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Microtubule-associated protein-4 (MAP-4) inhibits microtubule-dependent

distribution of mRNA in isolated neonatal cardiocytes

Dimitri Scholz a,c,*, Paul McDermott a,c, Maria Garnovskaya b,c, Thomas N. Gallien a,c,

Stefan Huettelmaier d, Christina DeRienzo a,c, George Cooper IV a,c

a Gazes Cardiac Research Institute, Cardiology Division, Medical University of South Carolina, United Statesb Medical and Research Services of the Ralph H. Johnson Veteran Affairs Medical Center, Nephrology Division,

Medical University of South Carolina, United Statesc Department of Veterans Affairs Medical Center, Charleston, SC 29401, United States

d Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, United States

Received 3 October 2005; received in revised form 27 March 2006; accepted 9 April 2006

Available online 5 June 2006

Time for primary review 23 days

by guest on June 1p://cardiovascres.oxfordjournals.org/

Abstract

Objectives: Active mRNA distribution in the form of ribonucleoprotein particles moving along microtubules has been shown in several cell

types, but not yet in cardiocytes. This study addresses two hypotheses: 1) a similar mRNA distribution mechanism operates in cardiocytes; 2)

decoration of microtubules with microtubule-associated proteins compromises this distribution.

Methods: To visualize ribonucleoproteins in cultured neonatal rat cardiocytes, they were transfected with vectors encoding zipcode binding

protein-1 and Staufen fused with GFP. The velocity of microtubular transport and elongation were calculated on time-lapse confocal pictures.

Results: ZBP-1 and Staufen labeled particles co-localized with each other and with microtubules and moved along microtubules over a

distance of 1–20 Am with a mean speed of 80 nm/s. The average speed decreased about 50% after decoration of microtubules by adenoviral

microtubule-associated protein-4 (MAP-4). The elongation speed measured using the GFP-tagged end-binding protein-1 exceeded 200 nm/s

and was not influenced by MAP-4.

Conclusions: We demonstrate for the first time ribonucleoprotein particles in cardiocytes, their microtubular-related movement, and its

inhibition (but not of the microtubular elongation), by the MAP-4 decoration of microtubules.

D 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

3, 2

013

Keywords: mRNA; Microtubule; Cardiocyte; Translocation

1. Introduction

Cardiocytes have a high rate of protein turnover, with

proteins of both the myofilament and extramyofilament

cytoskeleton being renewed to the extent of ¨20% per day

[1]. These processes are greatly enhanced during hypertro-

phy [2]. Thus, a consistent supply of translation-competent

mRNA for these structural proteins to the polysomes is

0008-6363/$ - see front matter D 2006 European Society of Cardiology. Publish

doi:10.1016/j.cardiores.2006.04.001

* Corresponding author. Gazes Cardiac Research Institute, Medical

University of South Carolina, 114 Doughty Street, Rm. 302 Charleston,

SC 29403, United States. Tel.: +1 843 876 5067, fax: +1 843 876 5068.

E-mail address: scholzd@musc.edu (D. Scholz).

needed for normal cellular homeostasis, and it is essential

for hypertrophic growth. However, several unique aspects of

cardiocyte cytoarchitecture challenge this biosynthetic

pathway. In terms of cardiocyte volume, myofibrils are

50–60%, mitochondria are 25–35%, nuclei are 3–5%, the

T-system is 1–2%, and the sarcoplasmic reticulum is 1–3%

[3–5]. ‘‘Free’’ cytoplasm (containing glycogen, ribosomes

and the extramyofilament cytoskeleton) makes up only

¨10%. This high concentration of organelles markedly

limits the free diffusion of macromolecules within these

large cells. Therefore, mRNAs for cardiocyte structural

proteins must be actively delivered to their sites of

translation rather than simply arriving via diffusion.

71 (2006) 506 – 516

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In 1993, James Wilhelm and Ron Vale [6] formulated a

hypothesis according to which the transport of mRNAs, and

not of the translated proteins, is responsible for the

localization of cytoplasmic proteins. This model, slightly

modified by Jansen [7], entails the binding of specific

shuttling factors to the so-called ‘‘zip-code’’ sequence of

nascent mRNA in the nucleus [8,9]. The term ‘‘zip code’’ as

a definition for mRNA localization signals within the

3VUTR was proposed by Singer in 1993 [10] and since

then has been broadly employed [7,10–12].

Zip code function is mediated by mRNA-binding proteins

[7,13–18]. The best described and the most promising for

the study of mRNA trafficking in cardiocytes are zipcode-

binding protein-1 (ZBP-1) [10–12,19,20] and Staufen [21–

28]. ZBP-1 was shown to participate in mRNA granule

formation and is needed for mRNA–cytoskeleton attach-

ment [19]. Further, ZBP-1 co-localizes with a h-actintranscription site [20]. Staufen, found also in skeletal muscle

[26], was co-immunoprecipitated with the microtubule-

based + end directed motor protein kinesin in Xenopus

oocytes and was proposed to represent a link between

specific mRNAs and the transportation machinery [29].

The active distribution of mRNA has been described in

several cell types, including ascidian embryos [30], mos-

quito salivary glands [31], Drosophila oocytes [9,32],

Xenopus oocytes [33], rat embryonic neurons [34,35], rat

oligodendrocytes [8], and chicken embryo fibroblasts [19].

The organization of mRNA in ribo-nucleo-protein (RNP)

granules in living cells and their movement along the

cytoskeleton was shown for the first time in dendrites of

mammalian neurons [36], then in oligodendrocytes [37] and

the dendrites of rat hippocampal neurons [38]. RNP

granules have been hypothesized to represent storage

containers for mRNA under translational arrest, which

could be poised for release to actively translated pools

[35]. However, RNP granules and their active distribution

have not been described in cardiocytes.

