The Myxovirus Resistance Protein, MX1, Interacts with Tubulin Beta In Uterine Glandular Epithelial...

The Myxovirus Resistance Protein, MX1, Interacts with TubulinBeta In Uterine Glandular Epithelial CellsKaren Racicot, Troy Ott

Department of Dairy and Animal Science, The Pennsylvania State University, University Park, PA, USA

Introduction

MX proteins are integral components of the innate

immune response. Their expression increases in

response to type I interferon (IFN).1 Two or three MX

proteins are expressed in response to IFN, and MX

proteins have been described in all species studied to

date, from humans to an invertebrate, the disk abo-

lone.2,3 MX proteins block replication of a wide range

of viruses in all species tested.1 The general mecha-

nism utilized by MX proteins is largely dependent

upon its cellular localization. For example, in rodents,

MX1 is localized to the nucleus and inhibits primary

transcription of influenza A virus, whereas human

MXA binds viral nucleocapsid protein and interferes

with viral genome replication (Bunya viruses) or

Keywords

Interferon, mitosis, myxovirus resistance

protein, protein interactions, trafficking

Correspondence

Troy L. Ott, The Pennsylvania State University,

324 Henning, University Park, PA, 16802, USA.

E-mail: [email protected]

Submitted February 8, 2010;

accepted May 3, 2010.

Citation

Racicot K, Ott T. The myxovirus resistance

protein, MX1, interacts with tubulin beta in

uterine glandular epithelial cells. Am J Reprod

Immunol 2011; 65: 44–53

doi:10.1111/j.1600-0897.2010.00885.x

Problem

MX proteins are upregulated during viral infection and during early

pregnancy in ruminants by type I interferons and exhibit a number of

characteristics that would suggest they function in basic cellular pro-

cesses. We hypothesize MX1 plays a role in intracellular trafficking and

secretion, and the objective of this study was to identify cellular proteins

that interact with MX1.

Method of study

Western blot and polymerase chain reaction were used to detect expres-

sion of MX1 and endogenous interferon (IFN), respectively. Affinity

chromatography and mass spectrometry identified proteins that inter-

acted with MX1. These interactions were confirmed and characterized

using co-immunoprecipitation and co-immunofluorescence.

Results

MX1 was expressed in ovine glandular epithelial cells without IFN treat-

ment, while another interferon-stimulated gene, ISG15, was not. MX1

was shown to interact with tubulin beta (TUBB) during interphase and

mitosis and nocodazole disrupted this interaction.

Conclusion

We propose that by tethering to TUBB, MX1 could be transporting pro-

teins or vesicles throughout the cell, such as those destined for secretion

or required for mitosis. This would be a novel role for an ISG, but one

that is consistent with the enhanced secretion occurring in the uterus

during early pregnancy in ruminants in response to conceptus-produced

IFN-tau.

ORIGINAL ARTICLE

American Journal of Reproductive Immunology 65 (2011) 44–53

44 ª 2010 John Wiley & Sons A/S

inhibits transport of viral nucleocapsids into the

nucleus, where they replicate (Thogoto virus).4

There has been little research on the function of

MX in the absence of virus or IFN. This is not sur-

prising considering the high level of MX expression

in response to IFN and its clear role in the antiviral

response. Despite this, MX proteins exhibit a num-

ber of characteristics that would indicate they have

a basic cellular function. For example, there are

structural similarities between MX and dynamin, a

protein thought to be involved in a variety of cel-

lular processes. Results from several laboratories

demonstrated that human MXA self-assembles into

high-order oligomers similar to conventional dyn-

amin.5,6 MXA was also shown to bind and tubulate

lipid droplets despite the fact that it lacks the

pleckstrin homology domain that is responsible for

lipid interactions of dynamins.6 The same research

also showed MXA colocalized with distinct subcom-

partments of the smooth endoplasmic reticulum; an

area thought not to be associated with viral replica-

tion.6 These results indicate that MXA could play a

role in membrane remodeling or trafficking in the

cell independent of its antiviral activity. Indeed,

others have demonstrated that human MX proteins

have roles in endocytic trafficking. Jatiani and Mit-

tal7 over-expressed human MXA in Chinese ham-

ster ovary cells and observed a transient

perturbation of trafficking of internalized transfer-

rin. The same article reported the co-immunopre-

cipitation of MXA with dynamin, lending further

support for a role for MX in intracellular traffick-

ing.7

Our laboratory has also provided evidence, indi-

cating MX1 has a role in cellular processes not tied

to viral infection. For example, it was shown that

MX1 expression in the endometrium and peripheral

blood leukocytes of sheep increased in response to

the presence of a conceptus (i.e. embryo and associ-

ated membranes).8–10 In addition, it was demon-

strated that MX1 was a secreted protein even

though it lacks a canonical secretion signal sequence.

