CEP120, CPAP or SPICE1 led to a decrease in the frequency of observed abnormal

centriole elongation affecting both centrioles and procentrioles (Figure 2.17B). In contrast,

SASS6 depletion effectively reduced procentriole formation but had no detectable impact on

centriole elongation (Figure 2.17A, B). Therefore, CEP120, CPAP and SPICE1 are all essential

to the elongation of both mature centrioles and procentrioles, even when elongation is driven by

chemical stabilization of centriolar microtubules themselves.

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2.5. Discussion

2.5.1. Placing CEP120 and SPICE1 in the Centriole Assembly Pathway

The results described here demonstrate that CEP120 and SPICE1 interact and are required for

centriole duplication in multiple assays and cell types (Comartin et al., 2013; Hutchins et al.,

2010). Independently, our collaborators have shown that SPICE1 is important for centriole

duplication, and during the preparation of this work another group validated our finding that

CEP120 is important for centriole duplication (Archinti et al., 2010; Mahjoub et al., 2010). We

show here that CEP120 and SPICE1 interact, and that this interaction is dependent on the C-

terminal most coiled-coil domain of SPICE1, but stronger when both coiled-coil domains are

present. The fragment of SPICE1 that contains both coiled-coil domains (444-856) localizes to

the centrosome but not the mitotic spindle (Archinti et al., 2010). Taken together with our

findings, this suggests that SPICE1 spindle localization is not mediated by CEP120 interactions

alone. Full-length CEP120 was required to co-IP SPICE1, whereas neither the CTD coiled coil

domain nor NTD domain of CEP120 were sufficient for the interaction. This suggests that either

the full CEP120 protein is required for the interaction, or more likely that the SPICE1 binding

region bridges both of the fragments used here, as is the case for the CPAP binding domain of

CEP120 (416-730) (Lin et al., 2013b). Future work could include co-IPs with different

fragments of CEP120 to more precisely refine the SPICE1 binding domain, for example those

used to define the microtubule binding domain of CEP120 (Lin et al., 2013b). Consistent with

an interaction, depletion of either CEP120 or SPICE1 strongly reduces the recruitment of the

other protein in centriole duplication assays. Thus CEP120 and SPICE1 interact and cooperate

in human centriole duplication.

How CEP120 and SPICE1 act in centriole duplication was not understood at the outset of this

work. Given the previous utility of PLK4 induced centriole overduplication in describing

aspects of the human centriole assembly pathway, I employed PLK4 overexpression to decipher

where CEP120 and SPICE1 acted in the pathway. CEP120 and SPICE1 were recruited

dependent upon CEP152, SASS6, STIL, CEP135 and CPAP. I further observed that CEP120

and SPICE1 were dispensable for SASS6, STIL and CPAP recruitment, consistent with other

reports about CEP120 function in this process (Lin et al., 2013b). I showed that CEP120 and

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SPICE1 were required for CEP97, CP110 and CEP135 recruitment to the site of procentriole

assembly. CPAP and CEP135 are required for CP110 localization during duplication, so it is

possible that CEP120, SPICE1 and CPAP are impacting CP110 localization indirectly through

localizing CEP135 (Kleylein-Sohn et al., 2007). This is unlikely, however, since in the presence

of colchicine CP110 localizes to the PCAR without CEP135. Alternatively, CEP120/SPICE1

could be directly involved in recruiting CP110 to procentrioles during assembly. Future work

should include investigating possible interactions between CEP120/SPICE1 and CP110/CEP97

by co-IP.

Both CEP120 and SPICE1 localize to procentrioles that are assembled in the absence of hPOC5,

consistent with reports that hPOC5 acts later in procentriole assembly but is important for

Centrin recruitment (Azimzadeh et al., 2009). POC1 depletion strongly affects SPICE1 but not

CEP120 localization to the procentriole regions. This result warrants further investigation, given

that other proteins studied here have similar effects on CEP120 and SPICE1 localization in our

assays. POC1 is important for S-phase elongation of procentrioles, so the requirement for POC1

in SPICE1 localization could explain why procentrioles fail to elongate or are unstable when

POC1 is depleted (Keller et al., 2009; Venoux et al., 2013). We were unable to obtain antibodies

for POC1 that were functional in immunofluorescence, but in future it will be worth

investigating whether CEP120 and SPICE1 are required for POC1A or POC1B localization to

procentrioles, as this may be relevant to the functions of CEP120 and SPICE1 in procentriole

microtubule assembly. In summary, CEP120 and SPICE1 are downstream of SASS6/STIL in

centriole duplication, but are required for CEP135 and CP110 recruitment, similar to CPAP.

2.5.2. CEP120 and SPICE1 Cooperate with CPAP in Centriole Elongation

Overexpression of CEP120, but not SPICE1, gave abnormal centriole elongation in G2 phase of

the cell cycle specifically, a phenotype reminiscent of CPAP induced elongation (Kohlmaier et

al., 2009; Tang et al., 2009a). Depletion of any of these three proteins was sufficient to perturb

this process, whereas SASS6 depletion blocked procentriole elongation presumably by blocking

procentriole formation, but not mature centriole elongation. The fact that we observed both

parental and procentrioles elongating upon CEP120 overexpression, similar to CPAP

overexpression, suggests that the levels of these proteins must be controlled to maintain

appropriate centriole length. Indeed, CPAP is a cell-cycle regulated protein, and an independent

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study has shown that CEP120 levels are regulated through the cell cycle similar to CPAP levels

(Lin et al., 2013b; Tang et al., 2009a). However, another study of CEP120 and CPAP in

centriole elongation showed that when one was over-expressed, both were localized to the

elongated centrioles (Lin et al., 2013b). This may indicate that both proteins are present at levels

sufficient to drive elongation, but not sufficient to initiate it. Alternatively, the presence of excess

of either protein may stabilize the other, effectively increasing levels of both when either is over-

expressed. It has been shown that Centrobin can stabilize ubiquitinylated CPAP at the centriole

by inhibiting its destruction, so it is possible CEP120 could likewise protect CPAP and vice

versa (Gudi et al., 2015). Further, CEP120 must be able to affect both proximal and distal

portions of the centriole microtubules, since CEP120/CPAP driven elongation occurs distal to

the appendage component CEP164 (see Figure 2.15) and procentrioles are shorter when

CEP120/CPAP/SPICE1 are depleted.

Given the functional interaction, we considered a possible interaction between CEP120 and

CPAP. We found that CEP120 could co-IP CPAP, suggesting they interact in vivo. The same

independent study defined the CPAP binding, microtubule binding, and dimerization domains of

CEP120 (Lin et al., 2013b). Disruption of the microtubule binding domain, but not the

dimerization domain, strongly inhibits CEP120 induced elongation following overexpression

(Lin et al., 2013b). A similar microtubule binding-dependence of CPAP in centriole elongation

was previously reported, suggesting that these domains are both involved in centriole elongation

(Lin et al., 2013b; Tang et al., 2009a). In general, the other published study of CEP120 and

CPAP cooperation is largely in agreement with our study, with the exception that they conclude

that SASS6/STIL are important for centriole elongation following CEP120 overexpression (Lin

et al., 2013b). There are several differences in the experimental protocols used, including that in

our study, we synchronized cells in G2 for a prolonged period, whereas in the other report cells

were asynchronous (Lin et al., 2013b). Since CEP120 and CPAP are primarily active in G2

centriole elongation, our prolonged cell cycle arrest could have sensitized our system to the

effects of overexpression, possibly explaining why we observed mature centrioles elongating

more frequently. It is worth noting that the authors of the other study report 19% of the cells had

elongated mother centrioles despite SASS6 depletion (Lin et al., 2013b). Based on the fact that

SASS6 should not be incorporated in the distal portions of mother centrioles, but CEP120

overexpression drives elongation of that region, I favor a model where SASS6 does not play a

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direct role in CEP120/SPICE1/CPAP mediated elongation of centrioles. In support of this,

taxol-induce centriole elongation was not inhibited by depletion of SASS6 in our study.

Procentrioles are not assembled when SASS6 is depleted, and SASS6 is required for CPAP,

CEP120 and SPICE1 to localize to these structures. Thus a decrease in overall percentage of

elongated centrioles is expected due to the impact on procentriole elongation alone, but is

probably an indirect effect upstream of CEP120/SPICE1 activity.

2.5.3. CEP120 and SPICE1 are Important for Microtubule Formation during

Procentriole Assembly

We used TEM with serial-sectioning and tomography in combination with the G2 PLK4 assay to

observe the state of microtubules in the procentriole assembly region following CEP120,

SPICE1 or CPAP depletion. Initially, with serial EM we found that in each case, parental

centrioles were surrounded by electron dense regions that either lacked detectable microtubules

or contained very short microtubules. We used tomography to measure the lengths of the

observed procentriole microtubules in three-dimensions, and found that indeed they were

significantly shorter when CEP120, SPICE1 or CPAP were depleted. These results, taken along

with the ring-like localization of centriole assembly proteins, support a model where CEP120

and SPICE1 are required for the assembly of procentriole microtubules or their stability, or both.

In either case, we would expect to see the appearance of some microtubules that are only capable

of reaching short lengths due to the dynamic nature of microtubule assembly. It will be of

interest to test the impact of recombinant CEP120 and SPICE1 on microtubules in vitro, to

determine whether they stabilize microtubules or alter polymerization rates. Combinations of

recombinant CEP120, SPICE1, CEP135 and CPAP could also be tested.

Previous studies have shown that there is a cell-cycle dependent elongation of procentrioles,

continuing through S and G2 until they reach full length in mitosis (Azimzadeh et al., 2009;

Kuriyama and Borisy, 1981). Under depletion of the distal centriole elongation protein hPOC5

for example, the procentriole remains short in G2 and forms only doublet microtubules

(Azimzadeh et al., 2009). While we were not able to detect whether procentrioles had doublets or

triplets, we certainly observed a shorter average length in CEP120/CPAP/SPICE1 depletion

conditions, and fewer microtubules were detectable in procentriole regions. In C. elegans, which

have only single microtubules instead of triplets, there are no identified CEP120, SPICE1 or

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CEP135 homologs (Carvalho-Santos et al., 2010; Mahjoub et al., 2010). In future, it will be

worth investigating the structure of procentrioles by EM under CEP120/SPICE1 depletion

conditions in more depth, to see if single microtubules, doublet or triplet microtubule are

observed in the absence of CEP120 and SPICE1. Overall, I conclude based on these results that

CEP120 and SPICE1, along with CPAP are important for very early stages of procentriole

microtubule assembly and/or stability. This is consistent with the localization of CEP120 and

SPICE1 along procentriole barrels, as reported here by 3D-SIM and previously by immuno-EM

for CEP120 (Mahjoub et al., 2010).

2.5.4. CEP135 Localization Requires CEP120 and SPICE1, and is a

Microtubule Dependent Event During Centriole Duplication

The application of 3D-SIM to the G2 PLK4 assay gave us the ability to observe features and

structures not normally resolvable by conventional fluorescence microscopy methods. Critically,

we were able to distinguish between localization on parental centrioles versus recruitment to the

region where procentrioles are being assembled, based on localization relative to Myc-PLK4 in

properly oriented centrosomes. This capability made possible the key observation that CEP135

recruitment to procentrioles is lost when CEP120 or SPICE1 are depleted. Using conventional

microscopy, the strong signal from CEP135 stably incorporated into the mother centrioles is not

resolvable from the newly recruited CEP135 participating in procentriole assembly, and this has

been elsewhere reported (Lin et al., 2013a). When we depleted CEP120, SPICE1, CPAP or

CEP135, the levels of CEP135 in the procentriole assembly region decreased significantly.

When colchicine was present, CEP135 levels were reduced in the procentriole assembly region,

but SASS6 levels were not affected. Additionally, CEP120 and SPICE1, along with CP110 and

CPAP were all localized properly despite the lack of procentriole microtubules. One possible

interpretation of this is that CEP135 recruitment/anchoring might be downstream of or dependent

upon procentriole microtubule elongation. CEP135 is thought to be a part of the cartwheel

linkage to the microtubule triplets, so it is possible that trace amounts of CEP135 are present to

establish the first cartwheel(s), but then procentriole microtubule elongation could either precede

or coincide with the incorporation of subsequent cartwheels. Consistent with this, SASS6

structures do not elongate in our CEP120/SPICE1 depletion conditions, but remain as rings or

small puncta, whereas in controls SASS6 becomes resolvable as within distinct procentrioles.

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Perhaps CEP135 is only stably retained in growing procentrioles once it is incorporated, whereas

other proteins are recruited in excess before assembly of a procentriole. Clearly, CEP135

recruitment to growing procentrioles is distinct from the other proteins examined here, being the

only protein that is strongly affected when microtubules are disrupted. Alternatively, it is

possible that CEP135 delivery to centrosomes is dependent upon a microtubule cytoskeleton.

CEP135 has been shown to bind to p50, a subunit of the dynactin complex which facilitates

microtubule motor-driven cargo transport along microtubules (reviewed in Schroer, 2004), and

importantly both CEP135 and p50 are co-dependent for centrosome localization (Uetake et al.,

2004). Thus CEP135 may be actively transported to centrosomes, but then incorporated or

retained only when procentrioles are being successfully assembled, in a CEP120/SPICE1

dependent process. CPAP also interacts with CEP135, but this is likewise not sufficient for

robust localization in the absence of a microtubule cytoskeleton under colchicine treatment (Lin

et al., 2013a). A model combining microtubule dependent CEP135 localization and

CEP120/SPICE1/CPAP dependent CEP135 incorporation/retention into procentrioles resolves

the observations from the colchicine and RNAi experiments. What is not clear yet is whether

CEP120 or SPICE1 are directly involved in CEP135 incorporation into procentrioles, or are

merely required to build/stabilize procentrioles which then retain CEP135. To date, no

interaction between CEP135 and CEP120 or SPICE1 has been published. Clearly, determining

whether CEP120 and/or SPICE1 bind CEP135 is a critical future experiment.

2.5.5. CEP120 and SPICE1 are Required for Tubulin Incorporation into

Procentrioles

Taxol is a microtubule stabilizing chemical, and upon taxol treatment in G2, U-2 OS cells show

abnormal centriole and procentriole elongation. CEP120 and SPICE1 are necessary for

procentriole microtubule assembly and elongation, and one possible mechanism for their

function could be the stabilization of microtubules. Taxol induced elongation is absent when

CEP120, SPICE1 or CPAP are depleted, but not when SASS6 is depleted. Therefore CEP120,

SPICE1, and CPAP are required for the induced elongation of both mother and procentrioles in

G2 arrested cells. From this, I conclude that CEP120 and SPICE1 have a function beyond

merely stabilizing procentriole microtubules, because taxol cannot facilitate centriole elongation

in their absence.

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Centriole elongation occurs under a ‘cap’ of CP110 that must be removed for ciliogenesis, but

CP110 remains localized to the plus ends of abnormally elongated centrioles (Kleylein-Sohn et

al., 2007; Schmidt et al., 2009; Spektor et al., 2007). Microtubule incorporation into centrioles

could be driven by diffusion and stabilization, or it could require a chaperoned or active delivery

system. In a simple diffusion model where tubulin is under free exchange and proteins provide

additional stability, taxol stabilization should affect the length of procentrioles regardless of the

presence or absence of other proteins. Thus, the incorporation of free tubulin into centrioles at

the centrosome is dependent upon CEP120, SPICE1 and CPAP. CEP120, CPAP and Centrobin

are all important for centriole elongation, and each of these proteins has a microtubule binding

domain (Gudi et al., 2011; Lin et al., 2013a, 2013b; Tang et al., 2009a). In Drosophila, SAS-4

(CPAP) is a tubulin binding protein that helps deliver PCM components to the centrosome

through a mechanism that depends on binding and release of tubulin by SAS-4 (Gopalakrishnan

et al., 2011, 2012). Perhaps a similar mechanism of tubulin dimer-delivery explains how human

CEP120 and CPAP function in centriole elongation. In CEP120 the microtubule binding domain

is essential for elongation upon overexpression, whereas the tubulin dimer binding domain of

CPAP is essential for elongation upon its overexpression (Lin et al., 2013b; Tang et al., 2009a).

It remains to be shown whether CEP120 binds tubulin dimers, versus microtubules exclusively,

as the assay used to demonstrate the interaction was done in the presence of taxol (Lin et al.,

2013b). SPICE1, although it localizes to the spindle, has not had a microtubule binding activity

demonstrated or characterized in vitro. Addressing how CEP120 and potentially SPICE1 interact

with microtubules and/or tubulin dimers will be a very important step towards understanding

their functions in procentriole elongation. Future experiments should be directed at determining

whether CEP120 and SPICE1 bind free tubulin dimers or polymerized microtubules in vitro, and

whether this is dependent upon the nucleotide bound by the tubulin (GTP vs. GDP), as it is in the

case of Drosophila SAS-4 (Gopalakrishnan et al., 2012). Further, the impact of

CEP120/SPICE1 on polymerized microtubules, specifically whether each protein can

stabilize/destabilize them, or affect polymerization dynamics, should be determined.

Many questions arise from our work. Most importantly, we have very little understanding of the

exact molecular mechanism(s) by which tubulin dimers are incorporated into centrioles. Are

CEP120, CPAP, Centrobin and other proteins each adding tubulin dimers in a redundant yet

robust system of microtubule incorporation? How do the various dependencies and interactions

69

among these proteins relate to their tubulin binding? How does the tubulin incorporation system

link with addition of core centriole or PCM proteins? How do CP110, CEP97 or other negative

regulators of centriole length counterbalance this system? Is it only the A-microtubule of the

triplet that is regulated this way, since it is capped by a -TuRC at one end and CP110 at the

other, whereas B and C microtubules assemble bi-directionally along the A-tubule and are not

nucleated by a -TuRC? (Guichard et al., 2010). Understanding this process is critical to

understanding how centrioles are built to consistent lengths.

While many questions remain, our work here reveals a role for CEP120 and SPICE1, in

cooperating with CPAP in procentriole assembly and elongation. Further, CEP120 and SPICE1

are essential for each other’s recruitment, along with recruitment of CEP135, CP110 and CEP97

during procentriole assembly.

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2.6. Figures

Figure 2.1.

Figure 2.1. CEP120 is required for centriole duplication in HeLa and U-2 OS cells.

(A) (Left) IF microscopy images of fixed HeLa cells at 72 hours post transfection with esiRNA

against firefly Luciferase (Control) or CEP120. Cells were labeled for DNA (blue), CETN2

(green) and PCNT (red). Boxed insets show 2x magnified centrioles as labeled by CETN2.

Scale bars 5 m. (Right) Histogram showing the percentage of mitotic HeLa cells in which either

0 or 1-4 centrioles were detectable. White histogram bars indicate Luciferase RNAi, black

histogram bars indicate CEP120 RNAi. (B) HeLa cells were treated with esiRNA as in (A), with

or without stable expression of mouse CEP120 fused to a LAP-tag from a BAC. The number of

centrioles were counted in mitotic cells for each condition based on CETN2 labeling, and results

used to generate the histogram shown. No significant differences in centriole number were

observed in the HeLa cells expressing mouse CEP120. (C) Bar graph showing the percentage of

U-2 OS cells in which more than 4 CETN2 foci were observed after 24 hours of RNAi followed

by 68 hours of arrest in S-phase by HU treatment. Error bars represent the standard deviation of

the mean. *p<0.05, **p<0.01 based on unpaired two-tailed student t-test. At least 50 cells were

counted per each replicate, with three replicates per experimental condition.

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Figure 2.2

72

Figure 2.2. CEP120 and SPICE1 are required for centriole duplication in cycling HeLa

cells.

(A) Immunofluorescence microscopy images of fixed mitotic HeLa cells at 72 hours post

transfection with siRNAs targeting CEP120 or SPICE1, or a control siRNA with no target

sequence. Cells were labeled with DAPI (blue in panels), and antibodies detecting SASS6

(green), CEP135 (red), or CETN2 (blue in insets). (B) Centrioles were counted by examining

CETN2 signal, and the frequency of observed mitotic cells with four or more centrioles is shown

for each condition. (C) Western blot of cell lysates collected from the same experiment showing

(top two rows) CEP120 protein levels under each condition versus tubulin levels under each

condition and (bottom rows) SPICE1 protein levels under each condition versus tubulin levels.

Markers indicate approximate molecular weights based on protein standards. Scale bar in (A) is

10 m, and insets are four-times magnified. (D) HeLa T-Rex cells with or without inducible

expression of siRNA resistant CEP120 were transfected with control or CEP120-si#1 for 24

hours before being induced by addition of tetracycline for 48 hours then fixed in methanol. Cells

were immunolabeled for Centrin (CETN2) and Pericentrin (PCNT) and mitotic cells were

examined. (D) Shows representative images of mitotic cells under the absence of siRNA

resistant CEP120 (left) or in the presence of siRNA-resistant CEP120 (right). Scale bar is 10

m, and insets are approximately 2.5-times magnification. (E) The frequency of mitotic cells

with un-duplicated centrioles was quantified in the experiment described in (D), and bar graph

shows the frequency of cells with failed duplication under each condition. Error bars in (B, E)

indicate SEM from three replicates in which at least 50 cells were counted per condition per

replicate.

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Figure 2.3.

Figure 2.3. SPICE1 interacts with CEP120 through its coiled-coil domains.

(A) Western blots from IP experiments using CEP120 protein truncations (residues 1-700 and

700-986) or full length CEP120 fused to HA tag as bait. Blot shown using antibodies against

(top) SPICE1 and (bottom) HA following co-IP and for inputs. A model of CEP120 is shown

below indicating the coiled-coil domain and the fragments used for IP. (B) Western blots from

IP experiments using SPICE1 truncations (residues 1-450, 350-750, and 450-856) or full length

SPICE1, each fused to GFP, as baits. (Top) Western blot using antibodies against CEP120 co-IP

in each condition and in respective inputs. (Bottom) Western blot using antibodies against GFP

for each sample. A model of SPICE1 indicating the coiled-coil domains and the fragments used

for IP is shown. Experiments performed by Dr. M. Archinti, Lab of Dr. J. Luders.

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Figure 2.4

75

Figure 2.4. CEP120 and SPICE1 Depletion blocks centriole overduplication induced by

PLK4 overexpression in U-2 OS cells.

(A) U-2 OS cells on coverslips were transfected with siRNA or esiRNA against target genes as

indicated, and at 48 hours post-transfection were arrested in S-phase with hydroxyurea and

induced to over-express PLK4 by addition of tetracycline. At 20 hours post induction and arrest,

cells were fixed and lysates were collected for Western blots. Stacked bar graph shows the

frequency of centriole overduplication (black bars) versus non-overduplication (grey and white).

White bars represent the fraction of centrosomes that failed to produce procentrioles and

simultaneously lacked SASS6 localization around the mother centriole. Grey-bars show the

portion of centrosomes where duplication failed but SASS6 was seen localized around the

mother-centrioles. Error bars represent the SEM from three replicates for which at least 100

cells were examined. (B) Representative IF images from each condition where either SASS6

was recruited or SASS6 was not recruited. In large panels, DNA (blue), SASS6 (green) and

CETN2 (red) labels are shown. Insets show CETN2, CEP135 and SASS6 at centrosomes at 2.5x

magnification. No cells were observed with SASS6 recruitment under SASS6 RNAi, so no

image is shown. Scale bar 10 m. (C) Western blots for CEP120 (top) and SPICE1 (third row)

with loading controls (tubulin blots, second and bottom rows respectively) from the same

experiment.

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Figure 2.5

77

Figure 2.5. CEP120 and SPICE1 depletion block centriole overduplication downstream of

SASS6 recruitment, but lead to abnormal SASS6 localization.