As one example of active intracellular distribution,

microtubules serve as rails for the transport of vesicles,

cytoplasmic particles [39,40], membrane receptors [41], and

translation-competent mRNAs [6,42–44]. Active, microtu-

bule-based mRNA transport has been described in Dro-

sophila oocytes, yeast, neurons, oligodendrocytes, and

fibroblasts [15,16,22,37,43–45].

A major impetus to this study was our finding that in

pressure-overload cardiac hypertrophy, cardiocytes contain

an increased amount of microtubules, heavily decorated

with and stabilized by the predominant cardiac microtubule-

associated protein, MAP-4 [46], which contributes to cell

viscosity and contractility deficits [47–56]. Depolymeriza-

tion of microtubules restores both the contractility of

cardiocytes and cardiac function to normal [54,56–58].

However, the present study was based not on cardiocyte

mechanics but on the question of whether the extensive

MAP-4 decoration of a dense microtubule network that we

find in pressure-overload cardiac hypertrophy might disrupt

important cellular functions by interfering with microtubule-

based intracellular transport of vesicles and cytoplasmic

particles [6,39,40].

Thus, the aim of present study was to initially test in

neonatal cardiocytes the hypothesis that cardiocyte mRNA

transport is microtubule-dependent and that excessive MAP-

4 decoration of microtubules compromises this transport

function, thereby affecting processes important for the

protein biosynthesis that is essential for the anabolic cellular

response to hemodynamic overloads.

2. Materials and methods

2.1. Ethical statement

This investigation conforms with the Guide for the Care

and Use of Laboratory Animals published by the US

National Institutes of Health (NIH Publication No. 85-23,

revised 1996).

2.2. Animals and isolation of cells

Primary neonatal rat heart cell cultures were prepared from

ventricular myocardium of 0- to 3-day-old rats as described

[59]. Briefly, the cardiocytes were dissociated in (Ca2+–

Mg2+)-free Hank’s salt solution buffered with 30 mM

HEPES, pH 7.4, in a Celstir apparatus (Wheaton Instruments)

at 37 -C by the addition of trypsin (Cooper Biomedical Inc.),

chemotrypsin, and elastase (Sigma) at concentrations of 2.4,

2.7 and 0.94 U/ml, respectively. After each of six successive

20-min incubations, the dissociated cells were mixed with

minimum Eagle’s medium (GIBCO) containing 10% new-

born calf serum, centrifuged at 500�g, and pooled. Cells

were transfected by nucleofection (see below) and plated at a

density of about 5�105 cells in 22 mm (170 Am thick-

bottomed) gelatin-coated WillcoWells\ culture dishes for

live cell microscopy [28]. The majority of cells remained

quiescent in vitro, and only a small percentage was beating at

a low frequency spontaneously or under the laser beam;

these cells were excluded from measurements.

2.3. Plasmids and transfection of cardiocytes

The vector encoding the fusion EYFP–Staufen protein

[21–28] was constructed in the lab of Dr. M. Kiebler, Max-

Planck-Institute for Developmental Biology, Tuebingen,

Germany. The vector encoding the fusion EYFP–ZBP-1

and the ECFP–ZBP-1 proteins [10–12,19,20] were con-

structed in the lab of Dr. R. Singer, Albert Einstein College

of Medicine, NY, USA. The vector encoding the fusion

EGFP–EB-1 protein [60–62] was constructed in the lab of

Dr. Y. Mimori-Kiyosue, Exploratory Research for Advanced

Technology, Japan Science and Technology Corporation,

Kyoto, Japan. The vector encoding the fusion EYFP–a-

tubulin [63] protein was purchased from Clontech.

Fig. 1. Distribution of Staufen–EYFP fusion protein 4 h (A) and 48 h (B)

after nucleofection of neonatal rat cardiocytes. Light diffuse labeling over

the entire cytoplasm corresponds to unbound protein; the nucleus appears as

a dark oval. Granular labeling, most likely representing RNP particles,

appears in the perinuclear region shortly after nucleofection (A) and later

populates the entire cytoplasm (B). Scale bar=10 Am.

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A Nucleofectori kit designed for transfection of freshly

isolated primary cardiocytes from newborn rats (Amaxa

Biosystems, Giessen, Germany) was used according to the

manufacturer’s protocol. About 2�106 cells and 2–5 Ag of

plasmid per transfection (single plasmid or double) were

used for each transfection. Up to 10% of cardiocytes were

transfected.

2.4. Adenoviral infection

Adenoviruses expressing bacterial h-gal and human

MAP-4 (Adh-gal and AdMAP-4) were generated in our

laboratory as described [64]. Neonatal rat cardiocytes were

infected in serum-free medium [64] at a multiplicity of

infection (MOI) of about 10 plaque forming units per cell,

thus infecting about 90% of the cardiocytes as determined

by immunofluorescence microscopy using either our poly-

clonal MAP-4 antibody or an X-gal reaction; there was no

observable cytotoxicity.

Preliminary results have shown that MAP-4 appears

mostly diffuse until day 3 after infection, after which it

decorates the microtubules. Therefore, all experiments were

conducted after day 3.

2.5. Immunofluorescent labeling

Neonatal heart cells were fixed in freshly prepared 4%

formaldehyde for 10 min, permeabilized in 0.1% Triton

X-100 and were covered with antibody against MAP-4

and then with the anti-rabbit antiserum tagged with Alexa

Fluor\ 488 (Molecular Probes, Eugene, OR). To generate

the MAP-4 antibody, we made a bacterial expression

construct using the 1–740 NH2-terminal residues of

human MAP-4 [41]. Briefly, the recombinant protein,

which had a hexahistidine tag inserted at the COOH

terminus, was overexpressed in Escherichia coli, purified

on a nickel–chelate affinity column, and submitted to

SDS–PAGE. The purified protein band was excised,

eluted from the gel, and sent to Lampire Biological

Laboratories for preparation of a rabbit polyclonal MAP-4

antibody.