When MX1 expression was blocked, secretion of

another protein, ISG15, was also reduced, suggesting

that MX1 may regulate some aspect of the secretory

process. It was later determined that MX1 was regu-

lating one of the, largely uncharacterized, unconven-

tional secretory pathways.11 We hypothesized that

MX1 has cellular roles in addition to the antiviral

response. The objective of this study was to identify

the cellular proteins that interact with MX1 and

characterize those interactions in the presence and

absence of type I IFN.

Materials and methods

Cell Culture and Immunofluorescence

Immortalized ovine glandular epithelial (GE) cells12

were cultured in 12-well plates on coverslips for

24 hr in Dulbecco’s modified Eagle’s medium

(15.63 g ⁄ L DMEM; Sigma, St Louis, MO, USA) with

10% fetal bovine serum (FBS) under 5% CO2 at

38.5�C. Wells were treated with nocodazole, a chem-

ical that disrupts the polymerization of microtubules

(Sigma) or vehicle (dimethyl sulphoxide) in the

presence or absence of IFN-tau (10,000 U ⁄ mL).

Briefly, following 24 hr of culture, cells (approxi-

mately 70% confluent) were cultured for 24 hr in

DMEM with 10% FBS in the presence or absence of

IFN-tau (10,000 U ⁄ mL; provided by Fuller W. Bazer,

Texas A&M University). Approximately 30 min prior

to harvest, 50 lm nocodazole or vehicle was added

to appropriate wells. The cells were rinsed with

1 mL of phosphate-buffered saline (PBS: 137 mm

NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, 1.4 mm

KH2PO4), and 1 mL of 3% formaldehyde (Poly-

sciences Inc., Warrington, PA) was added for

10 min. Next, 1 mL of permeabilization reagent

[PBS, 0.05% Triton X-100, 2% bovine serum albu-

min (BSA)] was added, and cells were rocked over-

night at 4�C. A 1:1000 dilution of an amino terminal

rabbit polyclonal ovine MX1 antiserum (90618 bleed

no. 2, prepared by Multiple Peptide Systems, San

Diego, CA, USA) and 1:1000 dilution of mouse

TUBB antibody (Abcam, Cambridge, MA, USA) were

added and rocked overnight at 4�C. Negative con-

trols included pre-immune rabbit serum at a dilution

of 1:1000 and mouse IgG, as well as exclusion of

primary antibody. After incubation with primary

antibodies, cells were washed and the fluorescent-

labeled secondary antibodies were added at a 1:2000

dilution (goat anti-rabbit Alexa 488, or goat anti-

mouse Alexa 555; Molecular Probes Inc, Eugene,

OR, USA.) separately for 1 hr at ambient tempera-

ture in the dark. Nuclei were stained with Hoechst

reagent (Sigma) for 5 min then each cover slip was

mounted on a glass slide. Immunofluorescence (IF)

signals were detected with a Zeiss Olympus Axioskop

DP71 microscope with DP controller software. All IF

data are representative from three independent

experiments.