U-2 OS cells expressing Myc-PLK4 under the regulation of the tetracycline-repressor were

transfected with siRNA or esiRNA targeting CEP120, SPICE1, CEP135, CPAP or no-target at all

(control) for 24 hours before being arrested in S-phase by addition of hydroxyurea and induced to

over-express Myc-PLK4 by tetracycline addition. 24 hours after S-phase arrest and induction,

cells were rinsed and arrested in G2 by addition of RO-3066 for 16 additional hours before

fixation and cell lysate collection. (A) Representative IF images of cells from each condition

showing DNA (blue) and SASS6 (green), CETN2 (red). Insets show centrosomes at 4x

magnification (monochromatic) and 8x magnification (colour). Scale bar 10 m. (B) Stacked

bar graph showing the relative frequency of centriole overduplication (black bars) versus non-

overduplication (grey and white). White bars represent the fraction of centrosomes that failed to

produce procentrioles and simultaneously lacked SASS6 localization around the parental

centriole. Grey-bars show the portion of centrosomes where duplication failed but SASS6 was

seen localized around the parental centriole. (C) Quantification of the percentage of cells from

each condition where the SASS6 localization appeared smoothed as a ring-like shape around the

parental centriole, versus punctate allowing distinction of individual procentrioles (see Control

versus CEP120 IF image insets). For (B) and (C), error bars represent the SEM from three

replicates for which at least 100 cells were examined. For (C), significance is indicated as a result

of an unpaired two-tailed student’s t-test against the control. (D) Western blots for proteins

depleted in this experiment were prepared using lysate from the same experiment by Sally

Cheung. Western blots for -tubulin as a loading control are immediately below their respective

blots.

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Figure 2.6

79

Figure 2.6. Three-dimensional SIM imaging of PLK4 induced centriole overduplication. (A) (Left, Top) Representative immunofluorescence image of S-phase arrested un-induced U-2

OS cells showing cells fixed and immunolabeled for Centrin (CETN2) and Myc-PLK4 versus

(right) representative images from induced U-2 OS cells over-expressing Myc-PLK from the

same experiment. (Bottom) Models showing the presumed orientation of the centrioles from the

un-induced cell (left) versus the easily defined orientation of the procentrioles surrounding

parental centrioles in the induced Myc-PLK4 over-expressing cell (right). (B) 3D-SIM images

of centrosomes after Myc-PLK4 overexpression in U-2 OS. Cells were labeled with antibodies

against CETN2, CP110 and SASS6 for immunofluorescence imaging. Centrosomes were

imaged where the parental centriole was oriented along the z-axis, and thus procentrioles are

seen side-on in the images. Centrosomes were selected at various stages of procentriole

elongation, increasing from left to right, as indicated by the space between CP110/CETN2 and

the mother centriole. (C) Immunofluorescence images of similarly oriented centrosomes from

the same conditions labeled for CEP120 (left) or SPICE1 (right), Myc-PLK4 and CETN2.

Dashed circles indicate the location of the Myc-PLK4 signal throughout the images. White “M”

indicates the location of the mature (parental) centriole for the CEP120 labeled images. Scale

bars are each 1 m.

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Figure 2.7

81

Figure 2.7. Impact of CEP120 and SPICE1 on the recruitment and localization of centriole

assembly proteins.

3D-SIM images of centrosomes from the G2 PLK4 assay where cells were treated with non-

targeting siRNA (Controls), or siRNA against CEP120 or SPICE1. Cells were labeled with

antibodies against CETN2 and the Myc epitope tag, along with one of the procentriole assembly

proteins CEP152, SASS6, STIL, CPAP, CEP97, CP110 or hPOC5. For each staining, the

specific marker is shown in green at the top of the first column, and the last column where the

inset shows that marker alone. Scale bars represent 1 m. Images are scaled so that intensities

are quantitatively comparable within a given 3x4 panel, with the exception of Myc-PLK4 signal

which has been brightened in each Control to facilitate visibility.

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Figure 2.8

83

Figure 2.8. Depletion of CEP120 or SPICE1 leads to failure of CEP135 recruitment during

centriole duplication. (A) Representative images of centrosomes observed in the G2 PLK4

assay when CEP120, SPICE1, CPAP or CEP135 were depleted. Centrosomes were labeled with

antibodies against Myc-PLK4, CETN2 and CEP135. (B) Quantification of total pixel intensity

of CEP135 in the procentriole assembly region (PCAR) of 3D-SIM images of correctly oriented

centrosomes in the absence of CEP120, SPICE1, CPAP or CEP135 compared to an un-depleted

control, from a G2 PLK4 assay. Top: Outline of the masking approached used for image

quantification in MatLab by Dr. Gupta. The dashed region defined by Myc-PLK4 signal (red)

was masked and that mask applied to the CEP135 signal (green). The Cep135 intensity within

the Myc-PLK4 defined region was quantified. Bottom: Box and whisker plot showing total pixel

intensities from CEP135 overlapping Myc-PLK4 signal for each centriole from the various

depletion conditions. Multiple centrosome images were chosen for each condition based on

orientation of the centrioles (n=62, 59, 104, 78, 60 from left to right across X axis). Red bar

shows the mean, light-gray represents the 95% confidence interval, and darker gray outlines one

standard deviation. (C) Cycling U-2 OS cells expressing SASS6-GFP were depleted of CEP120,

SPICE1 or CEP135 as indicated for 72 hours. Cells were labeled with antibodies against GFP,

CEP135, and CETN2. Representative centrosomes were selected in S-phase cells for each

condition. Yellow arrowheads indicate position of SASS6 positive procentrioles in each case,

and are positioned consistently in every image within each column. (D) U-2 OS cells were

synchronized as indicated (bottom) to be in S-phase with two mother centrioles at the time of

fixation. Cells were labeled with antibodies as in (C), and arrowheads similarly indicate SASS6

positive procentrioles within each column. (E) Bar graph of the fraction of procentrioles under

each condition from (D) that were observed to have SASS6 but not CEP135. Bar graph shows

mean with error bars indicating the SEM from three experiments, with at least 50 centrosomes

examined for each condition. (F) Bar graph of the mean CEP135 pixel intensity overlapping

SASS6 signal from SASS6 positive procentrioles in cells treated and fixed as in (D). Bar graph

shows the mean from three experiments (total number of centrioles quantified is 158, 164, 112,

166 respectively, from left to right on the x-axis of the graph). Error bars indicate the SEM. All

images are from 3D-SIM experiments acquired quantitatively and are scaled identically for each

channel within a given panel, except for Myc-PLK4 in (A) which is scaled for visibility in the

image from the control condition.

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Figure 2.9

85

Figure 2.9. Recruitment dependencies of CEP120 and SPICE1 during PLK4 induced

centriole overduplication.

For all three panels (A-D), G2 PLK4 assays were performed with RNAi as indicated and cells

were fixed and labeled with antibodies against CEP120 (left) or SPICE1 (right), CETN2, and

Myc-PLK4. Representative quantitatively imaged 3-D SIM micrographs of centrosomes are

shown with equal intensity scaling within each panel, with the exception of Myc signal which is

enhanced for visibility in controls. (A) The early centriole assembly proteins CEP152, STIL or

SASS6 were depleted in the G2 PLK4 assay. Parental centrioles are oriented differently in

images from CEP152 depleted cells to show CEP120 and SPICE1 localization along the parental

centriole barrel. (B) CEP120, SPICE1, CPAP or CEP135 were depleted in the G2 PLK4 assay.

(C) The distal centriole proteins CEP97 and CP110 were depleted in the G2 PLK4 assay. (D)

The centriole elongation proteins POC1 and hPOC5 were depleted in the G2 PLK4 assay. Scale

bars 1 m for each panel.

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Figure 2.10

87

Figure 2.10. Depletion of CEP120 or SPICE1 results in procentrioles with short or

undetectable microtubules following PLK4 induced centriole overduplication.

A G2 PLK4 assay was performed and the U-2 OS cells were fixed and prepared for serial

electron microscopy (see Materials and Methods). For each condition, two example centrosomes

are shown. From left to right, micrographs of 100 nm serial sections are shown for the same

centrosome. Yellow arrowheads point to approximate locations of procentrioles in controls, or

electron density at the proximal end of the mother centriole in depletion conditions. Scale bar

100 nm.

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Figure 2.11

89

Figure 2.11. Depletion of CEP120 or SPICE1 results in shorter procentriole microtubules

following PLK4 induced centriole overduplication. (A) Tomogram projections (average projections of 20-30 nm central volume sections) of mother

and procentrioles from cells collected and processed following a G2 PLK4 assay where either no

protein, or CEP120, SPICE1 or CPAP were depleted. (Left) Original tomograms are shown,

with duplicate images (right) where microtubules are highlighted in green. Scale bar represents

100 nm. Bar graph (B) shows mean lengths of individual microtubules of procentrioles and (C)

shows mean cross-sectional widths of detectable procentrioles measured from multiple

tomograms for each condition using IMOD software. For (B) n=16, 12, 16 and 14 microtubules

measured, for (C) n=6, 5, 6, and 3 procentrioles measured for controls, CEP120 depleted,

SPICE1 depleted or CPAP depleted conditions respectively. Widths were determined based on

widest detectable point in tomograms under all conditions. Error bars indicated standard error

of the mean. Tomography acquisition and reconstruction, as well as measurements in IMOD

were performed by Dr. Eden Fussner.

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Figure 2.12.

91

Figure 2.12. Colchicine treatment effectively depolymerizes cytoplasmic microtubules and

prevents procentriole microtubule assembly. (A) U-2 OS cells over-expressing Myc-PLK4

were arrested in S-phase and induced to over-express Myc-PLK4 for 24 hours before being

rinsed and released into G2 for a final 16 hours before fixation. Fixation was performed with

additional pre-extraction in PBS-triton to remove soluble tubulin, then labeled with antibodies

against CEP135, -tubulin and the Myc epitope tag. Throughout the entire experiment, cells

were maintained with or without 10 M colchicine. (Left) Two examples of cells without

colchicine added, where the top shows a clear microtubule network, and the bottom shows a cell

where the centrosome and centriolar microtubules are visible. (Right) Two examples of cells

maintained with colchicine where centrosomes are visible that include mother centrioles with

detectable microtubules, but lack detectable procentriole microtubules. Images are deconvolved

and projected from wide-field fluorescence microscopy. Scale bar 10 m, insets 4x

magnification. (B) 3D-SIM images of centrosomes from the same experimental setup and

labeling as in (A). Untreated (left) or colchicine treated conditions (right), scale bar 1 m.

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Figure 2.13

93

Fig 2.13. Localization of centriole assembly proteins to the procentriole assembly region in

the absence of microtubules.

(A) (Left) Representative images from 3D-SIM of U-2 OS induced to over-express PLK4 in the

presence or absence (control) of colchicine. Cells were induced and arrested in S-phase for 24

hours before being released into G2 for another 16 hours prior to fixation. Cells were labeled

with antibodies against CEP135, glutamylated tubulin and SASS6. Scale bar 1 m. (Right):

Box-and-whisker plots of the total integrated intensity of each of the three labels for the whole

centrosome for multiple centrosomes as prepared for (A). At least 50 3D-SIM images were used

for the measurement of each condition. Red bar shows the mean, light-grey the 95% confidence

interval and dark grey bar is 1 standard deviation above and below the mean. (B) Cells were

treated as in (A), but labeled with antibodies against tubulin and a centriole assembly protein

as indicated. Images are representative from 3D-SIM imaging, scale bar 1 m.

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Figure 2.14

95

Figure 2.14 CEP120 overexpression leads to abnormally elongated centrioles in G2. (A) U-2 OS cells were transiently transfected with plasmids expressing SPICE1, CEP120 or

CPAP as fusions to GFP for 24 hours before being fixed and labeled with antibodies against GFP

and acetylated tubulin. Representative images are shown for each condition. Scale bar 10 m,

insets are 2x magnified. (B) The frequency of abnormally elongated centrioles was quantified

following each overexpression experiment in (A), with three replicates performed per

transfection. Bar graph shows the mean from replicates, with at least 100 cells per condition per

replicate examined, and error bars show the standard error of the mean. (C) A U-2 OS Flp-In

TREx cell line was used for inducible expression of CEP120 fused to a FLAG tag. Cells were

induced to over-express FLAG-CEP120 for 24 hours in the presence of HU (S-phase, left panel)

or RO-3066 (G2, right panel). Cells were fixed and labeled for CEP120 and acetylated tubulin.

Representative images are shown of (top) un-induced cells or (bottom) induced cells in either S-

phase or G2 arrest. (D) Quantification of the frequency of abnormally elongated centrioles in

each condition from (C), with bar graph showing the mean of three replicates for each condition,

with at least 100 cells examined per condition. Error bars show the standard error of the mean,

and asterisks show result of an un-paired two-tailed student’s t-test comparing the induced and

un-induced conditions.

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Figure 2.15

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Figure 2.15 CEP120, CPAP and SPICE1 are all required for abnormal centriole elongation

following CEP120 or CPAP overexpression in G2.

U-2 OS cells were depleted of proteins as indicated for 24 hours before being transfected with a

plasmid driving expression of either GFP-CEP120 (A-C) or GFP-CPAP (D-F) for another 24

hours before being arrested in G2 for 48 hours then fixed. (A) Representative images of cells

labeled with antibodies against CEP164, GFP and glutamylated tubulin from each experimental

condition. Scale bars 10 m, insets are 2x magnifications. (B, E) Quantification of the

frequency of abnormal centriole elongation within cells expressing CPAP-GFP (B) or CEP120

GFP (E) from each experimental condition. (C, F) Quantification of the fraction of transfected

cells where the mother centriole was abnormally elongated following the experiment described

in (A) or (D) respectively. Bar graphs show the means from three replicates for which at least

100 cells were counted, for each experiment (B, C, E, and F). Error bars show standard error of

the mean, asterisks are results of unpaired two-tailed student t-test of each condition against the

control.

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Figure 2.16

Figure 2.16. CPAP interacts with CEP120 in vivo.

Co-immunoprecipitation experiments were performed in HeLa cells transiently transfected

with FLAG-CEP120 or FLAG-USP37 as a control, and one of GFP-USP37, SPICE1-GFP

or CPAP-GFP. Western blots were performed to detect (top) GFP epitope from FLAG-IPs,

(middle) FLAG-epitope from FLAG-IPs, and (bottom) GFP epitope in the lysates from the

experiment before IPs were performed. This experiment was performed by Sally Cheung.

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Figure 2.17

100

Figure 2.17. CEP120, SPICE1 or CPAP depletion inhibits taxol induced centriole

elongation in G2 arrested U-2 OS cells. U-2 OS cells were depleted of select proteins by RNAi for 24 hours before being arrested in G2

for 48 hours, where taxol was added to 10 M final concentration for the last 24 hours. (A)

Representative images of U-2 OS cells under each depletion condition. Cells were fixed and

labeled with antibodies against CEP164 and glutamylated tubulin. Scale bar represents 10 m,

insets are 4x magnified from the merged pseudo-coloured images. An experimental timeline is

shown underneath the representative images. (B) The fraction of cells under each condition

where at least one centriole or procentriole was elongated abnormally was quantified, bar graph

shows mean from three replicates, where at least 100 cells were examined per replicate per

condition. Error bars show standard error of the mean, asterisks show results of an un-paired

two-tailed student t-tests comparing each condition to the control condition.

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Figure 2.18

102

Figure 2.18 CEP120 and SPICE1 Functions Centriole Assembly.

(A) (Top) The process of procentriole assembly on a single parental centriole is shown (see text

for references). (From top-left) In G1 PLK4 is first recruited as a ring to CEP192 on the parental

centriole, then migrates farther away from the centriole barrel by interacting with CEP152 which

is newly recruited in G1. At the start of S-phase, the formation of a SASS6 cartwheel occurs

within the lumen of each disengaged parental centriole. The recruitment of SASS6 into the

parental lumen is mediated through binding to CPAP therein. Subsequently, dependent upon

PLK4 and STIL, SASS6 is released from the parental centrioles and localizes to the proximal

outer wall of the centrioles along with a single focus of PLK4 and STIL. From there, assembly

of a procentriole is initiated requiring the combined activities of -tubulin, /-tubulin,

Centrobin, CPAP, CEP120, SPICE1, CEP135 and CP110. In S-phase, the elongation of the

procentrioles is driven by CPAP, CEP120, SPICE1, Centrobin and Poc1, whereas in G2

elongation is further promoted by CPAP, Centrobin,CEP120, SPICE1, hPOC5 and OFD14. The

figure legend is shown below the pathway. (B) A summary of the proteins known to promote or

restrict centriole elongation is shown, with the affected portion of the centriole indicated. *OFD1

restricts the length of the parental centriole specifically. (Bottom) Legend is shown.

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2.7. Materials and Methods

2.7.1. Cell lines and Tissue Culture

U-2 OS and HeLa cells were grown in Gibco brand McCoy’s 5A media supplemented with fetal

bovine serum (10%) and 2 mM L-glutamine at 37°C. U-2 OS T-REx cells with Tet-inducible

Myc-tagged PLK4 were a kind gift from Dr. E. Nigg, and were maintained as above but with

certified tetracycline-free fetal bovine serum, and G418 at a concentration of 0.5 mg/ml for

selection. FLAG-CEP120 inducible U-2 OS T-REx cell lines were generated as per

manufacturer’s recommendation (Invitrogen). Briefly, Flp-In T-REx U-2 OS cells (Invitrogen)

in 6-well dishes at 80-90% confluence in 1 ml Opti-MEM were transfected with 2 g pOG44

and 200 ng of the pCDNA5-FRT-TO-FLAG-CEP120 plasmid using 2.5 l Lipofectamine-2000

in a total volume of 200 l Opti-MEM media. Reaction mixtures were added to each well for 3

hours before an equal volume (1.2 ml) of McCoy5A with 20% FBS was added. Cells were

incubated overnight, then transferred into 10 cm dishes for another 24 hours before Hygromycin-

B was added to the media (200 g/ml final concentration) for selection. Clones (pools) were

picked using colony-lifts into individual wells, then maintained in tetracycline-free media before

being tested for inducible CEP120 expression. To induce expression of Myc-PLK4 or FLAG-

CEP120 from T-REx cells, tetracycline was added to media to a final concentration of 2 g/ml.

2.7.2. RNA Interference

For a list of primer sequences for esiRNAs and commercially purchased siRNAs used in this

study, see Appendix I: Supplemental Methods. For depletion of CPAP and SASS6 in this study,

esiRNA were synthesized as previously described (Kittler et al., 2005). For all cell assays, cells

were seeded on sterilized glass coverslips in 6-well tissue culture dishes and allowed to settle for

at least 12 hours before treatment with siRNA or esiRNA. RNAi transfection mixes were

prepared using Invitrogen Lipofectamine RNAiMAX. For siRNA, we prepared stocks at

concentrations of 20 M, and for esiRNA we prepared stocks with concentrations of 100 ng/l.

For each well, we prepared one mix with 5 l of siRNA and 245 l Gibco OptiMem media or 15

l esiRNA with 235 l Gibco OptiMem, and mixed these with a separately prepared mixture of

2.5 l of Lipofectamine RNAiMAX in 247.5 l OptiMem. RNA was incubated with

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Lipofectamine for a minimum of 20 minutes before being added to 2.5 ml of McCoy 5A media

in each well (final volume 3 ml).

For the CEP120 siRNA rescue, either T-REx HeLa or T-REx HeLa expressing siRNA resistant

FLAG-tagged CEP120 were seeded. The next day cells were transfected with siRNA, and at 24

hours post transfection were treated with 1μg/ ml tetracycline for 48 hours before fixation. This

experiment, and generation of the T-REx HeLa cell line expressing FLAG-CEP120, were

performed by Deborah Pinchev.

2.7.3. Cloning of CEP120 siRNA Resistant Construct

CEP120 was mutated at wobble-codon positions using QuikChange II XL site-directed

mutagenesis kit (Agilent Technologies) by Deborah Pinchev. For CEP120 siRNA-1 resistance,

the primers used were 5’- GATGATTATTTGACTCGCCTGATAGAAGAAAGGGATACTTTT

ATGCGGACAGGCGTTTACAATCACGAGGATCGAATAATAAGTGAACTCGACCGACA

G-3’ and 5’- CTGTCGGTCGAGTTCACTTATTATTCGATCCTCGTGATTGTAAACGCCTG

TCCGCATAAAAGTATCCCTTTCTTCTATCAGGCGAGTCAAATAATCATC-3’.

2.7.4. PLK4 Induced Centriole Overduplication Assays

For the S-phase assay initially used (Figure 2.4), U-2 OS T-REx cells with inducible myc-PLK4

were seeded in 6-well dishes and treated with RNAi as described above. At 48 hours post-RNA

addition, cells were arrested in S-phase by addition of hydroxyurea to a final concentration of 8

mM, and induced to over-express PLK4 by addition of tetracycline (2g/mL) for 20 hours

before fixation.

For the ‘G2 PLK4 assay’, RNAi was as above, but at 24 hours post-RNA addition the cells were

arrested in S-phase using HU and induced by tetracycline addition. After 24 hours in S-phase

arrest, cells were rinsed twice with 3 mL sterile PBS (Gibco) and put into fresh McCoy 5A

media containing 10 M RO-3066 (Enzo Life Sciences) to arrest them in G2. At 16 hours post

G2 arrest, cells were fixed on coverslips for microscopy.

For the PLK4 assay in the presence of colchicine, cells were arrested in S-phase and induced to

express PLK4 as described for 24 hours in the presence of colchicine, then rinsed and arrested in

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G2 as described for 16 hours before fixation. Colchicine (Sigma) was maintained at 10 M from

the time of S-phase arrest until fixation.

2.7.5. Taxol Induced Centriole Elongation

For the taxol induced centriole elongation assay, U-2 OS cells were seeded and transfected with

RNA as described, and at 24 hours post-RNA addition, cells were arrested in G2 using RO-3066

as described. After 24 hours of G2 arrest, taxol (Sigma) was added to 10 M final concentration,

and cells were maintained for another 24 hours in taxol before fixation.

2.7.6. CPAP or CEP120 Induced Centriole Elongation

For inducible FLAG-CEP120 expression in testing of the cell-cycle specificity of elongation,

cells were seeded at equal densities and the next day induced with tetracycline (2g/mL) in the

presence of either 10 M RO-3066 (Enzo Life Sciences) to G2 arrest, or 8 mM HU, and

incubated under these conditions for a subsequent 24 hour period before fixation and preparation

for microscopy. For assays quantifying centriole elongation by CEP120, SPICE1 and CPAP, U-

2 OS cells were seeded on coverslips in 6-well dishes for at least 12 hours before plasmid

addition. 2 g of plasmid was combined with 3 L of Lipofectamine-2000 (Invitrogen) to a final

volume of 500 L in OptiMem, incubated for 20 minutes, then added to 2.5 mL of McCoy-5A

media in each well (final volume 3 mL). At 24 hours post-addition of plasmid, cells were fixed

for microscopy. For overexpression in combination with RNAi, cells were transfected first with

siRNA or esiRNA (described above) for 24 hours, then subsequently with plasmid as described.

At 24 hours post plasmid addition, cells were arrested in G2 using RO-3066 at 10 M (Enzo Life

Sciences) for 48 hours, then fixed as described below.

2.7.7. Immunofluorescence Microscopy (IFM) and 3D-SIM

For IFM, cells on coverslips were fixed by submersion into pure methanol maintained at -20°C,

and incubated at -20°C for a minimum of 15 minutes. Cells were blocked by submersion in PBS

with 0.2% fish-skin gelatin (Sigma) at room temperature for 10 minutes (hereafter PBS-FSG).

Primary antibody solutions were prepared in PBS-FSG, and glass coverslips were inverted onto

droplets of antibody-PBS-FSG solution on clean Parafilm (Parafilm M) in a humidified chamber

for 30-60 minutes at room temperature. Coverslips were then floated off the Parafilm by

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pipetting 500 l PBS-FSG and transferred back to PBS-FSG for 15 minutes to wash off excess

primary antibody. Secondary antibodies were mixed in PBS-FSG at 1/500 dilution, along with

DAPI or Hoechst 33342 (Sigma) at 1 g/mL, and coverslips were again inverted onto droplets

for 30-60 minutes on clean Parafilm in a humidified chamber at room temperature. Coverslips

were again floated off the Parafilm and submerged in fresh PBS-FSG for a final rinse for 15

minutes. Coverslips were then submerged in ultra-pure distilled water for 1 minute before being

inverted onto drops of ProLong Gold mounting media (Molecular Probes/Invitrogen). A list of

antibodies used is provided in Appendix I.

For any images being compared as quantitative or scaled quantitatively in terms of intensities,

acquisition conditions (exposure time, number of z-sections imaged, light intensity, microscope

used) were identical for each image, and imaging was scaled to avoid pixel saturation. Post-

acquisition, or post reconstruction for 3D-SIM images, images were exported as tiff files of

either 8 or 16 bit depth with identical scaling from SoftWorx, then adjusted identically in Adobe

Photoshop before being used in figures. Images were scaled to avoid saturation at all times.