2.6. Microscopy and time lapse photography

A Zeiss LSM510META microscope with Ar-Laser (458,

477, 488 and 514 nm) 30 mW, Plan-Apochromat 63/1.4

objective and a chamber maintaining 37 -C and 5% CO2

was used. The minimal possible laser intensity was chosen

to decrease the thermal damage to cells. Excitation

wavelength and emission filters used for imaging fluores-

cence: CFP-458 nm/BP475-525, GFP-488 nm/LP505, YFP-

514 nm/LP 530. CFP and YFP were visualized simulta-

neously on Figs. 2 and 4.

For Fig. 6, an Olympus IX71 microscope equipped with

a mercury lamp as a light source, and filter sets for DAPI

(Chroma, excitation S403/12, emission S457/50) and for

Alexa488 (Chroma, excitation S490/20, emission D528/38)

have been used.

Time-lapse pictures of about 100 frames gathered over

10 to 25 min without intervals between successive frames

were scanned. Fluorescence-labeled particles moving

straight in at least 3 frames in the row and over a distance

more than 1 Am were considered to be moving actively

and were measured using LSM 5 Image Browser software.

Contracting or laser-damaged cells were excluded from the

study. The movement speed was measured separately for

ZBP-1, Staufen, and EB-1 labeled particles in control and

in MAP-4 or h-gal infected cells for at least 60 particles

per group. Results were analyzed statistically using

Student’s T-test.

2.7. Radioautography

Radioautography was conducted as described [65],

applied here to neonatal cardiocytes. Briefly, cells were

cultured on cover slips in 35 mm culture dishes. 3H-uridine

(10 ACi/ml) was added to the culture medium for 1–6 h.

After that, cells were fixed with formaldehyde, dried in

alcohol and coated with undiluted Kodak NTB2 photo-

graphic emulsion at 42 -C. After 7 days of exposure at 4 -C,they were developed in Kodak D-19 developer as recom-

mended by the manufacturer, counterstained with toluidine

blue and mounted in Entellan.

Fig. 2. Co-localization of ZBP-1 (red) and Staufen (green) in the same granules. Four out of six visible ZBP-1 granules also contain Staufen. A small shift is

caused by color aberration of the lens at the critical magnification. Neonatal rat cardiocytes were double-nucleofected with plasmids encoding the CFP–ZBP-1

and EYFP–Staufen. Scale bar=10 Am.

Fig. 3. Linear movement of a single ZBP-1–EYFP granule (green, arrow) over a distance of about 20 Am. Scale bar=10 Am. A movie of this figure is available

in the Appendix. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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3. Results

3.1. Assembly of RNPs

In order to visualize mRNA-related proteins in rat

neonatal cardiocytes, we transfected freshly isolated cells

with plasmids encoding fluorescently tagged RNP proteins

Staufen and ZBP-1. The diffuse fluorescence became

detectable in the cytoplasm as early as 3–4 h after

transfection and persisted throughout the period of

observation (7 days). This most likely corresponds to

newly synthesized protein which is not yet bound to

mRNA. Fluorescent granules, most likely representing

RNP particles, appeared first in a perinuclear location

(Fig. 1A) and then populated after 24 h the entire

cytoplasm (Fig. 1B).

Fig. 4. Movement of two ZBP-1–EYFP granules (red, arrows) along microtubu

encoding: 1) a-Tubulin–EYFP (green) and 2) ZBP-1–ECFP (red). A–E represent

of this figure is available in the Appendix.

Double transfection of neonatal rat cardiocytes with

plasmids encoding two different and differently labeled

fusion proteins (Staufen–YFP and ZBP-1–CFP) revealed

their co-localization in many instances (Fig. 2).

3.2. Movement of RNPs

Time-lapse pictures were analyzed to measure the speed

of RNP movement. We found that in live cells the vast

majority of labeled particles were oscillating in place, and

only a small subpopulation moved straight over a distance

more than 1 Am, and only rarely up to 10–20 Am (Fig. 3

and Fig. 3 movie in the Appendix). The measured speed

of granules averaged 77.2T6.3 nm/s for ZBP-1 and 76.7T8.5 nm/s for Staufen (meanTS.E.M.). The close corre-

spondence of the transport rates for these two types of

les (green). Neonatal rat cardiocytes were nucleofected with two plasmids

sequential pictures with a time interval of 14 s. Scale bar=10 Am. A movie

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Fig. 5. Elongation of microtubules. Neonatal rat cardiocytes were

nucleofected with the plasmid encoding the microtubule+end-binding

protein EGFP–EB-1. Scale bar=10 Am. A movie of this figure is available

in the Appendix.

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proteins indicates that they may be transported by the

same cellular motor and (combined with their co-localiza-

tion) suggests indirectly that we may really be dealing

with two proteins being transported as part of the same

RNPs.

Fig. 6. Neonatal rat cardiocytes labeled with anti-MAP-4 antibodies (green)

after AdMAP-4 infection (A) and without infection (B). Nuclei are stained

with DAPI (red). Scale bar=20 Am.

tp://cardiovascres.

3.3. Co-localization of RNP and microtubules and move-

ment along the microtubules

To test our hypothesis that microtubules serve as a

railroad system for RNP movement, microscopic assess-

ment of co-localization of microtubules and RNPs was

undertaken. Cardiocytes double-transfected with the a-

tubulin–EYFP and the ZBP-1–CFP demonstrated consis-

tent co-localization: the RNP granules were localized close

to the microtubules (Fig. 4). In addition, RNP granules

moved along the microtubules from the nucleus towards the

cell periphery, as shown by Fig. 4 and Fig. 4 movie in the

Appendix.