MX1 INTERACTS WITH TUBULIN BETA


ª 2010 John Wiley & Sons A/S 45

RNA Extraction, cDNA Synthesis and Reverse

Transcription Polymerase Chain Reaction (PCR)

Ribonucleic acid was extracted and purified using

Trizol (Invitrogen, Carlsbad, CA, USA) according to

the manufacturer’s recommendations. For cDNA

synthesis, 5 lg of total RNA was incubated with

1 lL of RQ1 DNAse (Promega, Madison, WI, USA)

and 1 lL of Strata Script RT buffer (Stratagene, La

Jolla, CA, USA) in 8 lL total volume at 37�C for

30 min. One microliter of DNAse stop solution (Pro-

mega) was added, and samples were incubated at

65�C for 10 min. Three microliters of random prim-

ers (Invitrogen) and 27 lL of nuclease-free water

were added to each sample, and samples were incu-

bated at 65�C for 5 min followed by 25�C for

10 min. Nine microliters of a master mix containing

5 lL of Strata Script RT buffer (Stratagene), 1 lL of

RNAse inhibitor (Invitrogen), 2 lL of Strata Script

RT (Stratagene) was added to each sample, followed

by incubation at 42�C for 2 hr and 90�C for 5 min.

Samples were stored at )20�C. For PCR, 5 lL of

cDNA was added to 5 lL Advantage 2 PCR buffer

(Clontech, Mountain View, CA, USA), 1 lL dNTP

(Clontech), 1 lL of 1 nm forward and reverse prim-

ers specific for IFN-alpha and beta (Invitrogen) and

1 lL Advantage 2 PCR DNA polymerase (Clontech).

The reaction was placed in a thermocycler and PCR

was performed using the following protocol: 1) 30 s

at 95�C; 2) 30 s at 95�C; 3) 30 s at 68�C; 4) 10 min

at 72�C, repeat steps 2–4 40 times; 5) 10 min at

72�C. Reactions were then separated on a 1% aga-

rose gel supplemented with ethidium bromide, and

visualized using a Chemidoc XRS system (Biorad,

Hercules, CA, USA).

Expression and Purification of Recombinant

Protein

The MX1 coding sequence was cloned into the pTr-

cHis TOPO TA expression plasmid (Invitrogen). Bac-

teria were transformed (One Shot chemically

competent cells; Invitrogen) and colonies were

screened for positive clones using ampicillin. Positive

clones were then sequenced and determined to be

MX1 using the Basic Local Alignment Sequence Tool

from the National Center for Biotechnology Informa-

tion. Cells were then grown until they reached the

log phase of the growth curve and were induced

with IPTG. Uninduced, or basal, expression was

detected and is common when using derivatives of

the lac promoter, because the transcriptional activa-

tor protein, CAP, can bind upstream of the trc pro-

moter and activate transcription (according to

manufacturer’s literature). Uninduced expression

resulted in less total protein but greater expression

of full-length MX1, and therefore uninduced condi-

tions were used for the remaining experiments.

Protein purification was performed according to

manufacturer’s instructions. Briefly, bacteria con-

taining recombinant protein were lyzed with lyso-

zyme, sonicated (medium intensity, 3- to 5-second

pulses) and incubated with a Ni-NTA column (1 hr,

room temperature) (ProBond; Invitrogen). Bound

proteins were sequentially washed with 10, 20, and

80 mm imidizole and were then eluted with 250 mm

imidizole. Proteins were then concentrated in

50-kDa size-exclusion columns (Amicon, Millipore,

Billerica, MA) to remove low molecular weight

contaminants.

Development of Affinity Column and Mass

Spectrometry

Recombinant MX1 was used as ‘bait’ to identify pro-

tein interactions in GE cell lysates. The column was

made using the purification procedure described ear-

lier, excluding the final elution of 250 mm imidizole.

The column was incubated with IFN-tau-treated GE

cell lysates, washed and eluted with 250 mm imidiz-

ole to isolate interacting proteins. A column bound

with bacterial lysates lacking rMX1 was also incu-

bated with GE lysates as a negative control and was

used to identify non-specific protein bands. The

eluted proteins were dissolved in sample buffer

[stock: 7.5 mL deionized H2O, 760 mg Tris base, 2 g

sodium dodecyl sulfate (SDS), 10 mL glycerol, pH to

6.8, 5 mL 2-bmercaptoethanol, 300 lL bromphenol

blue], and proteins were separated on a 12% SDS–

polyacrylamide gel with 6% stacking gel in electrode

buffer (stock: 30.3 g Tris base, 144.2 g glycine, 10 g

SDS, pH to 8.3, add deionized H2O to 1.0 L) at a

constant current 70 mAmp for approximately 2 hr.

Gels were stained with Commassie blue protein stain

(Biorad), destained and candidate bands were

excised for mass spectrometry.