Image quantifications were performed using MATLAB software by Dr. Gagan Gupta, using

scripts authored by Dr. Gagan Gupta. For 3D-SIM image quantifications, reconstructed and

aligned, maximum-intensity projected non-saturated images were used. For each image set, the

centrosome was identified as being within a region where the maximum overall intensities were

present. Within the region of the centrosome, ‘masks’ were generated using consistent signal-to-

noise and size thresholds for each image channel specific for the protein of interest detected in

that channel. Recruitment to the centrosome for a given protein was quantified as the total pixel

intensity of its masked region. The PCAR mask was defined as the region at the centrosome that

was marked by the Myc-PLK4 label. Recruitment to the PCAR was quantified as the total pixel

intensity of a given label where that label overlapped the PCAR mask. For CEP135

quantification with the SASS6 signal in S-phase synchronized cells (Figure 2.8), SIM images

were exported as quantitatively scaled .tiff files, and opened in IMAGEJ (FIJI build). A defined

size region of interest (ROI) was overlaid on the SASS6 signal from each procentriole centriole,

and the mean CEP135 pixel intensity was obtained for that area from the software. All imaging

was performed on Delta Vision microscopes (GE Healthcare-Applied Precision), with a 40X NA

1.35, 60X NA 1.42, or 100X NA 1.4 plan apochromat oil objective (Olympus). Image scaling

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and .tiff files were generated by SoftWoRx proprietary software. 3D-SIM was performed using

a Delta Vision OMX microscope, with reconstruction and image alignment performed in

SoftWoRx (GE-Healthcare-Applied Precision) as previously described (Lawo et al., 2012) with

either 60X NA 1.42, or 100X NA 1.4 plan apochromat oil objectives (Olympus).

2.7.8. Cloning

All cloning was performed using KOD DNA polymerase (Toyobio EMD Millipore), restriction

enzymes from New England Biolabs, and T4 DNA ligase from Fermentas (Rapid Ligation Kit).

A complete list of plasmids used in this work is provided in Appendix I. Briefly, the SPICE1

EGFP constructs (full length and fragments) were provided by the lab of Dr. Jens Luders

(Archinti et al., 2010). HA-CEP120 fragments used in the Co-IP experiments were cloned into

pCS2-HA-FA, a pCs2plus based plasmid provided by Guowei Fang (Genetech). Mouse CEP120

plasmid used in rescue experiments was a generous gift from the lab of Dr. Tsai (Xie et al.,

2007).

2.7.9. Statistical Methods

All reported probabilities (p-values) are from two-tailed unpaired student t-tests, performed using

GraphPad Prism 5 software. Experiment sample numbers and replicates are as indicated in

figure legends. The following shorthand is used throughout: *** p<0.001, **p<0.01, *p<0.05.

2.7.10. Western Blots

For RNAi verifications, cell lysates were collected by addition of Laemmli buffer directly to

cells in tissue culture wells following removal of the corresponding coverslip for fixation and

imaging. Proteins were resolved using 8% SDS-Polyacrylamide gels subjected to

electrophoresis (SDS-PAGE), then transferred to PVDF membranes (Immobilon-P, Milipore).

PVDF membranes were dried after transfer as per manufacturer’s recommendations to improve

protein adhesion. Membranes were then incubated in primary antibodies in a Tris-Buffered-

Saline buffer containing 0.1% Tween-20 (TBST), with 5% skim-milk powder (BioShop) and 5%

BSA Fraction V (OmniPur). Primary antibodies were incubated with blots for 12-16 hours

overnight at 4°C. Following primary antibody treatment, membranes were rinsed four times for

ten minutes in TBST alone, then incubated with secondary HRP conjugated antibodies (Mouse,

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Rabbit, or Goat, Jackson ImmunoResearch) for 1-2 hours at room temperature followed by a

repeated set of four rinses in TBST. Western blots were developed using SuperSignal Pico or

Dura reagents (Pierce/Thermo Scientific) and Amersham-Hyperfilm HCL films were used to

detect signals (GE Healthcare). Blots were digitized using a Canon scanner as tiff images.

2.7.11. Electron Microscopy

Samples were prepared as described in previous publications (Ahmed et al., 2008; Fussner et al.,

2012). Cell samples were provided to Doug Holmyard in Mt. Sinai as pellets fixed in 2%

glutaraldehyde. Doug Holmyard prepared samples using 1% osmium tetroxide followed by

dehydration in an ethanol series, and subsequent infiltration in propylene-Quetol-Spurr resin and

polymerization. 70-100 nm sections were contrast enhanced with 1% uranyl acetate. For

tomography, 10 nm gold particles were added onto samples to act as fiducial markers for

alignment of tilt-series images, an d all samples were coated with 3-5 nm carbon film by direct

evaporation to increase sample stability. Transmission EM images were all collected using a

Tecnai20, FEI operated at 200 kV and collected using an AMT side mount camera. All

tomography data sets were collected by Dr. Eden Fussner based on centrosomes I selected from

each condition. For tomography, zero-loss images were acquired with a post-column Gatan

imaging filter at tilt angles +/- 55 degrees with 2 degree increments using SerialEM

(Mastronarde, 2005). The images were aligned and processed using the weighted back-projection

implementation within the IMOD software program (Kremer et al., 1996; Mastronarde, 2005)

Microtubule length and centriole width measurements were performed in averaged tomographic

slabs from central volumes of 20-30 nm thickness using IMOD. Projections of these central-

slabs are shown in figures.

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3. Chapter III:

Identification of CEP120 and SPICE1 Associated

Proteins Required for Centriole Duplication and

Elongation

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3.1. Statement of Contribution, Rights and Permissions

Some of the data in this chapter has been published as part of a report in the journal Current

Biology:

David Comartin*1,2, Gagan D. Gupta1, Eden Fussner3,4, Etienne Coyaud5,6, Monica Hasegan1,

Marco Archinti7, Sally W.T. Cheung1, Deborah Pinchev1, Steffen Lawo1,2, Brian Raught5,6,

David P. Bazett-Jones3,4, Jens Luders8, and Laurence Pelletier1,2. CEP120 and SPICE1 Cooperate

with CPAP in Centriole Elongation, Current Biology.2013 July 22. 23(14): 1360-1366.

This publication was a collaboration involving researchers from:

(1) The Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto Ontario Canada.

(2) The Department of Molecular Genetics at the University of Toronto, Toronto Ontario

Canada.

(3) The Department of Biochemistry at the University of Toronto, Toronto Ontario Canada.

(4) The Hospital for Sick Children, Toronto, Ontario, Canada.

(5) The Ontario Cancer Institute, Toronto, Ontario, Canada.

(6) The Department of Medical Biophysics at the University of Toronto, Toronto, Ontario,

Canada.

(7) ERA Biotec, Barcelona, Spain.

(8) Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain.

Sally Cheung (Pelletier Laboratory) prepared and processed all the samples for the BioID

experiment, including cloning BirA*-tagged constructs discussed, generating and validating

inducible BirA* cell lines and preparing cell extracts for mass spectrometry.

Stine Morthorst (Pedersen Laboratory, University of Copenhagen, Denmark) collaborated with

our lab, working on NAP1L1 functional characterization. Stine provided the NAP1L1

expression constructs used here, cloned one of the siRNA resistant NAP1L1 constructs, and

assisted in quantification of some of the PLK4 assays wherein NAP1L1 siRNAs were tested.

Dr. Etienne Coyaud (Raught Laboratory) collaborated to process and analyze all the BioID

experimental samples, including affinity purification and mass spectrometry of cell pellets for

FLAG-BirA* controls, FLAG-BirA*-CEP120 and FLAG-BirA*-SPICE1 samples.

Dr. Mariana Gomez- Ferreria (Pelletier Laboratory) designed PCR primers for amplifying

GAPDH in RT-PCR experiments as a control for total mRNA levels.

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3.2. Summary

Identification of proteins required for centriole duplication, elongation or mitosis is an important

step towards fully understanding these processes. Here, I describe the use of the BioID method

to identify proteins in the proximity of CEP120 and SPICE1. From these proteins, I identified

several as being required for centriole overduplication following PLK4 overexpression. The

proteins required for overduplication were further tested for possible roles in CEP120/SPICE1

localization. Depletion of several of these proteins led to CEP120 localization to cytoplasmic

microtubules that were abnormally glutamylated. Further, I identified several proteins that were

required for centriole elongation in a taxol induced centriole elongation assay. Overall, I present

here a set of proteins with previously uncharacterized roles in centriole duplication,

CEP120/SPICE1 localization, and centriole elongation. These proteins are strong candidate

interactors for CEP120 and SPICE1, and potentially critical for their function. This work

provides a foundation from which further investigation promises to improve our understanding

of how centriole elongation and duplication are achieved, and how CEP120 and SPICE1

participate in these processes.

In this chapter, we identify a novel role for one of two ubiquitously expressed NAP1L proteins,

NAP1L1, in centriole duplication. Human nucleosome assembly protein-like proteins (NAP1Ls)

chaperone histone proteins into and out of nucleosome core particles, with important roles in the

regulation of DNA organization. NAP1L1 was identified as a prey protein for both CEP120 and

SPICE1 in our BioID experiments. Subsequently, several small-interfering RNAs that targeted

NAP1L1 mRNA were found to prevent centriole duplication. Further, NAP1L4, the other

ubiquitously expressed NAP1L protein, was not required for this process. In support of a novel

role for CEP120/SPICE1 within the nucleus, both CEP120 and SPICE1 BioID identified nuclear

proteins including histones and a component of the INO80 chromatin remodeling complex.

Overall, these findings reveal a novel link between CEP120/SPICE1 and the nucleus, and a

requirement for a nucleosome-assembly protein in centriole duplication.

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3.3. Introduction

3.3.1. BioID

One of the keys to understanding how a protein functions is to identify its interacting partners.

The interaction of CEP120 with SPICE1 was identified by affinity purification and mass

spectrometry, a method widely used to identify proteins either directly interacting or in complex

with a protein of interest (Hutchins et al., 2010). This approach will robustly detect stable

interactions, but can miss transient or unstable interactions. In this chapter of the thesis, I

describe the characterization of potential CEP120 and SPICE1 interacting partners identified by

another method known as BioID (Roux et al., 2012). BirA is an Escherichia coli biotin ligase

which has strong specificity for its substrates, whereas a mutant (BirA-R118G, hereafter BirA*)

has been identified that abolishes the normal interactions of BirA, and releases a biotin-

derivative that rapidly reacts with amines in its proximity, such as those of lysine residues (Choi-

Rhee et al., 2004; Roux et al., 2012). When fused to a ‘bait’ protein, BirA* will cause

biotinylation of the lysine residues of proteins found around the bait protein (Choi-Rhee et al.,

2004; Roux et al., 2012). Adopted to mammalian cells, the BirA* fusion provides an alternative

method to identify both proteins stably interacting with the bait and proteins that transiently

interact with or are in the proximity of the bait briefly (Roux et al., 2012). The use of the BirA*

fusion to detect proximity interactions has been termed the “BioID” method, wherein a bait of

interest is expressed in fusion to BirA* in the presence of exogenous biotin, and Streptavidin

beads are used to affinity purify biotinylated proteins, which are then identified by mass

spectrometry (Roux et al., 2012). One of the major benefits of this approach is that the covalent

biotin linkage is stable under denaturing conditions, so centrosome proteins can be solubilized

before Streptavidin affinity purification (Firat-Karalar et al., 2014). The potential drawbacks of

BioID include the fact that acceptor proteins will be biased or missed based on the number of

accessible lysines, and the BirA* tag (35 kDa) must not interfere with the function or interactions

of the bait protein (Roux et al., 2012). Another drawback is that BioID detects proteins only

based on their proximity, so some identified proteins will have no functional relationship or

direct interaction with the bait (Roux et al., 2012). Despite the drawbacks, BioID has been

successfully applied since its development, including one study that used known centriole

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assembly proteins as baits to identify novel proteins required for centriole assembly (Chen et al.,

2015; Dingar et al., 2014; Firat-Karalar et al., 2014; Kim et al., 2014; Lambert et al., 2014).

3.3.2. Human Nucleosome Assembly Proteins

One of the CEP120 and SPICE1 associated proteins we study within this chapter is a nucleosome

assembly protein, NAP1L1. Histones are octamers of histone proteins, and within the nucleus

DNA is wrapped around histones to create structures known as nucleosomes (Akey and Luger,

2003; Burgess and Zhang, 2010; Khorasanizadeh, 2004; Kornberg and Lorch, 1999). Within a

histone octamer, there are two copies of each of the histone proteins (H2A, H2B, H3 and H4)

(Akey and Luger, 2003; Kornberg and Lorch, 1999). The assembly of histones onto newly

synthesized DNA, their removal during replication or transcription, and the exchange of variants

of the histone proteins having different properties are all mediated by histone chaperone proteins

(Burgess and Zhang, 2010; Okuwaki et al., 2005, 2010; Park and Luger, 2006a). One class of

chaperones critical to the function of histones in humans is the nucleosome assembly protein

family. The nucleosome assembly protein NAP1 is found in most eukaryotes, and humans have

five NAP1-like nucleosome assembly proteins (NAP1L1-5) (Attia et al., 2011; Park and Luger,

2006a; Zlatanova et al., 2007). Three of these proteins are restricted in their expression to

specific tissues (NAP1L2,3,5), whereas NAP1L1 and NAP1L4 are expressed in all tissues (Attia

et al., 2011; Okuwaki et al., 2010; Rodriguez et al., 1997; Rougeulle and Avner, 1996; Simon et

al., 1994). Purified NAP1L1 and NAP1L4 have been shown to be capable of chaperoning both

nucleosome assembly and disassembly in vitro, with NAP1L1 showing a stronger ability to

disassemble histones than NAP1L4 (Okuwaki et al., 2005, 2010). There is also evidence that

both proteins interact with H2A-H2B dimers in vivo, and that the NAP1 family proteins can

hetero- or homo-dimerize (Attia et al., 2011; Park and Luger, 2006b). Although the majority of

cellular NAP1L1/NAP1L4 are reported to be cytoplasmic, both proteins must be at least partly

nuclear to function in histone protein exchange, and both contain nuclear-export sequences and

nuclear localization sequences (Attia et al., 2011; Okuwaki et al., 2010; Park and Luger, 2006a,

2006b; Rodriguez et al., 1997). NAP1L1, and no doubt NAP1L4, have critical roles in cellular

function as a consequence of their roles in histone structure and transcriptional regulation (Park

and Luger, 2006a; Zlatanova et al., 2007). NAP1L1 levels increase in proliferating cells,

suggesting that it is a positive regulator of growth (Simon et al., 1994). To date, no published

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evidence has suggested a direct functional link between NAP1L family proteins and the

centrosome.

3.3.3. The Role of tubulin Glutamylation in Microtubule and Centriole

Stability

The function and stability of microtubules can be heavily regulated by the addition or subtraction

of post-translational modifications to and tubulins (Janke and Bulinski, 2011). Within this

chapter of the thesis, I examine changes to the post-translational modifications of microtubules

and centrioles. Centriolar microtubules are subject to extensive post-translational modification

(PTM), including detyrosination, conversion to 2 tubulin, acetylation and glutamylation (Janke

and Bulinski, 2011). These PTMs are typically observed on long-lived microtubules, and though

they do not directly alter microtubule stability, they can serve to regulate it by affecting the

binding of microtubule-associated-proteins (Janke and Bulinski, 2011). Of particular interest for

the centriole is glutamylation, the addition of short or long polymers of glutamic acid residues to

an existing glutamic acid within the CTD of tubulins (Janke and Bulinski, 2011; Janke and

Kneussel, 2010; Magiera and Janke, 2014; Wloga and Gaertig, 2011). This modification is

limited to cellular contexts where MT stability is critical, including neuronal axons, cilia, the

mitotic spindle and the centrioles (Bobinnec et al., 1998b; Janke and Bulinski, 2011; Janke and

Kneussel, 2010; Magiera and Janke, 2014; Song et al., 2013; Wloga and Gaertig, 2011). It has

been suggested that glutamylated tubulin is absolutely critical for centriole stability, as injection

of an antibody that binds glutamylated tubulin (GT335) completely destabilizes the centrioles,

leading to their destruction in late G2/M (Abal et al., 2005; Bobinnec et al., 1998a; Janke and

Bulinski, 2011). This stabilization may not correlate with elongation in all cases, as OFD1

depletion leads to elongation of centrioles without glutamylation (Singla et al., 2010). The

enzymes that catalyze the addition of glutamic acid residues to tubulins are the TTLL (tubulin-

Tyrosine-Ligase-Like) polyglutamylases, of which there are 13 members in mammals (van Dijk

et al., 2007; Janke and Bulinski, 2011; Janke et al., 2005). Within this family of enzymes, there

are specificities for glutamylation of tubulin versus tubulin, and initiation versus elongation

of glutamic acid chains (van Dijk et al., 2007; Janke and Bulinski, 2011). Of the 13 TTLL

enzymes, TTLL1, TTLL5, TTL6, TTL7, TTLL9 and TTL11 localize to basal bodies (centrioles)

in ciliated mammalian kidney cells (van Dijk et al., 2007). In interphase human cells,

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glutamylation is restricted to centrioles, and how this is achieved is not well understood. In

addition to being regulated in their expression levels, it was hypothesized that concentration at

the centrosome prevents them from activation at other microtubules in the cell (van Dijk et al.,

2007). The precise mechanisms governing their localization to the centrosome, and which

TTLLs are glutamylating the centrioles, are not well understood.

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3.4. Results

3.4.1. Identification of Potential Functional Interactors of CEP120 and

SPICE1 by BioID

CEP120 and SPICE1 cooperate with CPAP to drive centriole elongation, and we have shown

that CEP120 interacts with CPAP by co-IP (Comartin et al., 2013; Lin et al., 2013b). In order to

look for other proteins that might functionally associate with CEP120 or SPICE1, Sally Cheung

generated HEK293 cell lines with inducible expression of FLAG and BirA* double-tagged

CEP120 or SPICE1 (Figure 3.1). To validate the localization, expression and activities of the

Flag-BirA*-CEP120/SPICE1 proteins, Sally Cheung examined induced and un-induced cells in

the presence or absence of biotin by IF and Western blot (Figure 3.1). Clear evidence of correct

protein localization and active biotinylation was observed for both constructs, so cell pellets were

collected and sent to Dr. Etienne Coyaud for affinity purification of biotinylated proteins using

Streptavidin, and subsequent mass-spectrometry. I analyzed the BioID data sets using ProHits

(see Materials and Methods) and generated a table of high-confidence prey (hits) for each bait

protein (Table 3.1, Table 3.2, Figure 3.2). The table of CEP120 prey here differs slightly from

our published CEP120 associations (Comartin et al., 2013) due to the use of newer controls and

replicates for the current tables. The CEP120 table included here contains more prey proteins,

and is consistent with the more recent SPICE1 BioID table in terms of controls used for analysis.

From the BioID hits for CEP120 and SPICE1, I chose several proteins for further study.

NAP1L1, MAP7D3, CCDC138, CCDC77, CSPP1, KIAA1731, KIAA0753 and ZC2HC1A were

prey found in both CEP120 and SPICE1 BioID experiments (Table 3.1, Table 3.2). CCDC138,

CCDC77, KIAA0753 and ZC2HC1A had no published functions or characterization at the time

of selection, making them potentially exciting novel proteins involved in centriole biology. Of

the selected proteins, only KIAA1731 had a known role in centriole duplication published at the

time of these experiments, but the characterization was limited to demonstrating a requirement

for KIAA1731 for centriole duplication in RPE-1 cells (Knorz et al., 2010). MAP7D3/Mdp3

was known to play a role in microtubule stability, making it an attractive possible cofactor for

CEP120/SPICE1 function (Sun et al., 2011). CSPP1 (centrosome and spindle-pole associated

protein-1) was known to be localized to the centrosome and spindles, and to be important for

mitosis and for cilia formation, but had not been implicated in centriole duplication (Asiedu et

al., 2009; Patzke et al., 2006, 2010). NAP1L1 was among the strongest peptide hits for both

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CEP120 and SPICE1, and had no previously reported role in centrosome biology, making it a

protein of particular interest. Two proteins only identified in association with CEP120 were also

chosen; ANK2 because of its high-peptide counts, and KIAA0586 because of a reported role in

cilia formation but not centriole duplication (Bangs et al., 2011; Yin et al., 2009). Several

microtubule associated proteins were identified only in the SPICE1-BioID experiments, and

from these MAP7 and MAP9 were included as potentially important for the microtubule related

functions of SPICE1. No critical cellular function for MAP7/Ensconsin had been described,

whereas MAP9/ASAP (ASter-Associated Protein) is a spindle associated MAP required for

proper mitosis (Bulinski et al., 2001; Saffin et al., 2005; Venoux et al., 2007, 2008). Lastly,

microtubule-associated tumour suppressor protein 1 (MTUS1) was also included as a protein

without a known role in centriole duplication, but was known to be centrosome and microtubule

localized with a role in mitosis, and to be down-regulated in multiple human cancers (Rodrigues-

Ferreira et al., 2009; Yu et al., 2009; Zuern et al., 2010).

For the selected proteins, I first determined whether they were required for centriole duplication

by testing them in the S-phase PLK4 induced centriole duplication assay (see section 2.7.4)

(Kleylein-Sohn et al., 2007). From this primary screen, a subset of proteins were identified that

were required for centriole overduplication (Figure 3.3). The proteins were NAP1L1, ANK2,

KIAA1731, MTUS1, CCDC77, CSPP1, KIAA0586, MAP7D3, MAP7 and MAP9 (Figure 3.3).

These findings demonstrate the potential of the BioID approach to identify proteins with

functional relationships to the FLAG-BirA* fused bait proteins used, in this case CEP120 and

SPICE1.

3.4.2. Characterization of CEP120/SPICE1 Associated Proteins

Having identified CEP120 and SPICE1 proximal proteins required for centriole overduplication,

I sought to investigate how these proteins might be functionally related to CEP120 and SPICE1.

First, I depleted the proteins for 72 hours in U-2 OS cells using RNAi. I then immunolabeled the

cells for CEP120 or SPICE1 and glutamylated tubulin to identify centrioles. I included

Centrobin in these experiments, as I was curious whether Centrobin might impact CEP120 and

SPICE1, given that it is a well-characterized centriole duplication protein that acts upstream of

CPAP in centriole duplication and was found within the CEP120 BioID preys (Gudi et al., 2014)

(Table 3.1). Further, as controls I included SASS6, CPAP, CEP120 and SPICE1 for each

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condition. Two siRNAs targeting NAP1L1 (NAP1L1-si#1, NAP1L1-si#17) were included,

because as described later in this chapter, multiple siRNAS targeting NAP1L1 were found to

affect centriole duplication. Under normal (control) conditions, CEP120 localization is restricted

to the centrosomes. I observed that depletion of several of these proteins caused CEP120

localization to cytoplasmic microtubules (Figure 3.4). Depletion of MTUS1, MAP7, Centrobin,

CSPP1, ANK2, NAP1L1, CEP135 or SASS6 lead to re-localization of CEP120 onto

microtubules (Figure 3.4). The two NAP1L1 siRNAs used in these experiments gave distinct

phenotypes in terms of CEP120 localization (Figure 3.4). Similar to CEP120, SPICE1 is

localized to the centrosome in interphase when expressed at endogenous levels. Depletion of

KIAA1731, MTUS1, ANK2, CSPP1, KIAA00586, MAP7 or MAP7D3 led to abnormal SPICE1

structures in U-2 OS cells, either resembling aggregates outside the centrosomes, or as long

filaments sometimes associated with the centrosomes (Figure 3.5). From these results, I

conclude that multiple proteins found in proximity to CEP120 and SPICE1 can affect their

localizations. Notably, while some proteins are important for both CEP120 and SPICE1

localization (ANK2, NAP1L1, MTUS1, MAP7, and CSPP1), the re-localizations of CEP120 and

SPICE1 following depletion of some of these proteins appears to be distinct. Depletion of

Centrobin, CEP135, and SASS6 uniquely altered CEP120 without detectably affecting SPICE1,

whereas KIAA0586, KIAA1731, and MAP7D3 depletion appear to have effects on SPICE1

localization without major changes to CEP120 (Figure 3.4 and Figure 3.5).