3.4. Microtubule elongation is about 3-fold faster than RNP

transport

Elongation of microtubules in live rat neonatal cardio-

cytes was measured using the GFP-labeled EB-1, which

binds to the + end of growing microtubules (Fig. 5 and Fig.

5 movie in the Appendix). The mean speed of microtubule

elongation was calculated to be 204T6 nm/s, or about three

times as fast as the transport of the RNP particles (Fig. 7).

Thus, the RNP transport kinetics that we have measured are

not a function of the rate of microtubule polymerization-

based elongation.

Fig. 7. Average speed of labeled granules in live neonatal cardiocytes, nm/s,

meanTS.E.M.

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3.5. MAP-4 decoration slows down RNP transport but not

microtubule elongation

MAP-4 decoration of microtubules at day 3 after the

AdMAP-4 infection was documented in cardiocytes by

immunofluorescence using antibodies against MAP-4 (Fig.

6). Non-infected cardiocytes demonstrated no detectable

labeling whereas about 90% of infected cells were positively

labeled, just as we demonstrated before [51]. In AdMAP-4-

treated cells, the mean speed of the ZBP-1 and Staufen-

labeled particles was reduced to about 50% of the control

value (Fig. 7). On the other hand, h-gal infection did not

cause significant changes.

In contrast, the mean speed of the EB-1-labeled particles

in AdMAP-4-infected cells did not change (204T6 nm/s

control vs. 227T10 nm/s AdMAP-4, n.s.).

3.6. Radioautographic dispersion of RNA

The incorporated radioactive 3H-uridine first localized in

the nuclei, predominantly in the nucleoli (Fig. 8A). The

nuclear and nucleolar labeling remained strong during 1–

6 h (Fig. 8A–D) and up to 24 h (not shown), which reflects

the steady high level of RNA synthesis. One hour after the

onset of the experiment, labeling over the cytoplasm was

minimal (Fig. 8A). Cells incorporating 3H-uridine for 2.5, 4

and 6 h demonstrated gradual dispersion of labeling over

Fig. 8. Radioautography of neonatal rat cardiocytes 1 h (A), 2.5 h (B), 4 h (C) and 6

nucleoli) persists from A–D, indicating ongoing transcription. The labeling over

gradually in B–D, indicating ongoing cytoplasmic transport of nascent RNA. Sc

the entire cytoplasm. Since we could not follow individual

silver grains or establish a cytoplasmic starting point, we

roughly estimated that the wave front of labeled RNA

moves from the nucleus to the cell periphery over a

distance of 20–30 Am in 2.5 h to 6 h, which corresponds to

a mean velocity of about 1–3.5 nm/s.

4. Discussion

This study provides the first experimental evidence for an

active, microtubule-based mechanism of mRNA distribution

in heart muscle cells. We demonstrated microscopically for

the first time the existence of RNP particles in cardiocytes,

their co-localization with microtubules, and their microtu-

bule-related transport. We calculated the speed of this

transport and its inhibition when microtubules are decorated

by MAP-4. Finally, we found that in cardiocytes microtu-

bule-based transport occurs independently from microtubule

elongation, and that the latter is not influenced by MAP-4.

4.1. Nature of RNP particles in cardiocytes

In the present study, RNP particles ranged in size from

about 30 to 100 nm. However, this is only an estimate, since

we could not measure particle size precisely via fluores-

cence microscopy because of resolution limitations. More-

h (D) after 3H-uridine incorporation. The labeling over the nucleus (mostly

the cytoplasm is barely detectable over the background in A and increases

ale bar=50 Am.

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over, overexposed particles appear larger, and some studies

have reported RNP particle sizes of up to 500 nm [34] or

even 700 nm [66]. Electron microscopy studies [67]

described nuclear RNP particles of 20 to 50 nm. However,

electron microscopy data on RNP particles outside the

nucleus [22] have described them as loose and amorphous

particles associated with polyribosomes, about 200 nm in

diameter. While Oleynikov and Singer described ZBP-1

shuttling between nucleus and cytoplasm in chicken embryo

fibroblasts [20], we could not detect any nuclear ZBP-1 or

Staufen labeling.

Pre-RNP exported through the nuclear pores to the

cytoplasm [68,69] matures into RNP particle binding

proteins, recently classified [70] into 1) RNA-binding

proteins that associate with specific mRNAs such as Staufen

[42], ZBP-1 [12], and Vera [71]; 2) motor proteins that

associate with RNPs to transport them, such as dynein [45]

and kinesin [72]; 3) adaptor proteins that interact with other

elements of RNPs and are essential for their transport, such

as Barentsz [27,73], She3p [74] and Miranda [75]; 4)

cytoskeletal structures along which RNPs move, such as

microtubules [42] and actin thin filaments [76]; and 5)

specific repressors of protein synthesis that prevent trans-

lation before the target mRNA is properly localized, such as

Bruno [77]. Obviously, after passing the nuclear pore

bottleneck, tens or hundreds of particles may come together

and bind additional regulatory and transport proteins and

ribosomes [78]. They thereby grow about 10-fold in size

(which corresponds to a 1000-fold gain in volume), bind to

the microtubules, and move to their destination where the

translation may finally begin [79].

4.2. Speed of RNP movement

Twenty years ago, Lasek et al. [80] differentiated fast

(0.5–4 Am/s) axonal transport of vesicles from slow (10–

50 nm/s) transport of cytoplasmic proteins based solely on

radiolabeling, the only technique available at that time. The

measurements for mRNA showed even slower movement

than that for proteins [65].

Table 1

Literature data for the velocity of microtubule transport for RNP and other non-v

Speed (nm/s) Reference Species, tissue

0.6 [65] Rat embryonic hippocam

1.0–1.2 [87] Rat embryonic hippocam

1.17 [88] Xenopus oocyte

200 [6] Myelin basic protein (M

100–1000 [37] Mouse oligodendocytes

100 [34] Rat embryonic cortical n

200–400 [89] Yeast

70 [82] HeLa cells

500–1000 [39] Squid axon

600 [20] Fibroblasts, ZBP-1

540 [90] Human U2OS osteosarc

300–2000 [91] CHO cells

Direct measurements with fluorescent microscopy.