Excised gel pieces were washed with 25 mm

ammonium bicarbonate, dehydrated with 50% ace-

tonitrile, and dried under a vacuum. Samples were

incubated overnight at 37�C with trypsin

(12.5 ng ⁄ lL in 25 mm ammonium bicarbonate). Pep-

tides were then extracted twice with 5% formic acid

RACICOT AND OTT



and re-dried. Tryptic digests were analyzed by capil-

lary liquid chromatography–nanoelectrospray ioniza-

tion-tandem mass spectrometry. A Waters Q-Tof

Premier mass spectrometer coupled with a Waters

Cap LC high-performance liquid chromatography

unit (Waters Co, Milford, MA, USA) was used for

the analysis. To identify proteins, MS ⁄ MS ion

searches were performed on the processed spectra

against a locally maintained copy of the Swiss pro-

tein bank using MASCOT Daemon and search

engine (Matrix Science, Inc, Boston Mass). MASCOT

uses a probability-based algorithm to determine the

probability of a peptide match being random. A

number of factors are used to determine the proba-

bility-based ion score, which are then used to

determine the statistical significance. The ion score is

-10*log(P), where P is the probability that the match

is random. An ion score >50 indicates extensive

homology (P < 0.05). The ion score for TUBB was

450 and was determined to be significant (P < 0.05).

Co-Immunoprecipitation and Western Blot

Analysis

Glandular epithelial cells were cultured as described

previously, treated with IFN-tau for 24 hr, and har-

vested using Mammalian Protein Extraction Reagent

(MPER; Pierce, Rockford, IL, USA). These cell lysates

were then used for immunoprecipitation. The Sieze

X co-immunoprecipitation (co-IP) kit (Pierce) was

used for co-IP experiments. The co-IP was performed

according to the manufacturer’s directions with only

minor modifications. Briefly, 200 lg of TUBB anti-

body (Abcam) or mouse IgG control antibody

(Pierce) were bound to a Protein G spin column and

incubated with IFN-tau-treated GE cell lysate for

2 hr at ambient temperature. The column was

washed several times, and then bound protein was

eluted with the provided elution buffer (Pierce). The

co-IP was repeated in two independent experiments.

The same protocol was used for the MX1-IP experi-

ment, but with 200 lg of monoclonal MX1 antibody

(clone 29C) and mouse IgM as a negative isotype

control. Protein concentrations were determined for

all samples collected during the co-IP using a BSA

protein assay (Pierce), and 20 lg total protein for

each sample was used for Western blotting. Samples

were dissolved in sample buffer and proteins were

separated on a 12% SDS-PAGE gel with 6% stacking

gel in electrode buffer at a constant current

70 mAmp for approximately 2 hr. The proteins were

transferred to nitrocellulose membranes (Protran;

Schleicher & Schuell, Keene, NH, USA) in a Mini-

Protean II Cell apparatus (Bio-Rad) at a constant

70 V for 90 min with an ice pack. Non-fat milk

(5%) was used as a blocker (Fisher Scientific, Pitts-

burgh, PA, USA), and immunoblotting was per-

formed with a 1:1000 dilution of an amino terminal

rabbit polyclonal ovine MX1 antiserum (90618 bleed

no. 3) or 1:1000 dilution of rabbit TUBB-HRP-conju-

gated antibody (Abcam) with 2% BSA at 4�C over-

night. Membranes were washed and a 1:200,000

dilution of goat anti-rabbit IgG-horseradish peroxi-

dase conjugate (Pierce) was added as appropriate.

Membranes were rocked at room temperature for

1 hr, washed and incubated with Super Signal�

West Femto Maximum Sensitivity Substrate chemi-

luminescence kit (Pierce) to detect immunoreactive

proteins. Detection of the chemiluminescence signal

was performed and quantified using the Bio-Rad

Chemidoc-XRS Multiimager system and Quantity

One software (Bio-Rad).