In the process of examining CEP120 and SPICE1 localization following depletion of BioID

identified proximal proteins, I noted two interesting features of the microtubule landscape under

various depletion conditions. Firstly, while under normal conditions, glutamylation is restricted

to centrioles in U-2 OS cells (see controls in Figure 3.4 and Figure 3.5), depletion of several

proteins led to glutamylation of cytoplasmic non-centriolar microtubules. To quantify this, I re-

analyzed the cells from the CEP120 localization experiment and quantified the frequency of

cytoplasmic microtubule glutamylation (Figure 3.6). Depletion of a number of the proteins led

to abnormal glutamylation, including MAP7, MAP7D3, MTUS1, CSPP1, ANK2, NAP1L1,

CEP120, SPICE1, Centrobin and CEP135 (Figure 3.6). Therefore, along with CEP120 and

SPICE1, CEP135, Centrobin and a number of other proteins tested here are important for

restricting glutamylation of microtubules to the centrioles in U-2 OS cells.

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A second unexpected observation was that depletion of some of the CEP120/SPICE1 associated

proteins led to elongated stretches of CEP120-labeled glutamylated microtubules that appeared

to be elongated centrioles. I quantified the frequency of elongated centrioles within the

experiment and observed that depletion of KIAA0586, CSPP1, ANK2 and MAP7 each resulted

in the formation of elongated structures (Figure 3.7). The elongated structures observed emanate

from paired glutamylated tubulin foci with CEP120 staining, and are therefore most likely to be

elongated centrioles or cilia. To further test the possibility that the proteins depleted in these

studies might be critical for centriole elongation, I tested the impact of depleting these proteins

on taxol-induced centriole elongation in the G2 phase of the cell cycle (see Materials and

Methods, Section 2.7). Two analyses were carried out to examine centriole elongation, the first

was based on glutamylated tubulin structure elongation in the taxol assay (Figure 3.8).

Consistent with my observations in cycling U-2 OS cells (Figure 3.7), depletion of ANK2 and

MAP7 resulted in increased frequency of centriole elongation in the taxol assay above control

conditions (Figure 3.8). Conversely, KIAA0586 and CSPP1 had opposite effects on centriole

length in the taxol assay versus the depletion assay in cycling U-2 OS cells (compare Figure 3.7

and Figure 3.8). All the other proteins depleted in the taxol assay impacted centriole elongation

negatively, with the exceptions being SASS6 and NAP1L1 when si#1 was used. The strongest

effects were observed upon depletion of MAP7D3, CPAP, CEP120, CSPP1, MTUS1 and

NAP1L1 using si#17 (Figure 3.8). For the second analysis, elongation was quantified based on

CEP120 signal, rather than glutamylated tubulin (Figure 3.9). Comparison of the two analyses

yielded three groups of results; glutamylation-specific, CEP120-specific, and results observed

with both markers. MAP7 significantly increased frequency of elongation in both cases, as did

ANK2 though the effect was not quite significant in the case of CEP120-based detection.

SASS6 had no effect on glutamylation, but did have a slight but significant positive effect on

elongation as determined by CEP120 signal. NAP1L1 depletion by siRNA#1 did not

significantly affect elongation in either assay. Several proteins had impacts that were

glutamylation specific: CCDC77, KIAA1731, and CEP135 did not have a significant impact on

CEP120 elongation from centrosomes, but each had a negative impact on centriole elongation

based on examination of glutamylated tubulin structures. Finally, depletion of KIAA0586

promoted centriole elongation in the taxol assay when CEP120 structures were quantified, but

had the opposite effect when glutamylated structures were quantified. This observation was

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unexpected based on the fact that depletion of KIAA0586 in cycling U-2 OS cells for 72 hours

resulted in apparent elongation of glutamylated tubulin structure resembling centrioles (Figure

3.7). Overall, using the BioID approach we identified CEP120/SPICE1 associated proteins

important for their localizations, centriole elongation, and/or cytoplasmic microtubule regulation

(summarized in Table 3.3).

3.4.3. NAP1L1 is a CEP120 and SPICE1 Associated Protein Required for

Centriole Duplication

One of the proteins identified in the proximity of both CEP120 and SPICE1 using the BioID

approach was the nucleosome assembly protein like protein NAP1L1. In collaboration with

Stine Morthorst, a visiting Ph.D. candidate from the Pedersen lab (University of Copenhagen), I

investigated the potential role of NAP1L1 in centriole duplication. We first investigated whether

NAP1L1 was required for centriole overduplication following PLK4 overexpression, using a set

of four siRNAs from Dharmacon and three siRNAs from LifeTech (Stealth siRNAS). Depletion

of NAP1L1 in the S-phase arrested PLK4 centriole overduplication assay using three of the four

siRNAs from Dharmacon and two of the three siRNAs from LifeTech significantly inhibited

centriole overduplication (Figure 3.10). To validate the role of NAP1L1 in centriole duplication,

I tested the impact of depletion of NAP1L1 using the siRNAS that gave phenotypes in the PLK4

assay on centriole duplication in cycling U-2 OS cells. Depletion of NAP1L1 using Stealth si#1

and Dharmacon siRNA #17 both resulted in cells with less than 4 centrioles in mitosis at 72

hours post-RNAi transfection, whereas the three other siRNAs tested did not significantly impact

centriole duplication under these conditions (Figure 3.11). Initially, NAP1L1 was depleted for

72 hours in HeLa using these two siRNAs, but the impact on centriole duplication was mild.

Therefore, HeLa cells were depleted of NAP1L1 for 96 hours and the frequency of mitotic cells

with fewer than 4 centrioles was quantified (Figure 3.12B). In this approach, both NAP1L1

Stealth si#1 and Dharmacon si#17 effectively inhibited centriole duplication (Figure 3.12). For

this reason, these two siRNAs were used for the assays described earlier in this chapter. Overall,

our observations show that NAP1L1 is important for centriole duplication in cycling cells as well

as in the PLK4 induced centriole overduplication assay.

When siRNA#1 was used to deplete NAP1L1 in the PLK4 induced centriole overduplication

assay, CEP120 was seen on cytoplasmic microtubules specifically encircling the nucleus, and

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these microtubules were enriched for acetylated tubulin (Figure 3.13). To see if this effect was

specific to the PLK4 overduplication assay, I depleted cycling U-2 OS cells of NAP1L1 for 72

hours by Stealth siRNA#1 and other NAP1L1 siRNAs. NAP1L1 depletion using Stealth-

siRNA#1 caused an increase in CEP120 localization to microtubules in this assay, though

SPICE1 depletion and NAP1L1 depletion with other siRNAs gave minor effects (Figure 3.14B).

To determine whether the re-distribution of CEP120 following NAP1L1 siRNA#1 depletion

resulted from NAP1L1 depletion or an off-target effect, Stine Morthorst generated a NAP1L1-

GFP construct that was resistant to Stealth siRNA#1. I generated a U-2 OS cell line stably

expressing this Stealth siRNA#1 resistant GFP-NAP1L1, and tested whether the presence of

GFP-NAP1L1 could restore the normal localization of CEP120 in U-2 OS cells. I found that

even in cells expressing GFP-NAP1L1 resistant to Stealth siRNA#1, depletion using this siRNA

led to CEP120 mislocalization (Figure 3.15A, B). This indicates that either GFP-NAP1L1 was

not completely functional, was not expressed at levels required for normal cellular functions, or

else that the effect of siRNA#1 on CEP120 localization was due to an off-target effect of that

particular siRNA.

Since NAP1L1 depletion by Dharmacon siRNA#17 also gave strong centriole duplication

defects in all experimental systems tested, and given the failure of GFP-NAP1L1 Stealth-

siRNA# 1 to rescue the mislocalization of CEP120 in U-2 OS, I attempted to rescue the centriole

duplication defects by generating two GFP fusions to NAP1L1 that were resistant to Dharmacon

si#17. In one construct GFP was fused to the amino-terminal end of NAP1L1, whereas the other

construct had GFP fused to the carboxy-terminal end of NAP1L1. I generated stable U-2 OS cell

lines expressing these two constructs, and depleted NAP1L1 for 72 hours in these cell lines using

either Stealth siRNA#1 or Dharmacon siRNA#17 (Figure 3.16). In both cases, the GFP-

NAP1L1 construct was stably expressed and resistant to Dharmacon siRNA#17, but the

expression of these constructs failed to restore centriole duplication to control levels (Figure

3.16).

Given the failure of multiple attempts to rescue the phenotypes associated with NAP1L1

depletion by various siRNAs, I considered the possibilities that the rescue constructs used were

not of the correct isoform of NAP1L1, or that the siRNAs were cross-targeting other NAP1L

protein family members. The NAP1L-family in humans includes NAP1L1, NAP1L2, NAP1L3,

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NAP1L4 and NAP1L5 (Park and Luger, 2006a). NAP1L2, 3 and 5 are predicted to be expressed

only in neurons, whereas NAP1L1 and NAP1L4 are ubiquitously expressed (Attia et al., 2011;

Park and Luger, 2006a). Since NAP1L4 is ubiquitously expressed I considered whether the

siRNAs used might target NAP1L4 isoforms as well as NAP1L1 isoforms. Using UniProt-

knowledge base (http://www.uniprot.org), I collected the sequences of the two NAP1L1 and two

NAP1L4 isoforms, and used the ClustalW program to align all four sequences

(http://www.ebi.ac.uk/Tools/msa/clustalw2). The two proteins show remarkable similarity

across their sequences, with the only difference between NAP1L1 isoforms 1 and 2 being the C-

terminal region, and the major difference between NAP1L4 and NAP1L1 occurring in the

amino-terminal region where NAP1L4 is lacking two short stretches of conserved amino acids

seen in both NAP1L1 isoforms (Figure 3.17). Like the two NAP1L1 isoforms, the two NAP1L4

isoforms vary from each other only at their CTDs (Figure 3.17). I obtained the targeting

sequences of NAP1L1 siRNAs #1 and #17, and aligned them with the mRNAs for NAP1L1 and

NAP1L4 isoforms, to determine which sequences each siRNA would be predicted to target.

Both siRNAs show 100% alignment with NAP1L1 across both isoforms, whereas each has

limited sequence identity with NAP1L4 (Figure 3.18).

To address the possibility that NAP1L4 and the shorter NAP1L1 isoform were being targeted, I

generated primers for PCR to detect specific sequences unique to each mRNA (see Materials and

Methods). I depleted U-2 OS cells of NAP1L1 using both siRNAs, and in parallel treated

samples with non-targeting siRNAs or a published siRNA specifically targeting NAP1L4

(Okuwaki et al., 2010) for 72 hours. Following depletion, total-RNA was prepared from each

condition using a commercially available kit, RNA concentrations were normalized, and cDNA

was generated from the RNA using random primers. Using equal amounts of the resulting

cDNA as template, parallel PCR reactions were set up to examine relative transcript levels of

NAP1L1 isoforms, NAP1L4, and GAPDH under each depletion condition. I observed that

NAP1L1 siRNA#1 and siRNA#17 resulted in a reduction in the PCR products from both

isoforms of NAP1L1, and the short-isoform of NAP1L1 alone, but did not noticeably decrease

NAP1L4 mRNA abundance (Figure 3.19). NAP1L4 siRNA reduced the abundance of the

NAP1L4 transcripts, consistent with published reports of its specificity (Okuwaki et al., 2010).

NAP1L4 RNAi did, however, slightly reduce the abundance of the PCR product from the short

isoform of NAP1L1 in the assay, and this was unexpected since it targets a conserved sequence

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in both NAP1L1 isoforms and did not impact the PCR product from both isoforms overall

(Figure 3.19). GAPDH amplification was successful and comparable across all conditions.

Overall, I conclude that the siRNAs used can target the short NAP1L1 isoform resulting in a

decrease in relative abundance of this transcript which may explain the failure of the long-

isoform to rescue the phenotypes of NAP1L1 depletion in both centriole duplication and CEP120

localization. Since both NAP1L1 siRNAs targeted the short and long isoforms to a similar

extent, the unique effect of NAP1L1 siRNA#1 on CEP120 localization is not due to differential

targeting of a NAP1L1 isoform compared to siRNA#17. Importantly, the NAP1L1 siRNAs do

not appear to reduce the abundance of NAP1L4 transcripts.

To directly test the possibility that cross-targeting of NAP1L4 by NAP1L1 siRNAs might

explain the failed rescue of these phenotypes by siRNA resistant GFP-NAP1L1, I examined

whether NAP1L4 depletion affected CEP120 localization or centriole duplication. Using the

same siRNA against NAP1L4 as was used in the PCR experiment, I tested whether NAP1L4

depletion impacted PLK4-induced centriole overduplication or CEP120 localization in the PLK4

assay. I observed that NAP1L4 depletion did not affect CEP120 localization or centriole

overduplication in the S-phase PLK4 induced centriole overduplication assay (Figure 3.20).

Therefore, the phenotypes of NAP1L1 depletion following treatment with siRNA#1 and

siRNA#17 are not due to any cross-targeting of NAP1L4.

Given the failure of rescue experiments to demonstrate conclusively that NAP1L1 RNAi

phenotypes were specific to NAPL1 depletion directly, I obtained an additional, published

siRNA that specifically targeted NAP1L1 but not NAP1L4 (Okuwaki et al., 2010). When

NAP1L1 was depleted using this previously published siRNA reagent (NAP1L1-NEW),

centriole overduplication in the PLK4 assay was significantly inhibited, and although CEP120

was observed on cytoplasmic microtubules, the effect was not as strong as with Stealth siRNA#1

(Figure 3.21). Overall, despite the failure to rescue the effects of NAP1L1 depletion on centriole

overduplication by expression of GFP-NAP1L1, the fact that multiple siRNAs, including a

published siRNA, prevent centriole overduplication in the PLK4 assay strongly supports the

conclusion that one or both NAP1L1 isoforms are critical for centriole duplication (summarized

in Table 3.4).

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3.5. Discussion

3.5.1. Identification of CEP120 and SPICE1 Associated Proteins Critical for

PLK4 Induced Centriole Duplication

Here, we report the use of the BioID method to identify a panel of candidate interactors for

CEP120 and SPICE1. Several proteins without previously known roles in duplication were

found to be required for PLK4 induced centriole overduplication (Figure 3.3). Those proteins

were NAP1L1, MAP9, CCDC77, CSPP1, MAP7, MAP7D3, KIAA0586, KIAA1731, MTUS1

and ANK2. This work represents a starting point for characterization of these proteins as

centriole duplication proteins. Future work should focus on dissecting how each of these

proteins functions in centriole duplication. For example, where each protein functions in the

centriole assembly pathway, whether these proteins are localized to the centrosomes, and how

they impact CEP120 and SPICE1 recruitment during centriole duplication will be critical

questions to address for each. Additionally, whether or not these proteins are directly interacting

with CEP120 and/or SPICE1 in vivo will be of interest. Overall, the successful identification of

proteins required for centriole duplication by using BioID to identify proteins in the proximity of

CEP120 and SPICE1 demonstrates the potential of this approach to expand our knowledge of

proteins with roles in centriole duplication.

3.5.2. Multiple CEP120 and SPICE1 Associated Proteins Are Required for

CEP120 or SPICE1 localization

The CEP120/SPICE1 associated proteins identified as important for centriole overduplication

following PLK4 overexpression were examined in a variety of assays aimed at identifying

functional relationships with CEP120 and SPICE1. Firstly, each protein was depleted in U-2 OS

cells for 72 hours and CEP120 and SPICE1 localization examined (Figure 3.4 and Figure 3.5).

The observation in this assay that cytoplasmic microtubules were often abnormally glutamylated

led me to quantify the frequency of this phenotype for each condition (Figure 3.6). I also

observed occasional elongation of glutamylated structures from centrioles, which I interpret as

most likely reflecting abnormal elongation of centrioles (Figure 3.7). Strictly speaking, the

possibility that they are cilia cannot be ruled out, so future work should examine these structures

and look for known centriole and cilia proteins, as well as to examine their structure by electron

microscopy as was done for CPAP induced filaments when they were first reported (Kohlmaier

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et al., 2009; Schmidt et al., 2009; Tang et al., 2009b). For simplicity, I will refer to the structures

as elongated centrioles.

I note that while examining centriole number in mitotic U-2 OS cells was one of the goals of

these experiments, the lack of mitotic cells observed in a many of the conditions made this

impossible. This points to a strong possibility that many of the proteins studied here are critical

for mitosis and/or cell cycle progression, along with centriole duplication. SPICE1 is required

for mitosis and thus these proteins may be functionally linked with SPICE1 in that process

(Archinti et al., 2010). Future work should clearly include examination of the number of

centrioles in synchronized S-phase or G2 cells at 72 hours post RNAi for each condition.

Finally, I utilized taxol induced centriole elongation to test which proteins were required for

elongation, a key function of CEP120 and SPICE1 (Comartin et al., 2013). To determine

whether centrioles were elongated by taxol treatment, I examined both glutamylated tubulin and

CEP120 at the centrioles, and quantified the elongation based on each marker (Figure 3.8 and

Figure 3.9). In some cases, these two approaches yielded conflicting results. In such cases,

glutamylated tubulin is the more stringent/reliable marker for centriole elongation, since CEP120

could form elongated non-centriole structures, though this has not been reported. Alternatively,

it is possible that some proteins affect glutamylation but not centriole elongation, explaining the

difference between the two analyses. CCDC77 is a particularly strong candidate for this, given

the strong impact of its depletion when glutamylated tubulin is examined in the taxol assay,

versus the lack of effect when CEP120 is analyzed (Figure 3.8 and Figure 3.9). It remains

possible that some proteins could promote centriole elongation without glutamylation, in which

case these centrioles would likely be unstable but could persist in the arrested U-2 OS cells (Abal

et al., 2005; Bobinnec et al., 1998a).

The depletion of MTUS1 MAP7, ANK2, NAP1L1, and the well characterized centriole

duplication proteins Centrobin, CEP135 and SASS6 all led to abnormal localization of CEP120

on cytoplasmic microtubules in U-2 OS cells (Figure 3.4). Why does this occur? CEP135 and

SASS6 are required for CEP120 localization in centriole assembly (Comartin et al., 2013). This

suggests the simple possibility that CEP120 localization to microtubules is the result of a loss of

its normal centrosomal binding sites, leading to a lower-affinity interaction with cytoplasmic

microtubules. The fact that CPAP and SPICE1 depletion do not lead to this phenotype argues

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against this interpretation. Also, depletion of CEP135 or SASS6 has only a minor effect on

CEP120 localization, whereas MTUS1, MAP7, CSPP1, Centrobin, NAP1L1 and especially

ANK2 have more dramatic effects (Figure 3.4). This suggests that these other proteins have an

additional or alternative role in CEP120 localization. Below I will briefly summarize each

protein’s specific effects in our assays, and describe which proteins should be prioritized for

future study.

MTUS1 is an appealing protein for future study as it is a microtubule associated protein that has

not been well characterized functionally, and is clearly down-regulated in cancers (Rodrigues-

Ferreira et al., 2009; Yu et al., 2009; Zuern et al., 2010). In our experiments, MTUS1 depletion

led to significant glutamylation of the microtubule cytoskeleton, mislocalization of CEP120 and

SPICE1, and importantly prevented taxol induced centriole elongation (Figure 3.4, Figure 3.5,

Figure 3.8 and Figure 3.9). From these results, I suggest that MTUS1 is a strong candidate for a

novel protein required for centriole elongation, and possibly a co-factor for CEP120 and SPICE1

in this role. As such, it will be of particular importance to look for a direct interaction between

CEP120 or SPICE1 and MTUS1, as well as to examine in greater depth the role of MTUS1 in

centriole duplication. The impact of MTUS1 depletion on centriole elongation following

CEP120 or CPAP overexpression should also be tested. Whether MTUS1 overexpression alone

can drive centriole elongation in S-phase or G2 arrested cells is worth investigating.

MAP7D3 depletion affected SPICE1 localization, but did not appear to strongly affect CEP120

localization in U-2 OS cells (Figure 3.4 and Figure 3.5). Importantly, in our taxol assay,

MAP7D3 depletion yielded a stronger phenotype than CPAP depletion, strongly suggesting a

role in centriole elongation (Figure 3.8 and Figure 3.9). As mentioned, MAP7D3/Mdp3 has been

shown to stabilize microtubules in vitro, and it is also important for microtubule regrowth in vivo

(Sun et al., 2011). Notably, MAP7D3 contains coiled-coil domains that are a hallmark of

centrosomal proteins, and these domains are indispensable for its interaction with microtubules

in vivo (Sun et al., 2011). As a microtubule stabilizing protein that gave very strong phenotypes

in our centriole elongation assays, MAP7D3 should be prioritized for future study, starting with

determining whether it interacts with CEP120 or SPICE1, and is detectable on centrioles. The

same experiments proposed to test MTUS1 in centriole elongation should also be considered for

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MAP7D3, as one or both proteins could be important for CEP120 and SPICE1 mediated

centriole elongation.

CSPP1 is critical for proper mitotic spindle formation, cilia formation, and is associated with

mitotic spindle poles (Akizu et al., 2014; Asiedu et al., 2009; Shaheen et al., 2014a). CSPP1

depletion mislocalizes both CEP120 and SPICE1 in U-2 OS cells, and strongly drives

glutamylation of interphase microtubules in our assays (Figure 3.4, Figure 3.5 and Figure 3.6).

CSPP1 depletion leads to abnormal elongation of centriole-like structures in U-2 OS cells,

whereas in the taxol induced centriole elongation assay CSPP1 depletion reduces the frequency

of centriole elongation (Figure 3.7, Figure 3.8 and Figure 3.9). In spite of the seemingly

contradictory phenotypes, we can strongly infer that CSPP1 is important for centriole length

regulation and affects CEP120/SPICE1. The requirement for CSPP1 in centriole duplication and

possibly elongation would be consistent with the loss of cilia formation observed upon depletion

of CSPP1, and may be relevant to its role in Joubert Syndrome (a ciliopathy) (Akizu et al.,

2014). As such, it will be very important to determine how CSPP1 participates in centriole

assembly, and whether it is a legitimate interacting partner of CEP120 and SPICE1.

ANK2 and MAP7 were the only two proteins tested that when depleted gave abnormal centriole

elongation and cytoplasmic MT glutamylation in U-2 OS cycling cells, as well as in the taxol

assay when glutamylated tubulin was used as a marker for centriole length (Figure 3.6, Figure

3.7 and Figure 3.8). Both proteins are also important for correct localization of CEP120 and

SPICE1 (Figure 3.4 and Figure 3.5). In Drosophila, it has been recently discovered that MAP7

is important for centrosome separation and microtubule elongation within the spindle (Gallaud et

al., 2014). It is surprising that our results are consistent with an opposite interpretation; that

MAP7 depletion drives centriole elongation in multiple assays. ANK2 (ankryin-repeat

containing protein-2 or ANK-B) depletion also gave a strong phenotype in the centriole

overduplication assay. ANK2 is important for localization of membrane associated ion channels

in cardiac muscle cells (Cunha et al., 2011; Mohler et al., 2003). Mutations in ANK2

accordingly have been shown to be causal in cardiac arrhythmias (Cunha et al., 2011; Mohler et

al., 2003). Perhaps more relevant, ANK2 interacts with DCTN4, a subunit of the dynactin

complex and plays an important role in DCTN4 mediated transport along axons, as well as being

important for axon length (Ayalon et al., 2008; Lorenzo et al., 2014). It was reported, however,

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that microtubules were not affected in ANK2-null mouse neurons, so the regulation of axon

length is unlikely to be through microtubule stability (Lorenzo et al., 2014). The dynein-

dynactin complex is a microtubule motor (dynein) coupled with a cargo adaptor (dynactin) that

together play important roles in cellular structure (Kardon and Vale, 2009; Vallee et al., 2004).

Dynein has been implicated in transport of centriolar satellites to the centrosome, and both

CEP120 and SPICE1 had the satellite proteins PCM1 and CEP131 within their BioID preys

(Dammermann, 2002; Kubo et al., 1999; Prosser et al., 2009; Staples et al., 2012). One

possibility is that ANK2 may be linking CEP120/SPICE1 and/or satellites to dynactin for

transport to or from centrosomes. Future work should certainly test this hypothesis. This could

be achieved by live-cell imaging of GFP-PCM1, GFP-SPICE1 or GFP-CEP120 under ANK2

depleted conditions. Overall, these findings identify novel roles for MAP7 and ANK2 in

centriole duplication, and possibly as being important for the restriction of centriole length.