Since fluorescence study of live cells after microinjection

of labeled mRNA [37], labeling with mRNA-dyes [34] or

transfection with fluorescent fusion proteins [23,81] became

available, many investigators have followed intracellular

movement directly and calculated the speed; the results are

summarized in Table 1.

In the present study, we estimated an average speed of

RNPs in the cytoplasm of about 80 nm/s, which corresponds

closely to the literature data for RNP transport in neurons,

oligodendocytes and fibroblasts [20,34,37,38,82].

In cells infected with AdMAP-4, the mean speed of RNP

particles was significantly reduced. We excluded the

influence of adenoviral infection by using Adh-gal as a

negative control and suggest that the reason for the speed

reduction by MAP-4 could be the competition of the

molecular motor with the MAP for binding sites on

microtubules.

In contrast, the mean speed of microtubule elongation

was almost 3-fold higher, very close to that described

for HeLa cells [83], and it was MAP-4-independent.

Thus, microtubule elongation and microtubule-dependent

transport of RNPs are based on different molecular

mechanisms.

At any time point the majority of particles do not exhibit

net movement but instead oscillate in place. Similar

behavior of RNP-labeled granules using GFP–ZBP-1 or

GFP–Staufen has been reported in chicken embryo fibro-

blasts [20], chicken neurons [84], and rat hippocampal

neurons [23]. This transport pattern could be explained by

saltatory motion or by very short-term movement of

intracellular cargos along microtubules [85]. Further, RNPs

have been shown not to be bound to motor proteins or

microtubules most of the time [86].

To approximate the rate of movement of RNA pools over

a longer period of time, we used radiolabeling, taking into

account that the measurement could be contaminated by

rRNA movement but hoping that rRNA might be trans-

ported in the same RNP particles [22]. We roughly

estimated the mean velocity of newly synthetisized RNA

in cytoplasm to be about 1–3 nm/s or 20 to 80 times less

esicle cytoplasmic particles

Comments

pal neurons 3H-uridine incorporation

pal neurons EGFP–ZBP-1

BP) mRNA

eurons Prevented by microtubule depolymerization

slow plus-end-directed mitotic kinesin motor

oma cells Intranuclear

Vesicles, short measurements

D. Scholz et al. / Cardiovascular Research 71 (2006) 506–516514

than that measured by our fluorescence method, which

probably means that at any given time only one out of 20 to

80 granules is moving. This result is consistent with our

observation by fluorescence microscopy that at any given

time only a small fraction of the particles show substantial

vectorial movement.

5. Conclusions

We demonstrate here for the first time RNP particles in

cardiocytes, their microtubule-related movement, and the

inhibition of this movement, but not of microtubule

elongation, by MAP-4 microtubule decoration.

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Acknowledgments

This study was supported by Program Project Grant HL-

48788 from the National Heart, Lung, and Blood Institute

and by a Merit Award from the Research Service of the

Department of Veterans Affairs. We thank Dr. Michael

Kiebler and Dr. Bernhard Goetze from Max Planck Institute

for Developmental Biology, Tuebingen 72076, Germany

and Dr. Yuko Mimori-Kiyosue from Tsukita Cell Axis

Project, Exploratory Research for Advanced Technology,

Japan Science and Technology Corporation, Kyoto Re-

search Park, Shimogyo-ku, Kyoto 600-8813, Japan, for

providing vectors and for the fruitful critical discussion and

comments during the manuscript preparation.

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Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.cardiores.

2006.04.001.

013

References

[1] Simpson D, Decker M, Clark W, Decker R. Contractile activity and

cell –cell contact regulate myofibrillar organization in cultured cardiac

myocytes. J Cell Biol 1993;123:323–36.

[2] Imamura T, McDermott PJ, Kent RL, Nagatsu M, Cooper G IV,

Carabello BA. Acute changes in myosin heavy chain synthesis rate in

pressure versus volume overload. Circ Res 1994;75:418–25.

[3] Page E, McCallister LP. Quantitative electron microscopic description

of heart muscle cells. Application to normal, hypertrophied and

thyroxin-stimulated hearts. Am J Cardiol 1973;31:172–81.

[4] Anversa P, Loud AV, Vitali-Mazza L. Morphometry and autoradiog-

raphy of early hypertrophic changes in the ventricular myocardium of

adult rat: an electron microscopic study. Lab Invest 1976;35:475–83.

[5] Rumyantsev PP. Cardiomyocytes in the reproduction, differentiation

and gegeneration. Leningrad’ Nauka; 1982 [in Russian].

[6] Wilhelm JE, Vale RD. RNA on the move: the mRNA localization

pathway. J Cell Biol 1993;123:269–74.

[7] Jansen R. mRNA localization: message on the move. Nat Rev Mol

Cell Biol 2001;2:247–56.

[8] Hoek K, Kidd G, Carson JRS. hnRNP A2 selectively binds the

cytoplasmic transport sequence of myelin basic protein mRNA.

Biochemistry 1998;37:7021–9.

[9] Norvell A, Kelley R, Wehr KTS. Specific isoforms of squid, a

Drosophila hnRNP, perform distinct roles in Gurken localization

during oogenesis. Genes Dev 1999;13:864–76.

[10] Singer R. RNA zipcodes for cytoplasmic addresses. Curr Biol

1993;3:719–21.

[11] Oleynikov Y, Singer RH. RNA localization: different zipcodes, same

postman? Trends Cell Biol 1998;8:381–3.

[12] Ross AF, Oleynikov Y, Kislauskis EH, Taneja KL, Singer RH.