Results

MX1 is Expressed in GE Cells in the Absence of

Virus or IFN Treatment

Western blot analysis demonstrated that MX1 was

expressed in ovine GE cells in the absence of IFN or

virus treatment. However, as expected, MX1 concen-

trations were much less than in cells treated with

IFN-tau (Fig. 1). It was then determined whether

this expression was a result of endogenous expres-

sion of IFN by these cells. Polymerase chain reaction

(PCR) was performed using primers specific for IFN-

alpha and IFN-beta. Results showed that IFN-beta,

but not IFN-alpha, mRNA was present in untreated

GE cells (Fig. 2). Estrous cycle day 3 (D3) T lympho-

cytes (T cells), day 11 peripheral blood leukocytes

(D11 PBL), day 11 T cells (D11 T cells), day 19 T

cells (D19 T cells) and ewe peripheral blood leuko-

cytes (PBL) were all positive controls for IFN-alpha.

The different days were utilized to ensure at least

one would be positive for IFN-alpha. Uterine stromal

cell lysate was used as a positive control for IFN-

beta, and negative controls were incubated without

cDNA. These results can be interpreted to indicate

that MX1 is expressed as a result of low levels of

IFN-beta produced by oGE cells. To determine

whether another ISG was induced by low levels of

endogenous IFN-beta, ISG15 concentrations were




examined in untreated cells (Fig. 1). Interestingly,

there was no detectable ISG15 protein using Western

blot analysis.

Recombinant Protein Over Expression and

Purification

Bacteria were transformed with the pTrcHis-MX1

construct, and cells were either induced with IPTG

or not induced. Cultures were sampled every 30 min

for 2 hr to evaluate expression of recombinant pro-

tein. Western blot analysis using antibodies specific

for both MX1 and a recombinant protein-specific tag

(anti-Xpress) were used to evaluate protein expres-

sion (Fig. 3). Recombinant protein was present in

samples at 0 hr (time of induction) and expression

increased at every time point through 2 hr. Recom-

binant MX1 was present in both induced and unin-

duced cells, but was not present in the negative

controls that consisted of bacteria transformed with

pTrcHis plasmid lacking the MX1 insert or positive

controls that over-expressed LacZ. Inducing rMX1

expression with IPTG caused high levels of expres-

sion accompanied by apparent degradation of some

of the protein. Because of this, uninduced conditions

were utilized to obtain the greatest amount of full-

length protein. When rMX1 was purified using a

nickel column, small quantities of recombinant pro-

tein were observed in the lysates before incubation

with nickel column, while there was very little pro-

tein apparent in samples after elution through the

column and in the initial wash. The washes with

greater concentrations of imidizole (wash 2) eluted

some rMX1, while the elution with the greatest con-

centration (250 mm imidizole) showed one distinct

band migrating at approximately 75 kDa, consistent

with MX1. Using these conditions, rMX1 was puri-

fied and again analyzed using SDS-PAGE (Fig. 4).

The Commassie-stained gel indicated that rMX1 was

approximately 90% pure and present at mg ⁄ mL

concentrations (Fig. 4).

MX1 Interacts with TUBB

An affinity column prepared with rMX1 was incu-

bated with GE cell lysates, followed by elution of the

Fig. 2 PCR analysis of IFN-alpha and IFN-beta in GE cells lacking exog-

enous IFN treatment. Immune cells were collected from different days

of the bovine estrous cycle including; Day 3 T cells (D3 T cells), Day

11 peripheral blood leukocytes (D11 PBL), Day 11 T cells (D11 T cells),

Day 19 T cells (D19 T cells) and also non-pregnant ewe peripheral

blood leukocytes (PBL), all positive controls for IFN-alpha. Uterine stro-

mal cells were a positive control for IFN-beta, no template was the

negative control lacking cDNA.

Xpress Antibody

MW Neg 0 30I 30N 1I 1N 2I 2N

MX1 Antibody

MW + Neg 0 30I 30N 1I 1N 2I 2N

Fig. 3 Western blot analysis showing rMX1 expression. Western blot

using antibodies against the NH3 terminus tag (Xpress; top) and MX1

(bottom) showing expression from 30 min to 2 hr after induction. I,

induced with IPTG. N, uninduced expression. Neg, negative control

consisting of bacteria transformed with an empty vector. Positive con-

trol (+) was GE cell lysates treated with IFNtau.