MAP9/ASAP has a critical role in mitotic spindle formation and is localized to the centrosome

and spindles (Bulinski et al., 2001; Eot-Houllier et al., 2010; Saffin et al., 2005; Venoux et al.,

2007, 2008). In one study, the authors report that the spindle assembly defects upon MAP9

depletion were not due to centriole duplication defects (Eot-Houllier et al., 2010). Our

observation of a requirement for MAP9 in centriole overduplication could be a result unique to

the PLK4 assay, or to the siRNA used in our study. Notably, SPICE1 induces spindle defects

prior to the loss of centrioles due to failed duplication, and so the MAP9 phenotypes that are

published could be separable from a role in duplication (Archinti et al., 2010). Further work will

be required to validate the proposed role of MAP9 in centriole assembly. Specifically, it will be

critical to demonstrate a failure of centriole duplication in cycling human cells when MAP9 is

depleted, and to reverse that phenotype with an siRNA resistant MAP9 construct. This may be

challenging as depletion or overexpression of MAP9 can cause mitotic defects (Saffin et al.,

2005).

KIAA0586 and KIAA1731 are both required for centriole overduplication in the PLK4 assay.

Subsequent to my experiments with KIAA0586/Talpid3, it was published to be a CEP120

interacting protein and to affect CEP120 localization to the mother centrioles (Wu et al., 2014).

Additionally, KIAA0586 has been found to interact with CP110, and is involved in multiple

ciliopathies (Alby et al., 2015; Kobayashi et al., 2014; Roosing et al., 2015). We observed a

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larger effect of KIAA0586 depletion on the localization of SPICE1, and found that depletion of

this protein led to elongated centrioles in U-2 OS cells (Figure 3.4, Figure 3.5 and Figure 3.7).

Within the taxol assay, KIAA0586 caused a slight increase in the formation of CEP120

filaments, but also slightly inhibited the elongation of glutamylated tubulin (Figure 3.8 and

Figure 3.9). KIAA1731/CEP295 was initially described as being required for centriole

duplication, however it has recently been demonstrated that KIAA1731/CEP295 is dispensable

for centriole assembly, but critical for daughter centrioles to become competent for PCM

recruitment and accordingly their stability following loss of their cartwheels (Izquierdo et al.,

2014; Knorz et al., 2010). In the PLK4 assay, the loss of parental centrioles due to loss of

stability following CEP295 depletion could explain why overduplication was inhibited. In our

assays, KIAA1731 also had a slight effect on SPICE1 localization and taxol induced centriole

elongation (Figure 3.5 and Figure 3.8). The possibility of functional cooperation between

KIAA1731/CEP295 or KIAA0586 and CEP120 or SPICE1 warrant further investigation.

CCDC77 depletion did not have a significant impact on CEP120 or SPICE1 localization,

glutamylation of interphase microtubules, or lead to abnormal elongation of centrioles when

depleted in U-2 OS cells (Figure 3.4, Figure 3.5, Figure 3.6 and Figure 3.7). CCDC77 depletion

did moderately inhibit taxol induced centriole elongation based on glutamylated tubulin signal

(Figure 3.8). Additionally, having no published characterization, CCDC77 represents an

attractive protein for study. Understanding the role of this coiled-coil protein in centriole

duplication and potentially elongation, as well as whether it cooperates with CEP120 and

SPICE1, would be of great interest.

Overall, the diversity of proteins identified here and the effects that result from depleting them

point to the possibility of a number of different pathways impacting centriole duplication and

CEP120/SPICE1 localization. These proteins represent a rich data set for future projects that will

yield novel insights into CEP120 and SPICE1 function, and centriole duplication.

3.5.3. Depletion of CEP120 and SPICE1 Associated Proteins Affects the

Microtubule Cytoskeleton

In the experiments described here, there is a correlation between CEP120 re-localization and

abnormal glutamylation of cytoplasmic microtubules (Figure 3.4 and Figure 3.6). ANK2,

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CSPP1, MTUS1, MAP7, Centrobin and possibly NAP1L1 (*siRNA#1) depletion caused both

phenotypes concurrently in U-2 OS cells. Could recruiting TTLLs to drive glutamylation of

centrioles be a function of CEP120? This possibility should clearly be investigated, given the

paucity of information on how TTLLs are regulated with regard to their localization to the

centrosome. It is known that when expressed above normal levels in HeLa cells, several TTLLs

will bind to and glutamylate cytoplasmic microtubules, so simple release of TTLLs from

centrosomes might explain this effect (van Dijk et al., 2007). Could the loss of centrioles

following CEP120/SPICE1 and other proteins lead to a loss of TTLL restriction to centrioles?

The fact that CPAP and SASS6 depletion in these assays did not have this effect as strongly as

CEP120 or SPICE1 depletion, or the depletion of other proteins, argues against this (Figure 3.6).

CPAP and SASS6 gave the strongest inhibition of duplication in the PLK4 assay, so they would

have caused the strongest glutamylation of cytoplasmic microtubules were loss of centrioles the

driving factor (Figure 3.3). If CEP120 is important for targeting TTLLs to regulate microtubule

glutamylation at centrioles, mislocalized CEP120 on cytoplasmic microtubules could drive

aberrant glutamylation. In this case, the minor but significant glutamylation of cytoplasmic

microtubules following CEP120/SPICE1 depletion would be explained by loss of centrosome-

restriction of TTLL localization. This should be tested by examining whether double-depletion

of CEP120 along with ANK2 or CSPP1 reverses the glutamylation of cytoplasmic microtubules.

An alternative explanation for these results would be that depletion of one or more of the

proteins tested leads directly to glutamylation of microtubules, and CEP120 has a strong affinity

for glutamylated microtubules. The binding of recombinant CEP120 to glutamylated versus non-

glutamylated purified microtubules in vitro should be tested, to see if CEP120 has a higher

affinity for glutamylated tubulins. If TTLL overexpression recruits CEP120 to glutamylated

cytoplasmic microtubules in vivo, this will further support a model where CEP120 preferentially

binds glutamylated microtubules, or TTLL enzymes themselves. Lastly, it is possible that the

mislocalization of CEP120 onto cytoplasmic microtubules stabilizes them, and microtubules that

are long-lived then accumulate post-translational modifications including glutamylation (Janke

and Bulinski, 2011). The stability of microtubules under CEP120 over-expression, and depletion

of ANK2 or CSPP1, should be examined. Testing the resistance of microtubules to cold-shock

induced depolymerization in vivo under these conditions would be a valid approach. It will also

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be important to look for other microtubule PTMs such as acetylation, to determine whether

glutamylation is being affected specifically.

Depletion of proteins such as ANK2 and CSPP1 lead to accumulation of CEP120 on cytoplasmic

microtubules that were glutamylated, and failed centriole overduplication. It is possible that with

CEP120, other centriole assembly proteins might be localized to cytoplasmic microtubules under

these conditions. Future work should include analysis of the localization of CPAP, Centrobin,

SASS6, hPOC5, Poc1 and CEP135 under such depletion conditions. This could reveal proteins

cooperating with CEP120 in microtubule stabilization or glutamylation. Lastly, CEP120 is

recruited without SPICE1 to cytoplasmic microtubules under ANK2/CSPP1 depletion, indicating

that SPICE1 is dispensable for the glutamylation observed in this context.

In sum, the results presented here reveal a potential link between CEP120, several of the proteins

examined in this study, and the glutamylation of interphase microtubules. It will be of great

interest to understand this link as it may provide insight into the glutamylation of centrioles, as

well as novel functions for the proteins herein described.

3.5.4. NAP1L1 is Required for Centriole Duplication

NAP1L1 has an unreported role in centriole duplication. We have shown here that NAP1L1

depletion by multiple siRNAs, including a published siRNA shown to target NAP1L1, leads to

failed centriole duplication in cycling cells and in the PLK4 assay (Figure 3.21). The difference

in phenotypes between NAP1L1 siRNAs is unexplained, as is the failure of rescue experiments

aimed at validating their effects as being due to NAP1L1 depletion (Figure 3.15 and Figure

3.16). It is possible that the GFP-tag itself might interfere with folding or function of NAP1L1,

and while we mitigated this possibility by using GFP tags on either end of the protein in separate

rescue experiments, we cannot eliminate this possibility. The other possibility is that the

NAP1L1 minor isoform may be critical for centriole duplication, remains open based on the

results of my quantitative PCR experiment showing that the minor isoform is reduced in its

expression by treatment with siRNAs#1 and siRNA#17 against NAP1L1 (Figure 3.19). To

address these possible explanations, future rescue experiments should include use of both

NAP1L1 isoforms, with smaller epitope tags, under more carefully regulated expression.

Insertion of an siRNA resistant NAP1L1 protein at the endogenous locus under its native

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promoter using CRISPR based genome editing would be an excellent approach (Doudna and

Charpentier, 2014; Gilbert et al., 2014).

NAP1L1 and NAP1L4 are functionally related and may cooperate in their histone-chaperone

activities (Attia et al., 2011; Okuwaki et al., 2010). In our hands, NAP1L4 depletion using a

published siRNA did not affect centriole duplication (Figure 3.20). It will be important to test

whether NAP1L1 interacts with CEP120 or SPICE1 by co-IP, and this should be tested for both

isoforms of the protein. NAP1L1 and NAP1L4 are members of a select group of proteins other

than tubulin that can be glutamylated on their C-termini in vivo (van Dijk et al., 2008; Regnard,

2000). Could this glutamylation mimic a glutamylated microtubule, and facilitate CEP120

binding to regulate CEP120 localization to centrosomes? This is speculative, but since the

original identification of NAP1L1-glutamylation was due to pull-down by an antibody that

detects glutamylated tubulin, such a mechanism is not entirely impossible (Regnard, 2000; Wolff

et al., 1992). The glutamylation of NAP1L1 in Xenopus is essential to its function in mitotic

chromosome organization through histone exchange (Miller and Heald, 2015). NAP1L1

glutamylation sites conserved in the human protein are found within a region of the NTD that is

absent in NAP1L4, and a region of the CTD that is partly truncated in the short-isoform of

NAP1L1 (Miller and Heald, 2015). Therefore it is possible that the NTD glutamylation sites are

relevant to centriole duplication, since NAP1L4 differs only in that region and does not function

in centriole duplication. Testing the role of NAP1L1 glutamylation in centriole duplication

would be worthwhile, especially glutamylation of the NTD region of the protein. Once a rescue

experiment is successful for NAP1L1 depletion, it could be repeated with a NAP1L1 construct

where target glutamic acid residues have been substituted with aspartic acid residues (Miller and

Heald, 2015). This would answer the question of whether NAP1L1 glutamylation in the NTD is

required for centriole duplication. Likewise, if NAP1L1 is found to interact with CEP120 or

SPICE1, whether NAP1L1 glutamylation affected that interaction would be an important

question.

Depletion of NAP1L1 using multiple siRNAs caused CEP120 localization to cytoplasmic

microtubules (Figure 3.14), and preliminary evidence suggests NAP1L1 depletion reduced

CEP120 levels at centrosomes (see Figure 3.10 and Figure 3.11A). It will be important to

quantitatively analyze CEP120 and SPICE1 levels at centrosomes following NAP1L1 depletion,

133

once an siRNA has been successfully validated by rescue experiments. The levels of

CEP120/SPICE1 should also be measured by Western-blot, to determine whether NAP1L1 is

regulating their expression or stability.

The detection of NAP1L1 raises the possibility that a population of CEP120 and SPICE1 might

localize within the nucleus, where they could be associated with NAP1L1 and potentially link

nuclear processes to centriole duplication. The SPICE1 BioID preys included multiple Importin

proteins (IPO4, 5, 7, 8), histone variants (H2AFY, HIST3H2BB), DNA polymerase Eta (POLH)

and Topoisomerases 2A and 2B (Table 3.2 and Figure 3.2). IPO4, 5, 7 and 8 are involved in

transport of histone proteins through the nuclear pore complex, and one of the proposed

functions of NAP1 proteins is the transport of histone variants from the cytoplasm to the nucleus

(Cautain et al., 2015; Rodriguez et al., 1997; Zlatanova et al., 2007). Whether SPICE1 binds

importins, histones or NAP1L1 directly should be investigated. Whether SPICE1 is required for

NAP1L1 or histone import into the nucleus, or vice versa, are also important questions. A

truncated SPICE1 protein (residues 1-450) localizes to the nucleus, in support of this idea

(Archinti et al., 2010). The strongest peptide count among the CEP120 BioID preys was subunit

2 of the INO80 chromatin remodeling complex (INO80B), a complex involved in transcription

regulation, DNA repair and DNA replication (Table 3.1) (Conaway and Conaway, 2009;

Morrison and Shen, 2009). Several nuclear proteins were prey for both CEP120 and SPICE1 in

BioID experiments; NAP1L4, p53 (TP53), p53 binding partner 2 (TP53BP2), and basic

transcription factor 3 (BTF3). NAP1L1 binds multiple transcription factors, including p53, to

help facilitate transcription (Rehtanz et al., 2004). The association of INO80B, p53, NAP1L1

and NAP1L4 suggest that CEP120 and SPICE1 could play as yet unknown roles in

transcriptional regulation within the nucleus. Alternatively, p53 and P53BP2 (ASPP2) are both

associated with centrosomes in mitosis, which could explain their proximity to CEP120 and

SPICE1 (Ciciarello, 2001; Tritarelli et al., 2004; Zhang et al., 2015). Components of the NuRD

chromatin remodeling complex localize to the centrosome, and the CHD3 subunit specifically is

required for Pericentrin recruitment to centrosomes (Sillibourne et al., 2007; Torchy et al., 2015).

Centrin-2 and Pericentrin are also shuttled between the nucleus and the cytoplasm (Keryer et al.,

2003; Prosser et al., 2009). Thus there is precedent for centrosome proteins with nuclear

localizations, and chromatin remodeling complexes affecting centrosome function. Whether

NAP1L1 localizes to the centrosome should be examined carefully, although I failed to detect

134

GFP-NAP1L1 at centrosomes by IF in U-2 OS cells (see Figure 3.15A, right-panel). Further, the

possibility that CEP120 and SPICE1 shuttle into and out of the nucleus should be examined by

IF following inhibition of nuclear export with Leptomycin-B (Keryer et al., 2003; Kudo et al.,

1999; Prosser et al., 2009). A role for CEP120 or SPICE1 in chromatin remodeling, histone

chaperoning or transcriptional regulation in addition to centriole duplication would be a very

interesting discovery.

Overall, we identify NAP1L1 as a protein in the proximity of CEP120 and SPICE1 in vivo.

Depletion of NAP1L1 using multiple siRNAs including previously validated siRNAs inhibits

centriole duplication in multiple assays and cell types. The exact impact of NAP1L1 depletion

on CEP120 localization remains obscured by differences in siRNA effects and failure to rescue

them thus far. Clearly, however, there is an unreported role for NAP1L1 in centriole duplication.

This exciting discovery, and the possible interaction with CEP120 and/or SPICE1, warrant

further investigation.

135

3.6. Figures

Figure 3.1

136

Figure 3.1. Validation of FLAG-BirA*-CEP120 and FLAG-BirA*-SPICE1 biotinylation

activity and localization.

(A) IF images of HEK293 cells that have inducible expression of FLAG-BirA*-CEP120 (left) or

FLAG-BirA*-SPICE1 (right), labeled with antibodies against FLAG (green), PCTN (blue), and

biotin (Streptavidin, red). Cells were incubated for 24 hours in 50 M biotin and tetracycline (1

g/ml) to induce expression (top rows), or tetracycline alone (middle), or neither (bottom). Scale

bars 10 m. (B) Western blots show expression of a FLAG-BirA* peptide and FLAG-BirA*-

CEP120 (left panel), and FLAG-BirA*-SPICE1 (right panel). Bottom panels of each Western

blot show detection of biotinylation of cellular proteins by Streptavidin (SA-HRP) under each

condition. Sally Cheung provided the data shown in this figure, and carried out the experiments

described herein.

137

Table 3.1 CEP120 Associated Proteins Detected by BioID.

Prey Gene Counts Control Counts AvgP MaxP SAINT

INO80B 279|173 0|0|0|0|0|0|0|0|0|0 1 1 1

NAP1L1 194|201 25|24|10|12|12|10|34|36|37|40 1 1 1

PCM1 198|180 4|3|0|0|0|0|6|5|2|3 1 1 1

ANK2 247|99 0|0|0|0|0|0|1|0|0|0 1 1 1

XRN1 196|109 13|6|0|7|7|0|8|7|4|0 1 1 1

AZI1 121|59 0|0|0|0|0|0|0|0|0|0 1 1 1

ZC2HC1A 113|54 0|0|0|0|0|0|0|0|0|0 1 1 1

CENPJ 88|65 0|0|0|0|0|0|0|0|0|0 1 1 1

NAP1L4 85|65 9|8|0|12|12|4|14|13|15|14 1 1 1

OFD1 89|58 0|0|0|0|0|0|0|0|0|0 1 1 1

CEP170 87|58 5|8|0|0|0|0|12|18|13|9 1 1 1

SPICE1 62|55 0|0|0|0|0|0|0|0|0|0 1 1 1

CEP95 60|39 0|0|0|0|0|0|0|0|0|0 1 1 1

DVL3 49|48 18|11|8|5|2|8|13|15|0|4 1 1 1

MAP7D3 71|22 1|0|0|3|2|0|4|4|2|3 1 1 1

CCDC138 61|28 0|0|0|0|0|0|0|0|0|0 1 1 1

DVL2 59|29 4|0|0|0|0|8|3|5|2|2 1 1 1

HAUS6 51|36 1|2|0|0|0|0|0|0|1|6 1 1 1

KIAA0586 53|29 0|0|0|0|0|0|0|0|0|0 1 1 1

LRRC49 41|32 0|0|0|0|0|2|9|7|0|0 1 1 1

CSPP1 39|27 0|0|0|0|0|0|0|0|0|0 1 1 1

CEP192 37|28 0|0|0|0|0|0|0|0|0|0 1 1 1

KIAA1671 45|20 0|0|0|0|0|0|0|0|0|0 1 1 1

CEP350 24|41 0|0|0|0|0|0|0|0|0|0 1 1 1

LUZP1 26|33 1|2|0|3|3|0|2|2|0|0 1 1 1

TJP1 27|19 5|2|0|0|0|0|5|3|5|6 1 1 1

GPATCH1 26|20 0|0|4|0|0|3|1|3|3|2 1 1 1

PIBF1 30|14 0|0|0|0|0|0|0|0|0|0 1 1 1

PDE3B 13|27 0|0|0|0|0|0|0|0|0|0 1 1 1

HAUS3 27|12 0|0|0|0|0|0|0|0|0|0 1 1 1

KIAA0753 21|18 0|0|0|0|0|0|0|0|0|0 1 1 1

CCDC66 36|2 0|0|0|0|0|0|0|0|0|0 0.98 1 0.98

CEP97 18|19 0|0|0|0|0|0|1|1|0|0 1 1 1

ALMS1 21|13 0|0|0|0|0|0|0|0|0|0 1 1 1

TP53 17|17 3|4|0|0|0|0|0|0|0|0 1 1 1

HAUS5 18|15 0|0|0|0|0|0|1|1|0|0 1 1 1

SSX2IP 16|16 0|0|0|0|0|0|0|0|0|0 1 1 1

MPHOSPH9 17|11 0|0|0|0|0|0|0|0|0|0 1 1 1

HAUS8 19|8 0|0|0|0|0|0|0|0|0|0 1 1 1

CKAP2 15|12 0|1|0|0|0|0|0|0|0|0 1 1 1

KIAA1009 11|16 0|0|0|0|0|0|0|0|0|0 1 1 1

138

Table 3.1 Continued

Prey Gene Counts Control Counts AvgP MaxP SAINT

CCP110 15|12 0|0|0|0|0|0|0|0|0|0 1 1 1

CEP72 14|12 0|0|0|0|0|0|0|0|0|0 1 1 1

CAMSAP1 13|12 0|0|0|0|2|0|0|0|0|0 1 1 1

WDR67 17|8 0|0|0|0|0|0|0|0|0|0 1 1 1

ARHGAP21 13|12 0|0|0|0|0|0|0|0|0|0 1 1 1

CASC5 9|16 0|0|0|0|3|0|0|0|0|3 0.97 1 0.97

TP53BP2 13|11 0|0|0|0|0|0|1|1|0|0 1 1 1

C2orf44 14|10 0|0|0|0|0|0|0|0|0|0 1 1 1

CKB 4|20 1|0|0|0|0|0|0|0|1|1 0.92 1 0.92

CEP55 10|11 0|0|0|0|0|0|2|2|0|0 1 1 1

WRAP73 11|9 0|0|0|0|0|0|0|0|0|0 1 1 1

TTK 13|6 0|0|0|0|0|0|2|1|0|0 0.99 1 0.99

FGFR1OP 7|10 0|0|0|0|0|0|0|0|0|0 1 1 1

EXOC4 11|6 0|1|0|0|0|0|0|0|0|0 1 1 1

EIF2C2 11|5 0|0|0|0|0|0|0|1|0|0 1 1 1

YEATS2 11|5 0|0|0|0|1|2|0|0|0|0 0.98 1 0.98

CCDC77 9|6 0|0|0|0|0|0|0|0|0|0 1 1 1

UCHL1 10|5 0|0|0|0|0|0|0|0|0|0 1 1 1

UBE2I 9|5 0|0|0|0|0|0|0|0|0|1 1 1 1

CNTROB 6|8 0|0|0|0|0|0|0|0|0|0 1 1 1

EIF4E 8|6 0|0|0|1|0|0|2|0|0|0 0.99 1 0.99

NACAD 5|8 0|0|0|0|0|0|0|0|0|0 1 1 1

HAUS2 7|6 0|0|0|0|0|0|0|0|0|0 1 1 1

TNRC6B 9|4 0|0|0|0|0|0|0|0|0|1 0.99 1 0.99

RPAP2 8|4 0|0|0|0|0|0|0|0|0|0 1 1 1

KIAA1731 6|5 0|0|0|0|0|0|0|0|0|0 1 1 1

IBTK 5|6 1|1|0|0|0|0|0|0|0|0 0.99 1 0.99

AP4B1 2|9 0|0|0|0|0|0|0|0|0|0 0.98 1 0.98

SDCCAG3 4|6 0|0|0|0|0|0|0|0|0|0 1 1 1

SLC9A2 4|6 0|0|0|0|0|0|0|0|0|0 1 1 1

IL20RB 8|2 0|0|0|0|0|0|0|0|0|0 0.98 1 0.98

BTF3 6|4 0|0|0|0|0|0|1|0|0|1 0.97 1 0.97

KIF2A 4|6 1|0|0|0|0|0|0|0|0|1 0.97 1 0.97

IPMK 6|3 0|0|0|0|0|0|0|0|0|0 1 1 1

CEP152 4|4 0|0|0|0|0|0|0|0|0|0 1 1 1

HAUS1 4|4 0|0|0|0|0|0|0|0|0|0 1 1 1

SPATA2 6|2 0|0|0|0|0|0|0|0|0|0 0.98 1 0.98

KIF14 6|2 0|0|0|0|0|0|0|0|0|0 0.98 1 0.98

CDC27 6|2 0|0|0|0|0|0|0|0|0|0 0.98 1 0.98

TTC28 4|3 0|0|0|0|0|0|0|0|0|0 1 1 1

EPPK1 4|3 0|0|0|0|0|0|0|0|0|0 1 1 1

139

Table 3.1: CEP120 Associated Proteins Detected by BioID.

Results from two biological replicates of FLAG-BirA*-CEP120 expression in the presence of

biotin followed by Streptavidin affinity-purification and mass-spectrometry to identify

biotinylated peptides. Control counts are from 10 runs from four biological FLAG-BirA*

expression experiments prepared under the same conditions. (From left to right) Gene Symbol

for identified proteins, the spectral counts for each protein in each of the experimental replicates,

the spectral counts for each protein in the control replicates, the average probability of both

replicates (AvgP), the maximum probability between the two replicates (MaxP), and the overall

SAINT score for both replicates are shown (Choi et al., 2011). Spectra reflect the number of

spectral counts assigned to a prey polypeptide in each MS analysis. Samples were prepared by

Sally Cheung, and BioID-MS and was carried out by Dr. Etienne Coyaud.

140

Table 3.2 SPICE1 Associated Proteins Detected by BioID.