Characterization of a h-actin mRNA zipcode-binding protein. Mol

Cell Biol 1997;17:2158–65.

[13] Kislauskis E, Li Z, Singer R, Taneja K. Isoform-specific 3V-untranslated sequences sort a-cardiac and h-cytoplasmic actin

messenger RNAs to different cytoplasmic compartments. J Cell Biol

1993;123:165–72.

[14] Bashirullah A, Cooperstock R, Lipshitz H. RNA localization in

development. Annu Rev Biochem 1998;67:335–94.

[15] Bassell G, Oleynikov Y, Singer R. The travels of mRNAs through all

cells large and small. FASEB 1999;13:447–54.

[16] Farina K, Singer R. The nuclear connection in RNA transport and

localization. Trends Cell Biol 2002;12:466–72.

[17] Roegiers F. Insights into mRNA transport in neurons. Proc Natl Acad

Sci 2002;100:1465–6.

[18] Jockusch B, Huttelmaier S, Illenberger S. From the nucleus toward the

cell periphery: a guided tour for mRNAs. News Physiol Sci

2003;18:7–11.

[19] Farina K, Huttelmaier S, Musunuru K, Darnell R, Singer R. Two

ZBP1 KH domains facilitate h-actin mRNA localization, granule

formation, and cytoskeletal attachment. J Cell Biol 2003;160:77–87.

[20] Oleynikov Y, Singer R. Real-time visualization of ZBP1 association

with. Curr Biol 2003;13:199–207.

[21] Wickham L, Duchaine T, Luo M, Nabi IR, DesGroseillers L.

Mammalian Staufen is a double-stranded-RNA- and tubulin-binding

protein which localizes to the rough endoplasmic reticulum. Mol Cell

Biol 1999;19:2220–30.

[22] Kiebler MA, Hemraj I, Verkade P, Kohrmann M, Fortes P, Marion

RM, et al. The mammalian Staufen protein localizes to the

somatodendritic domain of cultured hippocampal neurons: implica-

tions for its involvement in mRNA transport. J Neurosci 1999;19:

288–97.

[23] Kohrmann M, Luo M, Kaether C, DesGroseillers L, Dotti CG, Kiebler

MA. Microtubule-dependent recruitment of Staufen–green fluores-

cent protein into large RNA-containing granules and subsequent

dendritic transport in living hippocampal neurons. Mol Biol Cell

1999;10:2945–53.

[24] Duchaine TF, Hemraj I, Furic L, Deitinghoff A, Kiebler MA,

DesGroseillers L. Staufen2 isoforms localize to the somatodendritic

domain of neurons and interact with different organelles. J Cell Sci

2002;115:3285–95.

[25] Martin S, Leclerc V, Smith-Litiere K, D.S.J.. The identification of

novel genes required for Drosophila anteroposterior axis formation in

a germline clone screen using GFP–Staufen. Development

2003;130:4201–15.

[26] Belanger G, Stocksley MA, Vandromme M, Schaeffer L, Furic L,

DesGroseillers L, et al. Localization of the RNA-binding proteins

Staufen1 and Staufen2 at the mammalian neuromuscular junction. J

Neurochem 2003;86:669–77.

[27] Macchi P, Kroening S, Palacios IM, Baldassa S, Grunewald B,

Ambrosino C, et al. Barentsz, a new component of the Staufen-containing

ribonucleoprotein particles in mammalian cells, interacts with Staufen in

an RNA-dependent manner. J Neurosci 2003;23:5778–88.

[28] Mallardo M, Deitinghoff A, Muller J, Goetze B, Macchi P, Peters C,

et al. Isolation and characterization of Staufen-containing ribonucleo-

protein particles from rat brain. Proc Natl Acad Sci U S A 2003;100:

2100–5.

D. Scholz et al. / Cardiovascular Research 71 (2006) 506–516 515

by guest on June 13, 2013http://cardiovascres.oxfordjournals.org/

Dow

nloaded from

[29] Yoon Y, Mowry K. Xenopus Staufen is a component of a

ribonucleoprotein complex containing Vg1 RNA and kinesin. Devel-

opment 2004;131:3035–45.

[30] Jeffery W, Tomlinson C, Brodeur R. Localization of actin messenger

RNA during early ascidian development. Dev Biol 1983;99:408–17.

[31] Mehlin H, Daneholt B, Skoglund U. Translocation of a specific

premessenger ribonucleoprotein particle through the nuclear pore

studied with electron microscope tomography. Cell 1992;69:605–13.

[32] Frigerio G, Burri M, Bopp D, Baumgartner S, Noll M. Structure of the

segmentation gene paired and the Drosophila PRD gene set as part of

a gene network. Cell 1986;47:735–46.

[33] Rebagliati M, Weeks D, Harvey R, Melton D. Identification and

cloning of localized maternal RNAs from Xenopus eggs. Cell

1985;42:769–77.

[34] Knowles R, Sabry J, Martone M, Deerinck TJ, Ellisman MH, Bassell

GJ, et al. Translocation of RNA granules in living neurons. J Neurosci

1996;16:7812–20.

[35] Krichevsky A, Kosik K. Neuronal RNA granules: a link between RNA

localization and stimulation-dependent translation. Neuron

2001;32:683–96.

[36] Tiedge H, Fremeau RJ, Weinstock P, Arancio O, Brosius J. Dendritic

location of neural BC1 RNA. Proc Natl Acad Sci U S A 1991;88:

2093–7.

[37] Ainger K, Avossa D, Morgan F, Hill SJ, Barry C, Barbarese E, et al.

Transport and localization of exogenous myelin basic protein mRNA

microinjected into oligodendrocytes. J Cell Biol 1993;123:431–41.

[38] Fredj N, Grange J, Sadoul R, Richard S, Goldberg Y, V.B..