MX1 MW NT IFN

ISG15 MW NT IFN

Fig. 1 Western blot analysis of MX1 and ISG15 with and without IFN-

tau. MW, molecular weight marker, NT, no interferon, IFN, interferon-

tau treated.

RACICOT AND OTT



rMX1 and interacting proteins. A column incubated

with bacterial lysates lacking rMX1 was also incu-

bated with GE lysates as a negative control (data not

shown), and Commassie staining was used to iden-

tify non-specific protein bands. One candidate inter-

acting protein migrated at approximately 50 kDa.

This band was excised, submitted for analysis by

mass spectrometry and was determined to be TUBB

(P < 0.05) (Fig. 5). Many of the other bands

observed were also detected in the column lacking

rMX1 and were therefore considered non-specific

proteins interacting with the nickel beads.

The interaction between MX1 and TUBB was con-

firmed using co-immunoprecipitation (co-IP). Co-IP

was performed using an antibody-specific for TUBB

and non-specific purified mouse IgG (negative con-

trol), or a monoclonal antibody specific for ovine

MX1 or non-specific purified mouse IgM (negative

isotype control). Fig. 6 shows the results of co-IP

with TUBB antibody (top panel) and with the MX1

antibody (bottom panel). Samples from multiple

steps of the procedure were analyzed using SDS-

PAGE and then Western blot analysis to determine

whether each antibody precipitated its cognate pro-

tein and the putative interacting protein. Cellular

lysates from before and after incubation with the

antibody, the washes from the column that included

non-specific proteins removed before elution and

the final elutions were analyzed. When the protein–

antibody complexes were eluted from the TUBB-spe-

cific IP, both TUBB and MX1 were present in the

elution as demonstrated by Western blot (Fig. 6, lane

4). Supporting those results, when the reciprocal

Purification steps MW Neg B A W1 W2 E

Large scale purification

MX1 (75 kDa)

MX1 (75 kDa)

(a)

(b)

Fig. 4 Purification of rMX1 from bacteria. (a) SDS-PAGE gels were

commassie stained and destained to show total protein in multiple

steps of purification. Neg, empty expression plasmid. B, lysate before

purification. A, lysate after purification. W1, W2, washes. E, eluted pro-

tein. (b) Large scale purification and SDS-PAGE analysis shows purity

of recombinant MX1.

Fig. 5 Identification of Tubulin beta (TUBB) following affinity purifica-

tion using mass spectrometry. Protein band was excised from gel

after affinity chromatography was used to isolate proteins that interact

with MX1. Proteins were removed from the gel, trypsinized, and iden-

tified using mass spectrometry.

Fig. 6 Western blot (WB) analysis of reciprocal MX1 and TUBB Co-

Immunoprecipitation (IP) Experiments. IP with TUBB antibody and con-

trol non-specific IgG (top two panels) and IP with MX1 antibody and

IgM isotype negative control (bottom two panels). Following each IP

experiment, proteins were detected on WB using MX1 and TUBB anti-

bodies. Lanes 1&5; GE cell lysate before IP. Lanes 2&6; lysates after IP

(i.e. flow through after incubation with antibody). Lanes 3&7; wash

(non-specific proteins washed off column before elution). Lanes 4&8;

elution of co-IP. Note the presence of bands in lane 4 that are absent

from lane 8, indicating specific reciprocal co-IP.




experiment was performed using an MX1-specific

antibody for IP, TUBB and MX1 eluted together,

confirming that they interact in IFN-treated oGE

cells (Fig. 6). TUBB was absent in both the mouse

IgG and IgM control co-IP elutions, while MX1 was

completely absent in the IgM control with very

slight staining in the IgG control (lane 8, compared

to lane 4). This staining is still much less than the IP

with the MX1 antibody, indicating that at least some

of the protein is retained on the column because of

the MX1 antibody (i.e. is specific). The lower molec-

ular weight band in lane 8 is thought to be non-spe-

cific (possibly IgG eluted from the column). These

results provide additional support for interaction

between MX1 and TUBB that was indicated by the

affinity chromatography and mass spectrometry

results.