Prey Counts Control Counts AvgP MaxP SaintScore

PCM1 172|208 4|3|0|0|0|0|6|5|2|3 1 1 1

NAP1L1 141|215 25|24|10|12|12|10|34|36|37|40 1 1 1

TOP2A 215|139 0|0|5|54|49|6|0|0|0|0 0.99 1 0.99

MAP7D3 115|142 1|0|0|3|2|0|4|4|2|3 1 1 1

IPO7 89|94 6|7|2|0|0|4|8|9|4|4 1 1 1

CEP120 75|95 0|0|0|0|0|0|0|0|0|0 1 1 1

AZI1 75|93 0|0|0|0|0|0|0|0|0|0 1 1 1

IPO5 72|93 0|0|0|0|0|0|4|4|3|2 1 1 1

KIAA1731 67|97 0|0|0|0|0|0|0|0|0|0 1 1 1

OFD1 65|97 0|0|0|0|0|0|0|0|0|0 1 1 1

CKAP2 72|79 0|1|0|0|0|0|0|0|0|0 1 1 1

TOP2B 95|55 0|0|4|0|0|5|0|0|0|0 1 1 1

LUZP1 63|86 1|2|0|3|3|0|2|2|0|0 1 1 1

LRRC49 69|75 0|0|0|0|0|2|9|7|0|0 1 1 1

NAP1L4 58|71 9|8|0|12|12|4|14|13|15|14 1 1 1

CCDC138 60|61 0|0|0|0|0|0|0|0|0|0 1 1 1

TPGS1 51|53 0|0|6|0|0|5|16|11|0|0 1 1 1

MTUS1 43|54 0|0|0|0|0|0|0|0|0|0 1 1 1

CEP135 41|55 0|0|0|0|0|0|0|0|0|0 1 1 1

KIAA0753 39|54 0|0|0|0|0|0|0|0|0|0 1 1 1

TRIM36 45|45 0|0|0|0|0|0|0|0|0|0 1 1 1

HAUS6 35|52 1|2|0|0|0|0|0|0|1|6 1 1 1

SKA3 35|50 0|1|0|5|0|0|4|2|0|1 1 1 1

SUPT16H 46|36 1|0|1|0|0|0|0|0|0|0 1 1 1

PLEKHA5 30|49 0|0|0|0|0|0|0|0|0|0 1 1 1

WDR67 36|42 0|0|0|0|0|0|0|0|0|0 1 1 1

H2AFY 38|33 15|13|5|3|2|5|0|4|0|11 0.94 1 0.94

KIF7 22|44 0|0|0|0|0|0|0|0|0|0 1 1 1

CAMSAP1 25|37 0|0|0|0|2|0|0|0|0|0 1 1 1

RBMX 34|28 0|0|0|0|0|0|0|0|10|12 0.95 0.97 0.95

SSX2IP 26|35 0|0|0|0|0|0|0|0|0|0 1 1 1

HAUS3 23|37 0|0|0|0|0|0|0|0|0|0 1 1 1

SSRP1 30|28 0|0|0|5|0|1|1|0|0|0 1 1 1

SKA2 24|31 0|0|0|6|4|0|1|1|0|0 1 1 1

NOP58 32|22 3|1|5|2|1|9|6|5|3|4 1 1 1

CSPP1 18|34 0|0|0|0|0|0|0|0|0|0 1 1 1

EML4 21|31 0|0|0|8|11|0|0|0|0|0 0.93 0.97 0.93

HIST3H2BB 26|20 0|0|0|0|0|0|0|0|0|0 1 1 1

WRAP73 22|24 0|0|0|0|0|0|0|0|0|0 1 1 1

PIBF1 20|25 0|0|0|0|0|0|0|0|0|0 1 1 1

HNRNPA3 21|22 2|3|5|1|1|5|3|2|6|7 1 1 1

141

Table 3.2 Continued


CKB 24|19 1|0|0|0|0|0|0|0|1|1 1 1 1

HAUS5 14|27 0|0|0|0|0|0|1|1|0|0 1 1 1

SKA1 14|20 0|0|0|0|0|0|1|0|1|0 1 1 1

PHIP 27|5 0|0|0|0|0|0|0|0|0|0 1 1 1

CEP55 14|18 0|0|0|0|0|0|2|2|0|0 1 1 1

CEP72 12|19 0|0|0|0|0|0|0|0|0|0 1 1 1

HAUS8 12|18 0|0|0|0|0|0|0|0|0|0 1 1 1

MAP7 10|20 0|0|0|0|0|0|0|0|0|0 1 1 1

POP1 14|15 0|0|7|2|4|6|1|1|1|0 0.96 0.98 0.96

RPS23 15|13 4|4|4|1|2|4|4|4|0|0 0.96 0.99 0.96

MAP7D1 12|15 0|0|0|0|0|0|0|0|0|0 1 1 1

ECH1 16|11 0|0|0|0|0|0|0|0|0|0 1 1 1

IPO8 12|14 0|0|0|0|0|0|0|0|0|0 1 1 1

CEP350 10|16 0|0|0|0|0|0|0|0|0|0 1 1 1

CLASP1 9|16 0|0|0|0|0|0|0|0|0|0 1 1 1

KIF2A 11|14 1|0|0|0|0|0|0|0|0|1 1 1 1

HMMR 11|14 0|0|0|0|0|0|0|0|0|0 1 1 1

TP53 14|11 3|4|0|0|0|0|0|0|0|0 0.97 0.99 0.97

SLC25A13 11|13 0|0|0|0|0|0|5|2|4|3 0.92 0.98 0.92

HAUS7 9|14 0|0|0|0|0|0|1|1|0|0 1 1 1

GTSE1 8|15 0|0|0|0|0|0|0|0|0|0 1 1 1

MAP9 7|15 0|0|0|0|0|0|0|0|0|0 1 1 1

GPI 15|7 0|0|0|0|0|0|0|0|0|0 1 1 1

KIF14 8|13 0|0|0|0|0|0|0|0|0|0 1 1 1

NEDD1 9|12 0|0|0|0|0|0|0|0|0|0 1 1 1

BANF1 10|10 0|0|0|0|1|0|0|0|0|0 1 1 1

SPAG5 6|13 0|0|0|0|0|0|0|0|0|0 1 1 1

MED4 6|13 0|0|0|0|0|0|0|0|0|0 1 1 1

UCHL1 10|9 0|0|0|0|0|0|0|0|0|0 1 1 1

C15orf23 8|10 0|0|0|0|0|0|0|0|0|0 1 1 1

CENPJ 4|14 0|0|0|0|0|0|0|0|0|0 1 1 1

SPC24 4|14 0|0|0|0|0|0|0|0|0|0 1 1 1

HAUS4 4|13 0|0|0|0|0|0|0|0|0|0 1 1 1

PRDX3 9|8 0|0|0|0|0|0|2|0|0|0 0.99 1 0.99

TP53BP2 3|14 0|0|0|0|0|0|1|1|0|0 0.91 1 0.91

RIN3 8|8 0|0|0|0|0|0|0|0|0|0 1 1 1

VPS33B 5|11 0|0|0|0|0|0|0|0|0|0 1 1 1

LOC100508808 8|7 0|0|0|0|0|0|0|0|0|0 1 1 1

ZC2HC1A 4|11 0|0|0|0|0|0|0|0|0|0 1 1 1

RACGAP1 8|7 0|0|2|0|0|3|0|0|0|0 0.94 0.96 0.94

PDZD11 7|7 0|0|0|0|0|0|0|0|0|0 1 1 1

142

Table 3.2 Continued


NDC80 7|7 0|0|0|0|0|0|0|0|0|0 1 1 1

CCNB1 5|9 0|0|0|0|0|0|0|0|0|0 1 1 1

LOC728307 7|7 0|0|0|0|0|0|0|0|0|0 1 1 1

TXLNG 5|9 0|0|0|0|0|0|0|0|0|0 1 1 1

CKAP2L 5|9 0|0|0|0|0|0|0|0|0|0 1 1 1

HAUS1 2|12 0|0|0|0|0|0|0|0|0|0 0.98 1 0.98

BTF3 7|6 0|0|0|0|0|0|1|0|0|1 1 1 1

MCTS1 6|7 0|0|0|0|0|0|0|0|1|0 1 1 1

KIAA1009 5|8 0|0|0|0|0|0|0|0|0|0 1 1 1

CAMSAP2 2|11 0|0|0|0|0|0|0|0|0|0 0.98 1 0.98

RALY 8|5 0|2|0|0|0|0|0|0|0|0 0.97 0.99 0.97

FAM161A 5|7 0|0|0|0|0|0|0|0|0|0 1 1 1

RPGRIP1L 3|9 0|0|0|0|0|0|0|0|0|0 1 1 1

HAUS2 4|8 0|0|0|0|0|0|0|0|0|0 1 1 1

COPA 8|4 0|0|0|0|0|0|0|0|0|0 1 1 1

CCDC66 7|5 0|0|0|0|0|0|0|0|0|0 1 1 1

TCHP 3|9 0|0|0|0|0|0|1|0|0|0 0.98 1 0.98

ARID1B 6|5 0|0|0|0|0|0|0|0|0|0 1 1 1

CEP44 4|7 0|0|0|0|0|0|0|0|0|0 1 1 1

CEP85 4|7 0|0|0|0|0|0|0|0|0|0 1 1 1

LTV1 5|6 0|0|0|0|0|0|0|0|0|0 1 1 1

HMGN1 6|5 0|1|0|0|0|2|0|0|0|0 0.97 0.99 0.97

DNM1L 7|4 0|1|0|0|0|0|2|0|0|0 0.93 1 0.93

MAP1S 4|6 0|0|0|0|0|0|0|0|0|0 1 1 1

IPO4 6|4 0|0|0|0|0|0|0|0|0|0 1 1 1

ZC3H15 3|7 0|0|0|1|0|0|0|0|0|0 0.98 1 0.98

TRA2B 4|6 0|0|0|0|0|0|0|0|1|2 0.92 0.99 0.92

DIAPH3 5|4 0|0|0|0|0|0|0|0|0|0 1 1 1

TROAP 3|6 0|0|0|0|0|0|0|0|0|0 1 1 1

CC2D1A 3|6 0|0|0|0|0|0|1|0|0|0 0.98 1 0.98

CCDC77 2|7 0|0|0|0|0|0|0|0|0|0 0.98 1 0.98

MPHOSPH9 2|6 0|0|0|0|0|0|0|0|0|0 0.98 1 0.98

ANAPC7 6|2 0|0|0|0|0|0|0|0|0|0 0.98 1 0.98

C1orf35 6|2 0|0|0|1|0|0|0|0|0|0 0.9 1 0.9

SRP72 4|3 0|0|0|0|0|0|0|0|0|0 1 1 1

CDC27 3|4 0|0|0|0|0|0|0|0|0|0 1 1 1

TEX9 2|5 0|0|0|0|0|0|0|0|0|0 0.98 1 0.98

MAP7D2 2|5 0|0|0|0|0|0|0|0|0|0 0.98 1 0.98

UBE2L3 4|3 0|0|0|0|0|0|1|0|0|0 0.97 0.99 0.97

DCLK1 2|5 0|0|0|0|0|0|0|0|1|0 0.9 1 0.9

POLH 2|5 0|0|0|0|0|0|0|1|0|0 0.9 1 0.9

143

Table 3.2: SPICE1 Associated Proteins Detected by BioID.

Results from two biological replicates of FLAG-BirA*-SPICE1 expression in the presence of

biotin followed by affinity-purification and mass-spectrometry to identify biotinylated peptides.

Control counts are from 10 runs from four biological FLAG-BirA expression experiments

prepared under the same conditions. (From left to right) Gene Symbol for identified proteins, the

spectral counts for each protein in each of the experimental replicates, the spectral counts for

each protein in the control replicates, the average probability of both replicates (AvgP), the

maximum probability between the two replicates (MaxP), and the overall SAINT score for both

replicates are shown (Choi et al., 2011). Spectra reflect the number of spectral counts assigned

to a prey polypeptide in each MS analysis. Samples were prepared by Sally Cheung, and BioID-

MS and was carried out by Dr. Etienne Coyaud.

144

Figure 3.2 Cytoscape map of CEP120 and SPICE1 BioID Preys.

The data from BioID experiments (see Tables 3.1 and 3.2) were input into the Cytoscape

Software program and used to generate a map of shared and unique prey for CEP120 and

SPICE1. Each circle within a cluster represents a prey protein, and the thickness of the

connecting line is proportional to the total peptide counts for each protein over the two

experimental replicates. Prey proteins shared by CEP120 and SPICE1 are shown (center), and

prey proteins unique to CEP120 (top) and SPICE1 (bottom) are shown.

Figure 3.2

145

Figure 3.3

Figure 3.3. BioID identified proteins in proximity to CEP120 or SPICE1 are important for

PLK4 induced centriole duplication in U-2 OS cells.

(A, C) In two separate experiments (A,C) U-2 OS cells on coverslips were transfected with

siRNA or esiRNA against target genes as indicated, where gene-symbol is given followed by si

or esi and a number to differentiate between multiple silencing triggers tested for each gene. At

48 hours post-transfection with RNA, cells were arrested in S-phase with 8 mM hydroxyurea and

induced to over-express PLK4 by addition of tetracycline to 2g/ml. At 20 hours post induction

and arrest, cells were fixed in methanol and immunolabeled for imaging and visual analysis. The

frequency of cells with overduplicated centrioles was quantified for each condition, with three

replicates per condition, 100 cells analyzed in each replicate. Bar graph shows frequency of

overduplication, grouped based on p-values from a two-tailed student’s t-test against controls.

Asterisks indicate significance as a result of an unpaired two-tailed student’s t-test against the

control. (B) Sample images of (top) overduplication and (bottom) CEP120 depletion preventing

centriole overduplication from the same assay as in (A).

146

Figure 3.4

147

Figure 3.4 Depletion of CEP120/SPICE1-proximal proteins involved in centriole

duplication results in CEP120 localization outside of the centrosome.

(A) U-2 OS cells were depleted of the indicated proteins for 72 hours by RNAi while cycling.

Cells were fixed and immunolabeled for CEP120 (green) and glutamylated tubulin (red). The

frequency of cells with CEP120 observed on non-centrosomal structures resembling

microtubules was quantified for each condition, and bar graph shows the frequency for each

depletion condition. The experiment was performed in triplicate, with at least 100 cells

examined per condition per replicate. P-values (*s) are results of student’s t-test, two-tailed

compared to control for each condition. (B) Representative micrographs showing CEP120

localization (green, insets) under each condition. Gene name above pictures indicates RNAi

treatment used. Scale bar 10 m.

148

Figure 3.5

149

Figure 3.5 Depletion of CEP120/SPICE1-proximal proteins involved in centriole

duplication results in abnormal SPICE1 structures.


Cells were fixed and immunolabeled with antibodies against SPICE1 (green) and glutamylated

tubulin (red). The bar graph shows the frequency of cells with SPICE1 forming abnormal

structures for each RNAi condition. The experiment was performed in triplicate, with at least 100

cells examined per condition per replicate. P-values (*s) are results of student’s t-test, two-tailed

compared to control for each condition. (B) Representative micrographs showing SPICE1

localization (green, insets) under each condition. Gene name above pictures indicates RNAi

treatment used. Scale bar 10 m.

150

Figure 3.6

151

Figure 3.6. Depletion of CEP120, SPICE1 and their associated proteins can lead to

cytoskeleton microtubule glutamylation.


Cells were fixed and immunolabeled for glutamylated tubulin (red) and CEP120 (green). Bar

graph shows the frequency of cells with glutamylated non-centriolar microtubules for each RNAi

condition. The experiment was performed in triplicate, with at least 100 cells examined per

condition per replicate. P-values (*s) are results of a two-tailed student’s t-test compared to

control for each condition. (B) Representative micrographs showing glutamylated tubulin

immunolabeling (red, insets). Gene name above pictures indicates RNAi treatment, scale bar 10

m.

152

Figure 3.7

153

Figure 3.7. Depletion of CEP120/SPICE1 associated proteins can lead to elongated

centriole-like structures.

(A) Bar graph showing the quantification of the frequency of elongated structures that emanated

from centrioles following 72 hours of depletion of the indicated proteins by RNAi in U-2 OS

cells. Quantification was performed in triplicate with at least 100 cells counted per replicate in

each condition. (B) Representative images of centrioles in each condition, with insets showing

glutamylated microtubules in the area of the centrioles (identified by a pair of brightly

glutamylated foci). Scale bars 5 m for both panels.

154

Figure 3.8

155

Figure 3. 8. CEP120/SPICE1 associated proteins are required for taxol induced centriole

elongation.

(A)U-2 OS cells were depleted of the indicated proteins for 24 hours before being arrested in G2

for 48 hours, with taxol added to 10 M for the final 24 of those hours. Cells were fixed and

immunolabeled with antibodies against CEP120, -tubulin and glutamylated tubulin. The

frequency with which elongated glutamylated tubulin structures were observed was quantified

for each condition. Bar graph shows frequency for each condition in decreasing order (from left

to right), with control bar in grey. P-values indicated are results of a two-tailed student’s t-test

compared to controls. For each condition, three replicates were tested and at least 100 cells were

examined per replicate. (B) Representative images from the experiment as described in (A), with

scale bar indicating 10 m and insets showing 4x magnification of centrosomes and

glutamylated tubulin staining.

156

Figure 3.9

157

Figure 3.9. CEP120/SPICE1 associated proteins are required for taxol induced centriole

elongation.

(A) The experiment was carried out as described in Figure 3.7, with the frequency of elongated

CEP120 structures being quantified for each condition. Bar graph shows (from left to right)

increasing inhibition of elongation based on frequency, with control bar in grey. P-values

indicated are results of a two-tailed student’s t-test compared to controls. For each condition,

three replicates were tested and at least 100 cells were examined per replicate. (B) Images used

are from Figure 3.7, with scale bar indicating 10 m and 4x magnified insets, with the exception

that insets in this figure show CEP120 antibody labeling.

158

Table 3.3 Summary of the Phenotypes of CEP120 and SPICE1 Associated Proteins in

Secondary Assays.

Abnormal

Localization Acentriolar MT Elongated

Taxol Induced

Elongation

RNAi CEP120 SPICE1 Glutamylation Structures Glut. Tub. CEP120

ANK2 Y Y Y Y ↑ − MAP7 Y Y Y Y ↑ ↑

CSPP1 Y Y Y Y ↓ ↓

MTUS1 Y Y Y N ↓ ↓

CENTROB Y N Y N ↓ ↓

CEP135 Y N Y N ↓ − NAP1L1-si1 Y N Y N − −

MAP7D3 N Y Y N ↓ ↓

KIAA0586 N Y N Y ↓ ↑

KIAA1731 N Y N N ↓ − SASS6 Y N N N − −

NAP1L1-si17 N N N N ↓ ↓

MAP9 N N N N ↓ ↓

CPAP N N N N ↓ ↓

CCDC77 N N N N ↓ −

Table 3.3. Summary of the Phenotypes of CEP120 and SPICE1 Associated Proteins in

Secondary Assays.

RNAi (Column 1): Names of the proteins including controls and those identified as prey in the

CEP120 and/or SPICE1 BioID datasets that were required for PLK-4 induced centriole

duplication, that were tested in secondary assays. (Columns 2-5): U-2 OS cells were depleted of

the indicated proteins (column 1) by RNAi for 72 hours before fixation.

Abnormal Localization (Columns 2 and 3): Is localization of CEP120 or SPICE1 to

cytoplasmic microtubules observed in this assay significantly increased under each condition

compared to the control (see Figures 3.4, 3.5)? Acentriolar MT Glutamylation (Column 4): Is

cytoplasmic microtubule glutamylation significantly increased under each condition in this assay

compared to the control (see Figure 3.6)? Elongated Structures (Column 5): Are elongated

centriole-like structures observed in more than 5% of cells under each condition (see Figure

3.7)? Taxol Induced Elongation (Columns 6 and 7): Is the taxol driven elongation of

centrioles inhibited (↓), increased (↑) or unchanged (−) by each RNAi compared to controls in

the taxol assay when either glutamylated tubulin (Glut. Tub) or CEP120 (CEP120) are used as

markers for centriole length (see Figures 3.8 and 3.9)?

159

Figure 3.10

Figure 3.10. NAP1L1 Depletion Reduces PLK4 induced centriole overduplication in U-2

OS cells.

In separate experiments, U-2 OS cells with inducible PLK4 expression on coverslips were

transfected with either LifeTech Stealth siRNA against NAP1L1 or Dharmacon siRNAs against

NAP1L1, or controls against CEP135 or CEP120, respectively. At 48 hours post-transfection

cells were arrested in S-phase with hydroxyurea and induced to overexpress PLK4 by addition of

tetracycline. At 20 hours post induction and arrest, cells were fixed and immunolabeled with

antibodies against CEP120 and CETN2. (Top) Representative images from the Stealth siRNA

experiment are shown, with scale bar representing 10 m and insets magnified 4x. (Bottom) Bar

graphs quantifying the frequency of centriole overduplication under each RNAi condition.

Asterisks over bars indicate significance for each sample as compared to control-transfection

based on results of a two-tailed unpaired student’s t-test.

160

Figure 3.11

161

Figure 3.11. NAP1L1 is required for centriole duplication in cycling U-2 OS cells.

U-2 OS cells were depleted of NAP1L1 using one of two LifeTech Stealth siRNAs (NAP1L1-

Stealth-1, NAP1L1 Stealth-2), or one of three Dharmacon siRNAs (NAP1L1-DHRM-2, 17, 18),

or CEP120 or SPICE1 for 72 hours before being fixed and immunolabeled with antibodies

detecting CETN2 and CEP120. Mitotic cells were examined, and the number of centrioles per

mitotic cell was counted based on CETN2 labeling. (A) Representative images are shown from

each RNAi condition, with scale bar representing 10 m and insets 2x magnified below. The

frequency of cells with four or more centrioles was determined under each condition and bar

graph (B) shows the percentage of cells for each RNAi condition, as the average from three

replicates for each of which at least 50 cells were counted. Asterisks indicate significance for

each sample as compared to the control, based on results of two-tailed unpaired student’s t-test,

with RNAi condition indicated on the X-axis.

162

Figure 3.12

163

Figure 3.12. NAP1L1 is required for centriole duplication in cycling HeLa cells.

HeLa cells were depleted of NAP1L1 using multiple siRNAs from LifeTech (NAP1L1-Stealth-1,

NAP1L1 Stealth-2) or Dharmacon (NAP1L1-DHRM-2, 17, 18), or depleted of CEP120 or

SPICE1 for 96 hours before being fixed and immunolabeled with antibodies detecting CETN2

and CEP120. Representative mitotic cells (A) are shown from each condition, with scale-bar

indicating 10 m and insets 2x magnified. The frequency of cells with four or more centrioles in

mitosis was quantified visually based on CETN2 signal, and bar graph (B) shows the average

percentage of cells meeting this criteria for each condition from three independent replicates for

each of which at least 50 mitotic cells were examined. Asterisks indicate the results of a

student’s t-test of significance (two-tailed, unpaired) comparing each condition to the control.

164

Figure 3.13

165

Figure 3.13. NAP1L1 depletion by Stealth siRNA#1 causes CEP120 localization to

perinuclear acetylated microtubules in the PLK4 centriole overduplication assay.

U-2 OS cells that express PLK4 upon treatment with tetracycline were depleted of CEP120 or

NAP1L1 using Stealth siRNA#1 for 48 hours before being arrested in S-phase and induced to

over-express PLK4. At 20 hours post-induction, cells were fixed and immunolabeled for (A)

CEP120 and CETN2, or (B) CEP135 and acetylated tubulin (Ac.Tub). Representative cells are

shown for each immunolabeling, and insets show (A) CEP120 or (B) acetylated tubulin staining

from the experiments. Scale bars 100 m.

166

Figure 3.14

167

Figure 3.14. Depletion of NAP1L1 by Stealth siRNA#1 leads to CEP120 localization on

cytoplasmic structures in U-2 OS cells.

U-2 OS cells were depleted of NAP1L1 using siRNA indicated, or CEP120 or SPICE1 for 72

hours before being fixed and immunolabeled with antibodies against CEP120 and CETN2.