Depolarization-induced translocation of the RNA-binding protein

Sam68 to the dendrites of hippocampal neurons. J Cell Sci 2004;

117:1079–90.

[39] Prahlad V, Helfand BT, Langford GM, Vale RD, Goldman RD. Fast

transport of neurofilament protein along microtubules in squid

axoplasm. J Cell Sci 2000;113:3939–46.

[40] Vale RD. The molecular motor toolbox for intracellular transport. Cell

2003;112:467–80.

[41] Cheng G, Iijima Y, Ishibashi Y, Kuppuswamy D, Cooper G IV.

Inhibition of G protein-coupled receptor trafficking in neuroblastoma

cells by MAP-4 decoration of microtubules. Am J Physiol Heart Circ

Physiol 2002;283:H2379.

[42] Ferrandon D, Elphick L, Nusslein-Volhard C, St Johnston D.

Staufen protein associates with the 3VUTR of bicoid mRNA to form

particles that move in a microtubule-dependent manner. Cell 1994;

79:1221–32.

[43] Carson J, Worboys K, Ainger K, Barbarese E. Translocation of myelin

basic protein mRNA in oligodendrocytes requires microtubules and

kinesin. Cell Motil Cytoskeleton 1997;38:318–28.

[44] Carson J, Kwon S, Barbarese E. RNA trafficking in myelinating cells.

Curr Opin Neurobiol 1998;8:607–12.

[45] Schnorrer F, Bohmann K, Nusslein-Volhard C. The molecular

motor dynein is involved in targeting swallow and bicoid RNA to

the anterior pole of Drosophila oocytes. Nat Cell Biol 2000;2:

185–90.

[46] Takahashi M, Shiraishi H, Ishibashi Y, Blade KL, McDermott PJ,

Menick DR, et al. Phenotypic consequences of h1-tubulin expression

and MAP4 decoration of microtubules in adult cardiocytes. Am J

Physiol Heart Circ Physiol 2003;285:H2072.

[47] Tsutsui H, Ishihara K, Cooper G IV. Cytoskeletal role in the contractile

dysfunction of hypertrophied myocardium. Science 1993;260:682–7.

[48] Tsutsui H, Tagawa H, Kent RL, McCollam PL, Ishihara K, Nagatsu

M, et al. Role of microtubules in contractile dysfunction of

hypertrophied cardiocytes. Circulation 1994;90:533–55.

[49] Tagawa H, Rozich JD, Tsutsui H, Narishige T, Kuppuswamy D, Sato

H, et al. Basis for increased microtubules in pressure-hypertrophied

cardiocytes. Circulation 1996;93:1230–43.

[50] Tagawa H, Wang N, Narishige T, Ingber DE, Zile MR, Cooper G IV.

Cytoskeletal mechanics in pressure-overload cardiac hypertrophy. Circ

Res 1997;80:281–9.

[51] Sato H, Nagai T, Kuppuswamy D, Narishige T, Koide M, Menick DR,

et al. Microtubule stabilization in pressure overload cardiac hypertro-

phy. J Cell Biol 1997;139:963–73.

[52] Koide M, Nagatsu M, Zile MR, Hamawaki M, Swindle MM, Keech

G, et al. Premorbid determinants of left ventricular dysfunction in a

novel model of gradually induced pressure overload in the adult

canine. Circulation 1997;95:1601–10.

[53] Zile MR, Richardson K, Cowles MK, Buckley JM, Koide M, Cowles

BA, et al. Constitutive properties of adult mammalian cardiac muscle

cells. Circulation 1998;98:567–79.

[54] Zile MR, Koide M, Sato H, Ishiguro Y, Conrad CH, Buckley JM, et al.

Role of microtubules in the contractile dysfunction of hypertrophied

myocardium. J Am Coll Cardiol 1999;33:250–60.

[55] Koide M, Hamawaki M, Narishige T, Sato H, Nemoto S, DeFreyte G,

et al. Microtubule depolymerization normalizes in vivo myocardial

contractile function in dogs with pressure-overload left ventricular

hypertrophy. Circulation 2000;102:1045–52.

[56] Schroder EA, Tobita K, Tinney JP, Foldes JK, Keller BB. Microtubule

involvement in the adaptation to altered mechanical load in developing

chick myocardium. Circ Res 2002;91:353–9.

[57] Cicogna AC, Robinson KG, Conrad CH, Singh K, Squire R, Okoshi

MP, et al. Direct effects of colchicine on myocardial function: studies

in hypertrophied and failing spontaneously hypertensive rats. Hyper-

tension 1999;33:60–5.

[58] Roos KP, Palmer RE, Miller TW. The role of microtubules in

structural remodeling and the progression to heart failure. J Card Fail

2002;8:S300.

[59] McDermott PJ, Morgan HE. Contraction modulates the capacity for

protein synthesis during growth of neonatal heart cells in culture. Circ

Res 1989;64:542–53.

[60] Mimori-Kiyosue Y, Shiina N, Tsukita S. The dynamic behavior of the

APC-binding protein EB1 on the distal ends of microtubules. Curr

Biol 2000;10:865–8.

[61] Mimori-Kiyosue Y, Tsukita S. ‘‘Search-and-capture’’ of microtubules

through plus-end-binding proteins (+TIPs). J Biochem (Tokyo)

2003;134:321–6.

[62] Piehl M, Tulu US, Wadsworth P, Cassimeris L. Centrosome

maturation: measurement of microtubule nucleation throughout the

cell cycle by using GFP-tagged EB1. Proc Natl Acad Sci U S A

2004;101:1584–8.

[63] Kimble M, Kuzmiak C, McGovern KN, de Hostos EL. Microtubule

organization and the effects of GFP–tubulin expression in Dictyoste-

lium discoideum. Cell Motil Cytoskeleton 2000;47:48–62.