Characterization of MX1-TUBB Interaction in GE

Cells During Interphase

To further characterize the interaction between

MX1 and TUBB, GE cells were grown on coverslips

and treated with IFN-tau or vehicle, and prepared

for immunofluorescence (IF). MX1 and TUBB were

identified by co-incubation with fluorescently

labeled antibodies and visualized using fluorescence

microscopy. In IFN-tau-treated cells, MX1 was

highly expressed and was localized throughout the

cell in a ‘net-like’ pattern (Fig. 7). While a portion

of MX1 co-localized with TUBB throughout the

cell, there were areas in which no co-localization

was observed (see Fig. 7). When the microtubule

network was disrupted by treatment with nocodaz-

ole, the staining pattern of MX1 became more scat-

tered and punctate, quite different from the

staining pattern in cells lacking nocodazole (Fig. 7).

In GE cells lacking IFN treatment, staining was

punctate, forming numerous small half-ring pat-

terns, and was more concentrated near the

nucleus, where it co-localized with TUBB (Fig. 7).

The ring-like pattern was small and cup-shaped

and was evident throughout the cell. Interestingly,

when cells not treated with IFN-tau were treated

with nocodazole, MX1 expression localized to a

perinuclear ring with only a small amount of MX1

protein remaining scattered throughout the cell

(Fig. 7). Nocodazole treatments efficiently disassoci-

ated the microtubule networks, as demonstrated by

lack of staining for TUBB in both IFN and

untreated GE cells (Fig. 7).

Characterization of MX1–TUBB Interaction in GE

Cells During Mitosis

To further characterize the interaction between MX1

and TUBB, cells were synchronized in their cell cycle

and prepared for IF as described earlier. Regardless

of IFN treatment, MX1 and TUBB co-localized dur-

ing mitosis (Fig. 8). The co-localization was most

apparent when there were distinct mitotic spindles

(during metaphase and anaphase). During this time,

localization of both MX1 and TUBB were very simi-

lar, which was apparent without merging the two

fluorescent labels (Fig. 8). When cells were treated

with nocodazole, the pattern of staining of both

MX1 and TUBB changed dramatically, and mitosis

did not occur because of the inability of mitotic spin-

dles to form (data not shown).

Discussion

It is widely accepted that MX proteins are only

expressed in the presence of IFN or viral infection.

This is understandable, considering their robust

upregulation during viral infections and IFN secre-

tion.1 Here, we used an immortalized uterine epithe-

lial cell line to identify proteins that interact with

MX1 in secretory epithelial cells.12 In preliminary

experiments, it was shown that MX1 was expressed

in GE cells in the absence of exogenous virus or IFN.

This finding was contrary to previous reports con-

cluding that human MXA was absent in untreated

cells.1 Interestingly, Ott et al.8 showed that MX1 was

expressed in the uterus of sheep during the estrous

cycle, in the absence of IFN produced by the early

embryo, and appeared to be regulated, at least in

part, by progesterone. In that study, they were not

able to rule out that basal levels of IFN were

expressed locally in the endometrium during the

estrous cycle. To determine whether the expression

of MX1 in vitro was as a result of endogenous

expression of IFN, PCR was used to assay for the

presence of IFN-alpha and IFN-beta transcripts.

Indeed, IFN-beta, but not IFN-alpha mRNA, was

present in GE cells. Therefore, endogenous IFN

expression could be responsible for low-level expres-

sion of MX1 in untreated cells, although other fac-

tors cannot be ruled out. Another classical

interferon-stimulated gene, ISG15, was not detect-

able in the same samples. This could indicate that

there are other factors that are specifically upregu-

lating MX1. MX1 has a number of transcription

RACICOT AND OTT



factor–binding sites in its promoter, including sites

for NF-jB, AP-1 and SP-1,13 all of which could be

playing a role in MX1 regulation. These observations

need to be further investigated using a more sensi-

tive assay like qPCR, because it cannot be ruled out

that the antibody for ISG15 is less sensitive than the

MX1 antibody, and it may simply not be possible

to detect the lower concentrations of ISG15 in

untreated cells.