Representative images are shown for each RNAi condition (A) with insets showing the CEP120

label, and scale bar approximating 10 m. The frequency of CEP120 on cytoplasmic

microtubules was quantified for each condition, and the average percentage of cells with CEP120

on cytoplasmic structures from three replicates for each of which at least 100 cells was counted

is shown in the bar graph. (B) Asterisks show significance of differences compared to controls

for each RNAi condition, as determined by unpaired two-tailed student t-test of significance.

168

Figure 3.15

169

Figure 3.15. NAP1L1-Stealth-siRNA#1 resistant GFP-NAP1L1 does not rescue the

phenotype of CEP120 distribution onto cytoplasmic structures.

U-2 OS stable cell lines expressing either GFP alone or a GFP-tagged NAP1L1 that is resistant

to Stealth siRNA#1 (GFP-NAP1L1-siRES) were generated. Both cell lines were treated with

control siRNA or Stealth siRNA#1 targeting NAP1L1 for 72 hours before being fixed and

immunolabeled with antibodies against CEP120 and GFP. Representative images are shown (A)

for each condition, scale bars indicate 100 m, and insets are 3X magnifications showing the

CEP120 signal. GFP-positive cells were analyzed under each condition, and the frequency of

CEP120 being observable on cytoplasmic microtubules was quantified for each condition. Bar

graph (B) shows the average frequency of the CEP120 redistribution phenotype from three

replicates of the experiment, where at least 100 cells were examined per replicate. (C) In a

separate experiment, the U-2 OS cell line described and shown in (A) (right-side) was treated

with Stealth siRNA#1, Dharmacon siRNA#17 (DHRM si#17), or esiRNA against GFP for 72

hours, before whole cell lysates were collected and analyzed by Western blot. Blot shows that

GFP-NAP1L1-siRES is sensitive to RNAi targeting GFP, or DHRM-si#17, but resistant to

Stealth siRNA#1 against NAP1L1.

170

Figure 3.16

171

Figure 3.16. SiRNA resistant NAP1L1-GFP and GFP-NAP1L1 do not restore centriole

duplication in U-2 OS cells treated with siRNA against NAP1L1.

(A, B, D, E) U-2 OS cells stably expressing either GFP or GFP fused to NAP1L1 that is resistant

to Dharmacon siRNA#17 were treated with control or Dharmacon siRNA#17 targeting NAP1L1

for 48 hours before being arrested in S-phase for 24 hours then fixed and immunolabeled with

antibodies against CEP135 and CETN2. (A) Representative images of U-2 OS from the

experiment expressing (left) GFP or (right) siRNA resistant NAP1L1 with GFP fused to its

carboxy-terminal end, under treatment with control or NAP1L1 siRNA#17. CEP135 was

immunolabeled with Alexa-488 and so the strong signal renders GFP signal below background

in the images. Scale bar 10 m, insets show 2X magnifications. (B) The number of centrioles

per cell was counted, and the frequency of cells with four or more centrioles was quantified over

three experiments where at least 100 cells were counted per replicate. Bar graph shows the

average from three experiments for each cell type. Asterisks indicate the result of an unpaired

two-tailed student’s t-test for significance against controls as indicated. (C) Cell lysates were

prepared from the same cell lines as in (A), following the same protocol as in (A). U-2 OS were

treated with Dharmacon siRNA#17, Stealth si#1, esiRNA targeting GFP, or control siRNA and

lysates were collected for Western blot. Western blot shows (top) GFP signal and (bottom)

GAPDH loading controls for each experiment. (D) Representative images from an experiment

as described for (A), but with the NAP1L1 expressed with GFP fused to its amino-terminal end

(right side). (E) The number of centrioles per cell was quantified, and the frequency of cells

with four or more centrioles was averaged from three experiments where at least 100 cells were

counted per replicate. Asterisks indicate the result of an unpaired two-tailed student’s t-test for

significance against controls as indicated. (F) Cell lysates were prepared from U-2 OS cells

treated as in (D), with Dharmacon siRNA#17, Stealth si#1, esiRNA targeting GFP, or control

siRNA. Western blot shows (top) GFP signal and (bottom) GAPDH loading controls for each

experiment.

172

Figure 3.17

N1L1-1 MADIDNKEQSELDQDLDDVEEVEEEETGEETKLKARQLTVQMMQNPQILA 50

N1L1-2 MADIDNKEQSELDQDLDDVEEVEEEETGEETKLKARQLTVQMMQNPQILA 50

N1L4-1 MADHSFSDG----VPSDSVEAAKNASN-------TEKLTDQVMQNPRVLA 39

N1L4-2 MADHSFSDG----VPSDSVEAAKNASN-------TEKLTDQVMQNPRVLA 39

*** . .: *.** .:: .. :.:** *:****::**

ALQERLDGLVETPTGYIESLPRVVKRRVNALKNLQVKCAQIEAKFYEEVH 100

ALQERLDGLVETPTGYIESLPRVVKRRVNALKNLQVKCAQIEAKFYEEVH 100

ALQERLDNVPHTPSSYIETLPKAVKRRINALKQLQVRCAHIEAKFYEEVH 89

ALQERLDNVPHTPSSYIETLPKAVKRRINALKQLQVRCAHIEAKFYEEVH 89

*******.: .**:.***:**:.****:****:***:**:**********

DLERKYAVLYQPLFDKRFEIINAIYEPTEEECEWKPD--EEDEISEELKE 148

DLERKYAVLYQPLFDKRFEIINAIYEPTEEECEWKPD--EEDEISEELKE 148

DLERKYAALYQPLFDKRREFITGDVEPTDAESEWHSENEEEEKLAGDMKS 139

DLERKYAALYQPLFDKRREFITGDVEPTDAESEWHSENEEEEKLAGDMKS 139

*******.********* *:*.. ***: *.**:.: **:::: ::*.

KAKIEDEKKDEEKE-DPKGIPEFWLTVFKNVDLLSDMVQEHDEPILKHLK 197

KAKIEDEKKDEEKE-DPKGIPEFWLTVFKNVDLLSDMVQEHDEPILKHLK 197

KVVVTEKAAATAEEPDPKGIPEFWFTIFRNVDMLSELVQEYDEPILKHLQ 189

KVVVTEKAAATAEEPDPKGIPEFWFTIFRNVDMLSELVQEYDEPILKHLQ 189

*. : :: :* *********:*:*:***:**::***:********:

DIKVKFSDAGQPMSFVLEFHFEPNEYFTNEVLTKTYRMRSEPDDSDPFSF 247

DIKVKFSDAGQPMSFVLEFHFEPNEYFTNEVLTKTYRMRSEPDDSDPFSF 247

DIKVKFSDPGQPMSFVLEFHFEPNDYFTNSVLTKTYKMKSEPDKADPFSF 239

DIKVKFSDPGQPMSFVLEFHFEPNDYFTNSVLTKTYKMKSEPDKADPFSF 239

********.***************:****.******:*:****.:*****

DGPEIMGCTGCQIDWKKGKNVTLKTIKKKQKHKGRGTVRTVTKTVSNDSF 297

DGPEIMGCTGCQIDWKKGKNVTLKTIKKKQKHKGRGTVRTVTKTVSNDSF 297

EGPEIVDCDGCTIDWKKGKNVTVKTIKKKQKHKGRGTVRTITKQVPNESF 289

EGPEIVDCDGCTIDWKKGKNVTVKTIKKKQKHKGRGTVRTITKQVPNESF 289

:****:.* ** **********:*****************:** *.*:**

FNFFAPPEVPESGD-LDDDAEAILAADFEIGHFLRERIIPRSVLYFTGEA 346

FNFFAPPEVPESGD-LDDDAEAILAADFEIGHFLRERIIPRSVLYFTGEA 346

FNFFNPLKASGDGESLDEDSEFTLASDFEIGHFFRERIVPRAVLYFTGEA 339

FNFFNPLKASGDGESLDEDSEFTLASDFEIGHFFRERIVPRAVLYFTGEA 339

**** * :.. .*: **:*:* **:*******:****:**:********

IEDDDD-DYDEEGEEADEEGEEEGDEENDPDYDPKKDQN-PAECKQQ 391

IEDDDD-DYDEEGEEADEVMFTK------------------------ 368

IEDDDNFEEGEEGEEEELEGDEEGEDEDDAEINPKV----------- 375

IEDDDNFEEGEEGEEEELEGDEEGEDEDDAEINPKKEPSQPAECKQQ 386

*****: : .***** : :

173

Figure 3.17. ClustalW Alignment of NAP1L1 and NAP1L4 isoforms. Isoforms for NAP1L1 and NAP1L4 were identified using the UniProt knowledge base online

(http://www.uniprot.org) (Operated by European Bioinformatics Institute (EMBL-EBI), Swiss

Institute of Bioinformatics (SIB), and the Protein Information Resource (PIR)). The FASTA

formatted sequences were then uploaded to ClustalW (http://www.ebi.ac.uk/Tools/msa/

clustalw2) (EMBL-EBI) and aligned using default parameters (slow alignment). Top row is

NAP1L1 isoform 1 (N1L1-1) (canonical), while the second row is NAP1L1 isoform 2 (UniProt

identifiers P55209-1, -2 respectively). Third row and fourth rows are NAP1L4 isoforms 1 and 2,

respectively (Uniprot identifiers Q99733-1,-2 respectively). Fifth row is the result of analysis of

the sequences by ClustalW, where “*” indicates conserved amino acid across all four proteins,

“:” indicates conservation between groups of strongly similar properties (scoring > 0.5 in the

Gonnet PAM 250 matrix), and “.” Indicates conservation between groups of weakly similar

properties (< 0.5 in the Gonnet PAM 250 matrix) (source: http://www.ebi.ac.uk/Tools/msa/

clustalw2/help).

174

Figure 3.18

Figure 3.18. Sequence Alignment of NAP1L1 siRNAs with NAP1L1 and NAP1L4 Long

isoforms.

The coding sequences of NAP1L1 and NAP1L4 long isoforms (NCBI Reference Sequence:

NM_139207.2 and NM_005969.3, respectively) were aligned using ClustalW

(http://www.ebi.ac.uk/Tools/msa/clustalw2) (EMBL-EBI). The target sequences of each

NAP1L1 siRNA were then manually identified within the NAP1L1 sequence. For (A) and (B),

top row of sequence show the NAP1L1 mRNA, bottom row the aligned NAP1L4 mRNA

sequence. Only the relevant regions of the mRNA alignment are shown. (A)The LifeTech

Stealth siRNA#1 (NAP1L1-si#1) target sequence is shown in bold (top), and in bold within the

NAP1L1 sequence. The mismatches with NAP1L4 mRNA are highlighted in red. (B)The

Dharmacon NAP1L1 siRNA #17 (NAP1L1-si#17) sequence is shown in bold. Alignment shown

as described in (A).

NAP1L1 si#1 Target Sequence: GGTAGAAACACCAACAGGATACATT

CAGATGATGCAAAATCCTCAGATTCTTGCAGCCCTTCAAGAAAGACTTGATGGTCTGGTA 180

CAGGTGATGCAGAATCCTCGAGTTCTGGCAGCTTTACAGGAGCGACTTGACAATGTCCCT 147

GAAACACCAACAGGATACATTGAAAGCCTGCCTAGGGTAGTTAAAAGACGAGTGAATGCT 240

CACACCCCTTCCAGCTACATCGAAACTTTACCTAAAGCAGTAAAAAGAAGAATTAATGCA 207

NAP1L1 si#17 Target Sequence: GCGTATAATCCCAAGATCA

TGAAGCTATCCTTGCTGCAGACTTCGAAATTGGTCACTTTTTACGTGAGCGTATAATCCC 1007

TGAATTCACATTAGCCTCTGATTTTGAAATTGGACACTTTTTCCGTGAGCGGATAGTCCC 986

AAGATCAGTGTTATATTTTACTGGAGAAGCTATTGAAGATGATGATGAT---GATTATGA 1064

GCGGGCTGTGCTGTACTTCACTGGGGAGGCCATAGAAGATGATGACAATTTTGAAGAAGG 1046

175

Figure 3.19

Figure 3.19. Relative quantification of NAP1L1 and NAP1L4 transcript levels by PCR.

PCR was performed on cDNA made from normalized amounts of total-RNA from U-2 OS at 72

hours following treatment with siRNA against either NAP1L1 (siRNA#1 or siRNA#17),

NAP1L4, or a control siRNA with no target. Primer sets were designed to target cDNA from

both NAP1L1 isoforms (left lanes), from NAP1L4 specifically (second lane), from the NAP1L1

short isoform (third lane), or GAPDH (fourth lane). (Top) A 2% agarose gel containing

ethidium bromide was run with equal sample loading of each PCR reaction. (Bottom) The lanes

for each sample from the gel (top) were isolated using Photoshop and labelled with their primers

(top) and sample experimental condition (left). Two different exposures of the same gel are

shown (Left versus right lower panels).

176

Figure 3.20

177

Figure 3.20. NAP1L4 is not required for PLK4 induced centriole over duplication or

CEP120 localization to centrosomes in U-2 OS cells.

(A) U-2 OS cells with inducible PLK4 expression were transfected with control siRNA, siRNA

targeting CEP120, or siRNA targeting NAP1L4, and at 48 hours post-transfection were arrested

in S-phase and induced to over-express PLK4 by addition of tetracycline. At 20 hours post

induction and arrest, cells were fixed and immunolabeled with antibodies against CEP120 and

CETN2. Representative images (A) are shown from each RNAi condition, with insets shown at

4X magnification and scale bar representing 10 m. (B) The frequency of centriole

overduplication was quantified for each condition, and bar graph shows the average percentage

of cells with centriole overduplication from three replicates for each condition, where at least

100 cells were counted per replicate. (C) U-2 OS cells treated as described for (A), but with

siRNA against NAP1L1 (Stealth si#1) instead of CEP120. (C) Representative images of

populations of cells showing CEP120 localization (insets), scale bar 100 m. (D) The frequency

of CEP120 observed on cytoplasmic structures was quantified for each condition, and the

average percentage of cells with CEP120 on cytoplasmic microtubules was calculated from three

replicates of each experiment with at least 100 cells being examined per replicate. For (B) and

(D), asterisks indicate results of a student’s t-test of significance comparing the condition to the

control condition.

178

Figure 3.21

Figure 3.21. NAP1L1 is required for PLK4 induced centriole overduplication.

(A) U-2 OS cells with inducible PLK4 expression were transfected with control siRNA, or one

of three siRNAs targeting NAP1L1 (Stealth-si#1, Dharmacon si#17, or a third siRNA (see text)).

At 48 hours post-transfection cells were arrested in S-phase and induced to over-express PLK4,

then at 20 hours post induction and arrest, cells were fixed and immunolabeled for CEP120 and

CETN2. Representative images (A) are shown from each RNAi condition, with insets shown at

8X magnification and scale bar representing 10 m. (B) The frequency of centriole

overduplication was quantified for each condition, and bar graph shows the average percentage

of cells with centriole overduplication from three replicates for each condition, where at least

100 cells were counted per replicate. (C) Representative images of populations of cells showing

CEP120 localization, scale bar 10 m. (D) The percentage of cells with CEP120 on cytoplasmic

microtubules was calculated for each of three replicates, with at least 100 cells being examined

per replicate. Bar graph shows average of three replicates for each condition. For (B) and (D),

asterisks indicate results of a student’s t-test of significance comparing the condition to the

control condition.

179

Table 3.4. Summary of the NAP1L1 siRNAs and Their Associated Phenotypes.

Dharmacon siRNAs Stealth siRNAs

Si#2 Si#17 Si#18 Si#1 Si#2 Si#3 Si-New

Isoforms Targeted BOTH BOTH BOTH BOTH BOTH BOTH BOTH

Centriole

Overduplication YES YES YES YES YES NO YES

Centriole Duplication

(72h RNAi, U-2 OS) NO YES NO YES NO NT NT

CEP120 Localization

(72h RNAi, U-2 OS) YES NO YES YES NO NT YES

MT Glutamylation

(72h RNAi, U-2 OS) NT NO NT YES NT NT NT

Table 3.4. Summary of the NAP1L1 siRNAs and Their Associated Phenotypes.

(First Column) Characteristics of different NAP1L1 siRNAs and their impacts in various assays.

Isoforms Targeted: NAP1L1 isoforms predicted to be targeted by the siRNAs based on manual

sequence alignments. Centriole Overduplication: Does siRNA indicated inhibit PLK4 induced

centriole overduplication (see Figures 3.10, 3.21)? Centriole Duplication: Does indicated

siRNA inhibit centriole duplication at 72 hours depletion in U-2 OS cells (see Figure 3.11)?

CEP120 Localization: Does indicated siRNA lead to abnormal CEP120 localization in U-2 OS

at 72 hours (see Figures 3.14 and 3.21)? MT Glutamylation: Does indicated siRNA lead to

abnormal glutamylation of cytoplasmic microtubules in U-2 OS cells at 72 hours RNAi. ‘YES’

indicates statistically significant phenotype in the assay as described. ‘NO’ indicates no

phenotype, or a non-significant phenotype in the assay as described. ‘NT’ indicates the siRNA

was not tested in the assay as described. (Top Row) Indicated siRNAs targeting NAP1L1 that

were used in this study (see Materials and Methods).

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3.7. Materials and Methods

3.7.1. Cell Culture, RNAi, PLK4 Assay, and Microscopy Sample Preparation

and Imaging

Cell culture conditions, RNA transfections, the PLK4 and taxol assays, and microscopy methods

used in this chapter are as described in detail in Section 2.7 of this thesis (Chapter 2 Materials

and methods).

3.7.2. BioID Analysis

All mass-spectrometry was performed by Etienne Coyaud from the lab of Dr. B. Raught as

previously reported (Comartin et al., 2013). Briefly, analytical columns and pre-columns (75 nm

and 150 nm inner diameters, respectively) were made from silica capillary tubing (InnovaQuartz)

and packed with 100 Angstrom C18-coated silica particles (Magic, Micrhom Bioresources).

Peptides were subjected to liquid chromatography electrospray ionization-MS/MS using 120

minute reversed-phase (100% water 100% acetonitrile, 0.1% formic acid) buffer gradient at 250

nl/min on a Proxeon EASY-nLC pump in line with a hybrid LTQ-Orbitrap Velos mass

spectrometer (Thermo Fisher Scientific). A parent ion scan was performed using a resolving

power of 60,000, then up to the forty most intense peaks were selected for MS/MS (minimum

ion count of 1000 for activation), using standard collision-induced dissociation fragmentation.

Fragment ions were detected in the LTQ. Dynamic exclusion was activated such that MS/MS of

the same m/z (within 15 ppm; exclusion list size =500) detected twice within 15 seconds were

excluded from analysis for 30 seconds. For protein identification, Thermo .RAW files were

converted to the .mzXML format using Proteowizard (Kessner et al., 2008), then searched using

X!Tandem (Craig and Beavis, 2004) against the human RefSeq database. X!Tandem search

parameters were: 15 ppm parent mass error; 0.4 Da fragment mass error; complete

modifications, none; cysteine modification, none; potentials modifications, +16@M and W,

+32@M and W, +42@N-terminus, +1@N and Q. For both CEP120 and SPICE1 datasets, two

technical replicates were used for analysis. Samples were analyzed using the trans-proteomic

pipeline (TPP) in the ProHits software suite (Choi et al., 2011, 2012; Deutsch et al., 2010;

Pedrioli, 2010). Proteins identified were analyzed with the SAINT algorithm, using cut-off

values of AvgP <0.90, MaxP<0.95, and with pre-filtering against heat-shock proteins, ribosomal

proteins, keratins, albumin, and translation elongation factors (Choi et al., 2012). Ten runs of

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FLAG-BirA* peptide expression pellets were used as controls to remove non-specific

interactions in SAINT, and control counts are reported for each protein.

3.7.3. PLK4 Screen

As described previously in Chapter 2, and similar to the originally published protocol (Kleylein-

Sohn et al., 2007), U-2 OS T-REx cells with inducible Myc-PLK4 expression were seeded on

coverslips in 6-well tissue culture dishes and transfected with siRNA or esiRNA as described in

Materials and Methods Section 2.7.2. At 48 hours post-RNA addition, cells were arrested in S-

phase by addition of hydroxyurea (HU) to 8 mM, and induced to over-express PLK4 by addition

of tetracycline (2g/mL) for 20 hours before fixation.

3.7.4. Analyses of CEP120 and SPICE1 localization, Microtubule

Glutamylation and Centriole Elongation following Depletion of CEP120 and

SPICE1 Associated Proteins in U-2 OS

For the U-2 OS assays wherein the localization of CEP120 or SPICE1were analyzed (Figure 3.4,

Figure 3.5), or wherein the fraction of cells with glutamylated cytoplasmic microtubules or

elongated centrioles were analyzed (Figure 3.6 and Figure 3.7), U-2 OS cells were treated with

siRNA or esiRNA for 72 hours before fixation by ice-cold methanol (-20°C) for at least 10

minutes. Microscopy sample preparation and image processing were as described in Materials

and Methods section 2.7.6. Taxol assays were performed as described in Materials and Methods

section 2.7.4.

3.7.5. Bioinformatic Analysis of NAP1L-family members and Isoforms

UniprotKB (http://www.uniprot.org) (EMBL-EBI, SIB, and the PIR) was used to obtain

information about NAP1L1 and NAP1L4 predicted isoform protein sequences in June 2014. The

FASTA sequences were then uploaded to ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2)

(EMBL-EBI) and aligned using default parameters (slow alignment) for protein alignment. For

mRNA alignment of NAP1L1 vs NAP1L4 long isoforms, the NCBI reference sequences

(NM_139207.2 and NM_005969.3) were obtained, the UTRs manually removed and the

sequences double-checked against the protein sequences of the appropriate isoforms by first

translating the mRNA to amino-acid sequence (http://web.expasy.org/translate/, SIB), then

aligning them against the appropriate FASTA sequence from UniProtKB using ClustalW. To

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align the mRNA sequences of NAP1L1 and NAP1L4, ClustalW was again used (DNA

alignment, default settings, slow alignment). NAP1L1 siRNAs target sequences were manually

identified from within the NAP1L1 sequence, and mismatches were manually identified. For

comparison of the two NAP1L1 isoforms and the NAP1L4 isoforms, all four FASTA protein

sequences were obtained from UniProt KB, and aligned simultaneously in ClustalW using

default parameters for protein alignment (slow method).

3.7.6. Cloning of Rescue Constructs

The NAP1L1 long isoform was mutated at wobble-codon positions using QuikChange II XL

site-directed mutagenesis kit (Agilent Technologies). For NAP1L1 siRNA#1 resistance, the

primers used were 5’- TTTAACTACCCTAGGCAGGCTTTCAATATAACCGGTAGGCGT

CTCGACCAGACCATCAAGTCTTTCTTGAAG G-3’ and 5’-CCTTCAAGAAAGACTTGAT

GGTCTGGTC GAGACGCCTACCGGTTATATTGAAAGCCTGCCTAGGGTAGTTAAA-3’.

For NAP1L1 si#17 resistance, the primers used were 5’- CTTCAATAGCTTCTCCAGTAAAA

TATAACACCGACCTGGGAATGATGCGCTCACGTAAAAAGTGACCAATTTCGAAGT-3’

and 5’-ACTTCGAAATTGGTCACTTTTTACGTGAGCGCATCATTCCCAGGTCGGTGTTA

TATTTTACTGGAGAAGCTATTGAAG-3’.