[64] Cheng G, Qiao F, Gallien T, Kuppuswamy D, Cooper G IV. Inhibition

of h-adrenergic receptor trafficking in adult cardiocytes by MAP4

decoration of microtubules. Am J Physiol Heart Circ Physiol 2005;

288:H1193–202.

[65] Davis L, Banker G, Steward O. Selective dendritic transport of RNA

in hippocampal neurons in culture. Nature 1987;330:477–9.

[66] Barbarese E, Koppel D, Deutscher M, Smith CL, Ainger K, Morgan F,

et al. Protein translation components are colocalized in granules in

oligodendrocytes. J Cell Sci 1995;108:2781–90.

[67] Stevens BJ, Swift H. RNA transport from nucleus to cytoplasm in

Chironomus salivary glands. J Cell Biol 1966;31:55–77.

[68] Daneholt B. Look at messenger RNP moving through the nuclear

pore. Cell 1997;88:585–8.

[69] Levesque L, Guzik B, Guan T, Coyle J, Black BE, Rekosh D, et al.

RNA export mediated by tap involves NXT1-dependent interac-

tions with the nuclear pore complex. J Biol Chem 2001;276:

44953–62.

[70] Villace P, Marion RM, Ortin J. The composition of Staufen-containing

RNA granules from human cells indicates their role in the regulated

transport and translation of messenger RNAs. Nucleic Acids Res

2004;32:2411–20.

[71] Deshler J, Highett M, Schnapp B. Localization of Xenopus Vg1

mRNA by Vera protein and the endoplasmic reticulum. Science

1997;276:1128–31.

D. Scholz et al. / Cardiovascular Research 71 (2006) 506–516516

http://cardiovascresD

ownloaded from

[72] Brendza R, Serbus L, Duffy J, Saxton W. A function for kinesin I in

the posterior transport of oskar mRNA and Staufen protein. Science

2000;289:2120–2.

[73] van Eeden F, Palacios I, Petronczki M, Weston M, St Johnston D.

Barentsz is essential for the posterior localization of oskar mRNA and

colocalizes with it to the posterior pole. J Cell Biol 2001;154:511–23.

[74] Takizawa PA, Vale RD. The myosin motor, Myo4p, binds Ash1

mRNA via the adapter protein, She3p. Proc Natl Acad Sci U S A

2000;97:5273–8.

[75] Shen C, Knoblich J, Chan Y, Jiang M, Jan L, Jan Y. Miranda as a

multidomain adapter linking apically localized Inscuteable and basally

localized Staufen and Prospero during asymmetric cell division in

Drosophila. Genes Dev 1998;12:1837–46.

[76] Takizawa PA, Sil A, Swedlow JR, Herskowitz I, Vale RD. Actin-

dependent localization of an RNA encoding a cell-fate determinant in

yeast. Nature 1997;389:90–3.

[77] Kim-Ha J, Kerr K, Macdonald P. Translational regulation of oskar

mRNA by Bruno, an ovarian RNA-binding protein, is essential. Cell

1995;81:403–12.

[78] Brendel C, Rehbein M, Kreienkamp H, Buck F, Richter D, Kindler S.

Characterization of Staufen 1 ribonucleoprotein complexes. Biochem J

2004;384:239–46.

[79] Huettelmaier S, Zenklusen D, Lederer M, Dictenberg J, Lorenz M,

Meng X, et al. Spatial regulation of h-actin translation by Src-

dependent phosphorylation of ZBP1. Nature 2005;438:512–5.

[80] Lasek R, Garner J, Brady S. Axonal transport of the cytoplasmic

matrix. J Cell Biol 1984;99:212s–21s.

[81] Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D. Green fluorescent

protein as a marker for gene expression. Science 1994;263:802–5.

[82] Abaza A, Soleilhac JM, Westendorf J, Piel M, Crevel I, Roux A, et al.

M phase phosphoprotein 1 is a human plus-end-directed kinesin-

related protein required for cytokinesis. J Biol Chem 2003;278:

27844–52.

[83] Perez F, Diamantopoulos G, Stalder R, Kreis T. CLIP-170 highlights

growing microtubule ends in vivo. Cell 1999;96:517–27.

[84] Zhang HL, Eom T, Oleynikov Y, Shenoy SM, Liebelt DA, Dictenberg

JB, et al. Neurotrophin-induced transport of a h-actin mRNP complex

increases h-actin levels and stimulates growth cone motility. Neuron

2001;31:261–75.

[85] Sheetz MP. Motor and cargo interactions. Eur J Biochem 1999;262:

19–25.

[86] Kloc M, Etkin L. RNA localization mechanisms in oocytes. J Cell Sci

2005;118:269–82.

[87] Tiruchinapalli DM, Oleynikov Y, Kelic S, Shenoy SM, Hartley A,

Stanton PK, et al. Activity-dependent trafficking and dynamic

localization of zipcode binding protein 1 and h-actin mRNA in

dendrites and spines of hippocampal neurons. J Neurosci 2003;23:

3251–61.

[88] Yisraeli J, Sokol S, Melton D. A two-step model for the localization of

maternal mRNA in Xenopus oocytes: involvement of microtubules

and microfilaments in the translocation and anchoring of Vg1 mRNA.

Development 1990;108:289–98.

[89] Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, Long

RM. Localization of ASH1 mRNA particles in living yeast. Mol Cell

1998;2:437–45.

[90] Shav-Tal Y, Darzacq X, Shenoy SM, Fusco D, Janicki SM, Spector

DL, et al. Dynamics of single mRNPs in nuclei of living cells. Science

2004;304:1797–800.

[91] Mundy D, Machleidt T, Ying Y, Anderson R, Bloom G. Dual control

of caveolar membrane traffic by microtubules and the actin cytoskel-

eton. J Cell Sci 2002;115:4327–39.

.ox

by guest on June 13, 2013

fordjournals.org/