To understand how MX1 functions in epithelial

cells that have not been infected with virus, affinity

chromatography was used to identify proteins that

GE Cell, No IFN

GE Cells, No IFN, nocodazole treated

MX1 TUBB Overlay

(a)

(b)GE Cells with IFN

GE Cells with IFN and nocodazole

MX1 TUBB Overlay

Fig. 7 MX1 and Tubulin beta (TUBB) protein

expression in glandular epithelial (GE) cells

during interphase, with and without nocodaz-

ole, in the absence (a) and presence of IFN

(b).




interact with MX1. Using a column enriched in

rMX1, interactions that are weak or rare have an

increased probability of being detected compared to

other techniques. Unfortunately, non-specific inter-

actions may also be detected; therefore, other tech-

niques are needed to validate proteins discovered

using this approach. Here, TUBB was found to inter-

act with MX1 in GE cells, and this interaction was

confirmed using reciprocal co-IP and dual labeled

immunofluorescence microscopy. Co-immunofluo-

rescence was then used to further characterize the

interaction between TUBB and MX1. Networks of

microtubules are present throughout the cell and

serve as highways for transport of molecules and

vesicles, as well as providing structural support for

the cell. When cells were treated with IFN and

stained during interphase of the cell cycle, MX1 was

dispersed throughout the cytoplasm along micro-

tubules. Interestingly, in cells that were not treated

with IFN, MX1 was localized throughout the cell in

a punctate pattern and formed small ring-like struc-

tures that did not appear to be associated with

microtubules (Fig. 7). Presently, there is not enough

information to determine the purpose of these for-

mations, but it is tempting to speculate that these

may be endosomal vesicles in some step of the secre-

tory pathway. Our working hypothesis is that MX1

regulates secretion of unconventionally secreted pro-

teins, and in these cells MX1 could be transporting

proteins or vesicles along the microtubule networks.

It was previously shown that MX1 was itself secreted

via an unconventional secretory pathway and regu-

lated the secretion of another unconventionally

secreted protein, ISG15 in IFN-treated GE cells.11

This would be vital for the ruminant (from which

the cell line is derived) during early pregnancy

because the embryo spends several days in the

uterus prior to firm attachment and placentation

and requires uterine secretions to survive.14 Interest-

ingly, the ruminant conceptus secretes a type I IFN

that maintains pregnancy and induces uterine secre-

tions,15 further supporting a potential role for MX1

as a regulator of secretion.

Because the spindle fibers responsible for binding

chromosomes during metaphase and anaphase are

also composed of TUBB, cells were synchronized and

GE Cells without IFN

GE Cells with IFN

GE Cells without IFN

MX1 TUBB Overlay

Fig. 8 MX1 and Tubulin beta (TUBB) protein

expression in glandular epithelial (GE) cells

during mitosis with and without IFN.

RACICOT AND OTT



MX1 and TUBB were identified using fluorescently

labeled antibodies during mitosis. Interestingly, MX1

was highly co-localized with TUBB in cells undergo-

ing mitosis. It is not known whether the MX1 that

was previously bound to the microtubule network

was maintained and concentrated during mitosis or

whether MX1 specifically bound to spindle fibers at

the time of assembly. If MX1 has a role in protein

trafficking, it could be transporting proteins or vesicles

along spindle fibers that are vital to mitosis or cytoki-

nesis. The interaction detected during mitosis was not

different between IFN-treated and non-treated cells.

Therefore, it appears that the role of MX1 in the

mechanism of cell division and cytokinesis takes pre-

cedence over IFN-induced roles or there is simply

enough MX1 expressed to perform the dual functions.

Conclusion

MX1 was expressed in epithelial cells in the absence

of exogenous IFN or virus, probably under the regu-

lation of endogenously produced IFN-beta. Our

working hypothesis is that by tethering to TUBB,

MX1 could be transporting proteins or vesicles

throughout the cell, such as those destined for secre-

tion or required for the process of mitosis. This

would be a novel role for an ISG, but one that is

consistent with the enhanced secretion that occurs

in the uterus during early pregnancy in ruminants.

Ruminants could have evolved an IFN as an

embryo-derived pregnancy signal to take advantage

of a protein (or proteins) that would support uterine

secretions that are required for embryo survival. This

may be particularly important in these species

because ruminant conceptuses spend a relatively

long period entirely reliant on histotrophic nutrition

supplied by uterine glands compared to rodents or

humans. Besides this putative role during early preg-

nancy, MX1 could also be regulating secretion in a

variety of un-induced epithelial cells.

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