3.7.7. Quantitative PCR to Detect NAP1L1 and NAP1L4 Transcripts

Following treatment with siRNAs Against NAP1L1

Equal amounts of U-2 OS cells were plated and treated with siRNAs as described in Materials

and Methods section 2.7.2. Cells were harvested and RNA prepared using and OMEGA kit

(OMEGA Bio-Tek) as per manufacturer’s protocols. RNA samples were treated with RNAse-

free DNAse-I (Qiagen) as per manufacturer’s recommendation for 15 minutes at room

temperature before being inactivated by incubation at 65°C in the presence of EDTA for 10

minutes. The concentration of RNA in each sample was quantified using a NanoDrop

spectrophotometer (Thermo-Fischer Scientific), normalized by addition of RNAse-free (DEPC

treated) water using RNAse free pipette tips. The concentrations were then checked again using

the NanoDrop. The SuperScript-3 (LifeTechnologies) reverse transcriptase kit was used

according to manufacturer’s protocols to synthesize cDNA using random oligos provided. For

the semi-quantitative PCR, equal amounts of each cDNA stock were distributed carefully to PCR

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tubes. A master-mix of PCR reagents (not including primers) was made and distributed evenly

among the wells. Primer mixes were made as pools for each PCR reaction, and equal amounts

distributed to the appropriate wells. PCR was performed using Taq polymerase (LifeTech) as

per manufacturer’s protocols. Equal amounts of each PCR reaction were then carefully loaded

into 2% agarose (BioRad) gel with ethidium bromide, electrophoresis was performed and the

resulting gel was imaged and photographed using UV illumination in an AlphaInnotech

AlphaImager gel imaging system. Printed gel images were then scanned at maximum dpi

greyscale using a Cannon scanner, then contrast adjusted uniformly across all wells in Adobe

Photoshop. Primers were custom designed and checked by alignment against each isoform of

NAP1L1 and NAP1L4 using ClustalW. Control GAPDH primers were designed by Dr. Mariana

Gomez-Ferreria, formerly of the Pelletier Lab. Primers used were:

NAP1L1 (both isoforms): Forward: 5’- GGTGAAGAAACAAAACTCAAAGC-3’

NAP1L1 (both isoforms): Reverse: 5’-CAATGTATCCTGTTGGTGTTTC-3’

NAP1L4 (both isoforms): Forward: 5’- TTCAGATGGGGTTCCTTCAG-3’

Nap1L4 (both isoforms) Reverse: 5’- GAGGGACATTGTCAAGTCGCT-3’

NAP1L1 (short isoform): Forward: 5’- CTTCGAAATTGGTCACTTTTTACG-3’

NAP1L1 (short isoform): Reverse: 5’- TCATTTGGTAAACATCACCTCATC-3’

GAPDH (Control): Forward: 5’- GAAGGTGAAGGTCGGAGTCA-3’

GAPDH (Control) Reverse: 5’-GTTAAAAGCAGCCCTGGTGA-3’

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Table 3.5 Primary Antibodies Used in this Work.

Primary antibodies Source

Rabbit Anti-CEP120 (Xie et al., 2007)

Rabbit Anti-SPICE1 (Archinti et al., 2010)

Rabbit Anti-CENPJ ProteinTech Group, Inc. 11517-1-AP

Goat Anti-SASS6 Santa-Cruz Biotechnology sc-82360

Rabbit Anti-SASS6 Pelletier Lab

Rabbit Anti-STIL Bethyl Laboratories A302-441A

Rabbit Anti-CP110 Bethyl Laboratories A301-343A-1

Rabbit Anti-CEP152 Bethyl Laboratories A302-480A-1

Mouse Anti-CETN2 Salisbury Laboratory, Mayo Clinic

Mouse Anti-Acetylated tubulin Sigma T 6793

Goat Anti-MYC Abcam Ab19234

Rabbit Anti-GAPDH Sigma G9545

Rabbit Anti-PCNT Abcam Ab4448

Rabbit Anti-FLAG Sigma F7425

Rat Anti--tubulin Serotec MCA77G

Mouse Anti-FLAG Sigma F3165

Mouse Anti-Glutamylated Tub.

(GT335) Gift of Dr. C. Janke,(Bobinnec et al., 1998a)

Mouse -tubulin Sigma T6199

Rabbit anti-CEP135 (Bird and Hyman, 2008)

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Table 3.6 Secondary Antibodies Used for Microscopy in this Work.

Fluorescent antibodies Source

Donkey Anti-Rabbit Alexa 488 Invitrogen




Donkey Anti-Mouse Alexa 488 Invitrogen




Donkey Anti-Mouse Dylight 405 Jackson ImmunoResearch

Donkey Anti-Goat Alexa 488 Invitrogen

Donkey Anti-Goat Alexa 594 Invitrogen

Donkey Anti-Goat Dylight 647 Jackson ImmunoResearch

Donkey Anti-Rat Dylight 647 Jackson ImmunoResearch

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Table 3.7 Sequences of Small Interfering RNA Sequences Used in this Work.

Gene Target Sequence (5`-3`) Company, Catalogue Number

Control AUGAACGUGAAUUGCUCAA GE-Dharmacon D-001210-04

CEP135 SMARTpool GE-Dharmacon M-014548-01

SPICE1-si1* GCUGAGAACAAAUGAGUCA Archinti et al, 2010.

SPICE1-si2 GAAAGCGGAGCCACAGGUA GE-Dharmacon siGenome d-017060-03

CEP120-si1* GAUGAGAACGGGUGUGUAU GE-Dharmacon siGenome d-016493-01

CEP120-si2 AAACCGAGCGACAAGAAUU GE-Dharmacon siGenome d-016493-04

STIL SMARTpool GE-Dharmacon M-019717-01

CEP152 SMARTpool GE-Dharmacon M-022241-01

NAP1L1-si18 UAACCAUAGUUCAUCGAAA GE-Dharmacon D-017274-18

NAP1L1-si17* GCGUAUAAUCCCAAGAUCA GE-Dharmacon D-017274-17

NAP1L1-si2 GACCAGAAAUUAUGGGUUG GE-Dharmacon D-017274-02

NAP1L1-si1* GGUAGAAACACCAACAGGAUACAUU LifeTech HSS106946

NAP1L1-si2 AAUGUAUCCUGUUGGUGUUUCUACC LifeTech HSS106946

NAP1L1-si3 CCUGCAAGUUAAAUGUGCACAGAUA LifeTech HSS106946

NAP1L1-New ACCTGCAAGTTAAATGTGCACAGAT (Okuwaki et al., 2010)

CSPP1-si1 GGAAAGGACUAGACAUUGA GE-Dhamacon D-015485-01

CSPP1-si2 GAACGAAUGCGAAGACUGA GE-Dhamacon D-015485-02

CSPP1-si3* AGACAUAUCCUGCCAUUGA GE-Dhamacon D-015485-03

CSPP1-si4 CAUCCCAAGUGCUAAAGUA GE-Dhamacon D-015485-04

MTUS1 SMARTpool GE-Dharmacon M-006848-00

KIAA1731 SMARTpool GE-Dharmacon M-029941-01

MAP9 SMARTpool GE-Dharmacon M-014470-01

MAP7D3-si5* UUACAGGCGUCACCAAUUA GE-Dharmacon J-024948-05

MAP7D3-si6 CUACAUUCGUCUACUGAUA GE-Dharmacon J-024948-06

MAP7D3-si7 UAAGAGUACUGCAGGUAUU GE-Dharmacon J-024948-07

MAP7D3-si8 GAAAGACCAAGCUCCAAUA GE-Dharmacon J-024948-08

NAP1L4 UCAGGUGAUGCAGAAUCCUCGAGUU LifeTech HSS106954

MAP7 SMARTpool GE-Dharmacon M-011563-00

ANK2-si5* GCAUGUAGCAGCCAAGUAU GE-Dharmacon D-0084187-05

ANK2-si2 GAGCAAAUGUUAACGCAAA GE-Dharmacon D-0084187-02

ANK2-si3 GAAAUUAUCUGCGUACCUU GE-Dharmacon D-0084187-03

ANK2-si4 GAAAUUGAACAGACCAUUA GE-Dharmacon D-0084187-04

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Table 3.7 Sequences of Small Interfering RNA Sequences Used in this Work.

Gene Target Sequence (5`-3`) Company, Catalogue Number

CENTROBIN AGUGCCAGACUGCAGCAACTT (Zou et al., 2005)

POC1 Proprietary Qiagen Flexitube siRNA S103097696

hPOC5 SMARTpool GE-Dharmacon L-016919-02

Table 3.7 Sequences of Small Interfering RNA Sequences Used in this Work. For genes targeted with multiple siRNAs, the * indicates siRNAs used in follow-up experiments.

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Table 3.8 Primers for esiRNA Used in this Study.

Gene Esi-Forward Primer (5’-3’) Esi-Reverse Primer (5’-3’)

CPAP atgttcctgatgccaaccctc gctccaactgttcggtcttc

SASS6 aagatgttggacagtctcttcg cagggtggctggtatttttg

CEP97 tgccagatatcccaggaaag attgttggctcaggacaagg

CP110 cgagctgccttgtacggtat aagtcttgggcagggaaaat

KIAA1731 esi1 gatgaagcagttaagctgaagg gttctgccgctgataaatatcttg

KIAA1731 esi2 gtttatcacatcttgttttacctcaac tgagggaagagtatcattacttttgg

MTUS1 esi1 tccctgcatgtgtcaacattg cagcagggaacaacacaaga

MTUS1 esi2 tttaaaaggaattgaagaagaaataaaatggcag cctacaagtgcaggacact

MAP7 esi1 aacaaacgctccgggaaatg ttaaatttcagctttggttcttcagaag

MAP7esi2 tttaatccaggcatatggtctttagta aaacagccaaaatacacctactgc

CSPP1 esi1 ttttgcacagttttgtcataaattaggg atacaagtaattacagttcccttcatttc

CSPP1 esi2 ttttgatagacggtatcatagaccag tctgttgctcagccatttgttc

KIAA0753 esi1 acaggtgctgttgagcatga gggctttcgctgtgtttact

KIAA0753 esi2 cagaaccagctgcagtttaatag gacaggctgcttgactctt

CCDC138 esi1 tggactggatttcggatcatc atgcctgagccaggtaatca

CCDC138 esi2 tttatcagtctaagtataagagaagaactc ctgacaggtttcaaaggagag

ZC2HC1A esi1 atactcgaaataccatttccagttaattttg taggcaccaataatacaaatgtttatcc

ZC2HC1A esi2 tttgtttatgattctgatcaactataagacac aaaacagtgctcagtattagataaaacaag

ANK2 esi2 gtgggaagatattacaggaactac ccacttttagttaatggtaccaagttg

CCDC77 esi1 cccgttatgcatgagagtca tatgtgcccgtctattgctg

CCDC77 esi2 gaagatcgtctggccaaac taggtcacttctccagcatct

KIAA0586 esi atcagaagaatcagagaaccaagaaag tacttgggctgaacaaatattgctg

Table 3.8. Primers for esiRNA Used in this study. Note that the forward primers have a non-

target specific lead in sequence of (5’-3’) TCACTATAGGGAGAG, and the reverse primers

have a non-target-specific lead in sequence of (5’-3’) TCACTATAGGGAGAC. These have

been omitted from the above table to show only the gene-specific primer complementary

sequence.

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Table 3.9 Plasmids Used in this study.

Gene Backbone Source

CENPJ PCDNA5/FRT/TO, PeGFP-N1 Generous gift of Dr. T.K. Tang

SPICE1 PCS2, PCDNA5/FRT/TO,PeGFP-N1 (Archinti et al., 2010)

CEP120 PCS2, PCDNA5,Pegfp-N1,PCDNA5-FLAG/FRT/TO-hBirA* This study.

NAP1L1 pEGFP-C1, pEGFP-N1 This study

USP37 PCDNA5/FRT/TO This study.

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4.

Chapter IV: Conclusion and Future Directions

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4.1. The Functions of CEP120 and SPICE1 in

Procentriole Assembly and Elongation

At the outset of my project, CEP120 and SPICE1 were proteins without known roles in centriole

duplication, though CEP120 had been shown to be important in neurons for interkinetic nuclear

migration in mouse neurons, a microtubule dependent process (Xie et al., 2007). My work here

showed for the first time that CEP120 was essential for centriole duplication as part of a

collaboration describing the CEP120/SPICE interaction (Hutchins et al., 2010). Essential roles

for CEP120 and SPICE1 in centriole duplication were subsequently confirmed (Archinti et al.,

2010; Mahjoub et al., 2010). I further characterized CEP120 and SPICE1 function in centriole

duplication, showing that they were required for CEP135 incorporation, as well as CP110/CEP97

and Centrin incorporation into procentrioles. Procentrioles assembled in the absence of SPICE1,

CEP120 or CPAP have shorter than normal microtubules when measured in tomograms. I

showed that CEP120 overexpression induced centriole elongation in G2, further supporting a

role in procentriole microtubule elongation. CPAP and CEP120 cooperated in this process with

SPICE1, as depletion of any of these blocked CEP120 or CPAP induced centriole elongation.

Further, we showed that CEP120 and CPAP interact in vivo. Overall, these results represented

the first characterization of how CEP120 and SPICE1 cooperate with CPAP in procentriole

assembly and elongation. Highlighting the importance of CEP120, mutations have been found in

patients with Jeune asphyxiating thoracic dystrophy (JATD) (Shaheen et al., 2014b). The

mutation in CEP120 identified in these patients leads to shorter cilia and fewer centrioles in

model systems, and recapitulates the JATD patient phenotypes in zebrafish development

(Shaheen et al., 2014b). In addition, mutations in CEP135 and CPAP have been found in

patients with microcephaly (Faheem et al., 2015).

Many questions remain as to how centriole assembly and elongation are achieved, and as to how

exactly CEP120/SPICE1 function in these processes. Specifically of interest is the question of

how exactly tubulin is incorporated into growing procentrioles, since my results are consistent

with the hypothesis that it is a chaperoned process. How do CEP120/SPICE1, along with other

factors like CPAP and Centrobin, cooperate to include tubulin under the cap of CP110 and

CEP97? This will be an important avenue of investigation. Future work should focus on testing

whether CEP120 binds free tubulin dimers or microtubules, whether SPICE1 binds either, and

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whether nucleotide status of those tubulin dimers is important as it is for SAS-4 (Gopalakrishnan

et al., 2012). How CEP120, SPICE1 and CPAP interactions relate to tubulin binding will be

important to study in vitro. Attempting to drive the elongation of purified centrioles in vitro by

incubation with tubulin and combinations of recombinant CEP120, SPICE1, CPAP, Centrobin

and CEP135 would be a challenging but potentially powerful experimental approach to gain

insights into how centrioles are elongated by these factors. Recently, purified Drosophila

centrioles stripped of PCM have been used with recombinant SAS-4 and tubulin to recapitulate

PCM assembly onto centrioles in vitro, illustrating the potential of such experiments

(Gopalakrishnan et al., 2012). Understanding how CEP120/SPICE1 interact with microtubules

and tubulin in vitro, then determining how they cooperate with other factors to elongate

centrioles in vitro, would provide answers to many of the questions posed in this thesis and

significantly advance our understanding of centriole elongation. To understand the relationship

between CEP120, SPICE1 and CEP135/CP110/CEP97 incorporation during procentriole

assembly, efforts should also include looking for interactions between CEP135, CP110, CEP97

and CEP120/SPICE1. This would be achieved by co-IP experiments immunoprecipitating

epitope-tagged CEP135/CP110/CEP97 proteins, and blotting for CEP120 and SPICE1 within the

immunoprecipitate. Lastly, SPICE1 is important for mitotic spindle formation, and mitosis is

critical for genome stability. Whether CEP120 might cooperate with SPICE1 in regulating

spindle assembly and genome stability, is not well studied and worth examining. The impact (if

any) of CEP120 on spindle-assembly should be examined, for example by looking at mitotic

cells with 4 centrioles following CEP120 depletion as was previously done for SPICE1 (Archinti

et al., 2010). A potential problem with that approach is that younger centrioles could be shorter

in those mitotic cells, so to overcome this the duration of RNAi before fixation could be reduced,

or the cells could be arrested throughout the RNAi to prevent centriole duplication under

CEP120 depleted conditions.

Whether CEP120 and SPICE1 have a microtubule stabilizing activity in addition to aiding

tubulin incorporation is also an important question. In the third chapter of this thesis, I show that

depletion of a variety of proteins found in the proximity of CEP120/SPICE1 leads to CEP120 re-

localization to cytoplasmic microtubules, and the abnormal glutamylation of cytoplasmic

microtubules. How the enzymes responsible for glutamylation localize to the centrosome is not

well studied, nor are the mechanisms that regulate centriole glutamylation (Janke and Bulinski,

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2011). From these results, I conclude that along with CEP120, tubulin glutamylation enzymes

(TTLLs) are localized to microtubules when the proteins studied here are depleted. Whether this

is direct, or a result of CEP120 increasing microtubule stability leading to long-lived cytoplasmic

microtubules that accumulate PTMs, remains to be tested. Whether microtubules become

stabilized following CEP120/SPICE1 over-expression should be investigated, for example by

exposing cells to cold-shock or colchicine treatment to see if microtubules are resistant to

depolymerization. Whether CEP120 binds glutamylated microtubules following TTLL

overexpression should also be tested in vivo. Can overexpression of CEP120 or SPICE1 drive

abnormal glutamylation of the cytoplasmic microtubules? Are CEP120 and/or SPICE1 required

for the glutamylation of cytoplasmic microtubules observed following depletion of proteins such

as ANK2 (see Figure 3.5)? Are CEP120 and/or SPICE1 required for the glutamylation of

cytoplasmic microtubules observed following over-expression of TTLL enzymes? These are

important experimentally tractable questions that should be addressed using combinations of

protein over-expression and depletion in tissue cultures, with analysis by microscopy.

4.2. Identification of CEP120 and SPICE1 Associated

Proteins with Uncharacterized Roles in Centriole

Assembly

The discovery of novel proteins involved in centriole duplication is a particularly heavy area of

research in centrosome biology, as identifying and characterizing such proteins leads to a more

complete picture of the critical processes of centriole assembly and elongation. Using a

proximity dependent biotinylation assay, we identified proteins that come into close proximity

with CEP120 and SPICE1 in human cells (Roux et al., 2012). From this list of proteins, I

showed that several were important for PLK4 induced centriole overduplication. I identified for

the first time roles for NAP1L1, ANK2, MTUS1, CCDC77, CSPP1, KIAA0586, MAP7D3,

MAP7 and MAP9 in centriole duplication in this assay. These proteins will require extensive

further validation as proteins involved in this process, beginning importantly with testing their

requirement for centriole duplication in non-PLK4 overexpression systems. Each of these

proteins represents a potential CEP120 or SPICE1 interacting protein, and as such could yield

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insights into the mechanisms by which CEP120/SPICE1 function. Interactions with these

proteins should be tested by expressing them as fusions to epitope tags, and using

immunoprecipitation followed by Western blots to look for co-purified CEP120 and SPICE1 (co-

IPs). Notably, MTUS1 is a tumour suppressor protein that is under-expressed in cancer, and is

required for CEP120 and SPICE1 localization and centriole elongation (Rodrigues-Ferreira et al.,

2009; Yu et al., 2009; Zuern et al., 2010). Thus I have here identified a potential function of an

important tumour suppressor protein in centriole duplication. ANK2 similarly affects both

CEP120 and SPICE1 localization, and may negatively regulate centriole elongation. Future

work should focus on how MTUS1 and ANK2 impact centriole assembly, and whether each

interacts with CEP120 or SPICE1 to facilitate this function. How CEP120 or SPICE1 function

in MTUS1-deficient cancer tissues from patients would also be a particularly interesting and

important question to address.

Several of the proteins identified in my screens as important for centriole duplication have

effects on CEP120 localization, microtubule glutamylation, or cause apparent centriole

elongation. It is important to consider the possibility that these phenotypes could be the result of

a general stress response. Centriolar satellites are molecular complexes containing centriolar and

ciliary proteins, with roles in regulating both centriole duplication and cilia formation (Bärenz et

al., 2011; Kubo, 2003; Kubo et al., 1999; Prosser et al., 2009; Tollenaere et al., 2015). A variety

of cellular stresses such as heat shock and DNA damage impact the function of centriolar

satellites (Löffler et al., 2012; Villumsen et al., 2013). One of the responses to stress is a loss of

AZI1 and PCM1 from satellites, initiated by either p38 activity or inactivation of MIB1, which

can promote ciliogenesis (Villumsen et al., 2013). Several satellite proteins are found in the

BioID prey list for CEP120 and/or SPICE1, including AZI1 and PCM1. It is possible then that

the elongated structures observed following depletion of ANK2, MAP7, CSPP1 or KIAA0586

are actually cilia formed as a result of stress or satellite disruption. To address this possibility,

future work should include staining for cilia-specific proteins such as Arl13b, and ultra-structural

analysis of the structures by electron microscopy (Cevik et al., 2010; Duldulao et al., 2009). The

other phenotypes, such as abnormal glutamylation of cytoplasmic microtubules or CEP120 mis-

localization could also be non-specific. To determine whether stress responses underlie the

phenotypes of depletion of the CEP120/SPICE1 BioID preys, future work should include

simultaneous inhibition of p38 (a stress response kinase) in these assays (Duch et al., 2012;

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Villumsen et al., 2013). The cell cycle profiles of cells under each RNAi condition should be

analyzed as well, to reveal possible arrests. Most importantly, it should be determined whether

experimentally stressing U-2 OS cells with UV radiation or heat-shock directly causes interphase

mis-localization of CEP120 or SPICE1, or microtubule glutamylation (Villumsen et al., 2013).

Lastly, testing the remaining BioID preys in the PLK4 assay using an siRNA library would be of

particular interest, as yet more novel roles for proteins in centriole duplication are no doubt

waiting there to be discovered. In sum, by identifying CEP120/SPICE1 proximate proteins, we

have generated a set of proteins with potential roles in centriole duplication worthy of further

study.

4.3. A Role for NAP1L1 in Centriole Duplication

The human nucleosome-assembly-like-proteins play important roles in chromatin structure, with

important implications for transcription, and NAP1L1 plays a role in some cancers (Kidd et al.,

2006; Park and Luger, 2006a; Schimmack et al., 2014). We show that NAP1L1, through

depletion with multiple siRNA in multiple assays, is required for centriole duplication. This is a

previously unreported role for NAP1L1, and a link between NAP1L1 and the centrosome is

novel. It will be important to see how the changes in NAP1L1 expression levels reported in

cancer would impact centriole duplication when introduced into non-cancerous cells such as

RPE-1 (Kidd et al., 2006; Park and Luger, 2006a; Schimmack et al., 2014). Understanding how

NAP1L1 functions in centriole duplication is going to be a critical task. Firstly, does NAP1L1

interact with CEP120 or SPICE1 directly? The results of the BioID experiments described

herein suggest that CEP120 and SPICE1 may have nuclear localizations. First, both CEP120 and

SPICE1 are associated with nuclear proteins including NAP1L1, NAP1L4, Histones, Importins,

and INO80B. Second, an amino-terminal fragment of SPICE1 is restricted to the nucleus when

exogenously expressed as GFP-fusion in human cells (Archinti et al., 2010). Third, SPICE1 is

spindle associated in mitosis and plays a role in faithful mitotic chromosome segregation

independent of centriole number (Archinti et al., 2010). The possibility that CEP120 and

SPICE1 shuttle to and from the nucleus should be tested, for example by treatment of cells with

Leptomycin-B to inhibit nuclear export (Kudo et al., 1999), or careful biochemical isolation of

nuclei followed by Western blots for CEP120 and SPICE1. Might the roles of CEP120/SPICE1

in centriole duplication and mitotic spindle assembly be regulated by interactions with NAP1L1?

196

This is an exciting possibility that clearly warrants further investigation. It will be important to

test whether NAP1L1 interacts directly with CEP120 and SPICE1, and to determine where this

interaction occurs within the cell (nuclear, cytoplasmic or both?). This can be achieved by co-IP

experiments using cytoplasmic and nuclear extracts separately. Whether CEP120/SPICE1 are

associated with nucleosomes and DNA should be tested. This can be achieved by chemical

cross-linking of DNA to proteins and subsequent immunoprecipitation from nuclear extracts

using antibodies against histone proteins (i.e. chromatin immunoprecipitation) (Solomon et al.,

1988). These extracts would then be analyzed by Western blot for CEP120 and SPICE1.

Alternatively, chemical cross-linking of proteins to DNA, extraction of nuclei and

immunoprecipitation using antibodies against CEP120/SPICE1 could be attempted. These

extracts could be analyzed for DNA content using ethidium bromide gel electrophoresis, or

tested for the presence of histones or NAP1L1 by Western blot. How does changing the cellular

levels of CEP120/SPICE1 affect NAP1L1 function in transcription, or its localization? One of

the NAP1L1 siRNAs tested here affected CEP120 localization, supportive of a functional link

between the two proteins. This effect, however, was not reversed by the presence of GFP-

NAP1L1. Future work should address these issues, and focus on validating the possible

functional relationship between NAP1L1, CEP120 and SPICE1. Importantly, we report here a

novel link between a cancer-associated transcription regulating histone chaperone and two

proteins involved directly in centriole assembly. In sum, the results described within this thesis

represent significant advances in the field of centrosome biology and centriole duplication, and

open up several new research directions warranting further investigation.

197

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