1 Carmen M. Pérez-Ferreiro§, Carlos M. Luque+ and Isabel Correas

1

4.1R PROTEINS ASSOCIATE WITH INTERPHASE MICROTUBULES IN HUMAN T

CELLS. A 4.1R CONSTITUTIVE REGION IS INVOLVED IN TUBULIN-BINDING

Carmen M. Pérez-Ferreiro§, Carlos M. Luque+ and Isabel Correas#

Centro de Biología Molecular “Severo Ochoa” (CSIC / UAM).

Departamento de Biología Molecular. Facultad de Ciencias.

Universidad Autónoma de Madrid.

E-28049. Madrid. Spain

#To whom correspondence should be addressed

telephone number +34 91 397 8044

fax number +34 91 397 8087

e-mail [email protected]

September 25, 2001

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 28, 2001 as Manuscript M107369200 by guest on February 3, 2018

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Association of 4.1R proteins with T cell microtubules

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SUMMARY

Red blood cell protein 4.1 (4.1R) is an 80 kDa protein that stabilizes the

spectrin-actin network and anchors it to the plasma membrane. To contribute to

the characterization of functional roles and partners of specific nonerythroid

4.1R isoforms, we analyzed 4.1R in human T cells and found that endogenous

4.1R was distributed to the microtubule network. Transfection experiments of T

cell 4.1R cDNAs in conjunction with confocal microscopy analysis revealed the

colocalization of exogenous 4.1R isoforms with the tubulin skeleton.

Biochemical analyses using taxol-polymerized microtubules from stably

transfected T cells confirmed the association of the exogenous 4.1R proteins

with microtubules. Consistent with this, endogenous 4.1R immunoreactive

proteins were also detected in the microtubule-containing fraction. In vitro

binding assays using GST-4.1R fusion proteins showed that a constitutive

domain of the 4.1R molecule, one which is therefore present in all 4.1R

isoforms, is responsible for the association with tubulin. A 22-amino acid

sequence comprised in this domain and containing heptad repeats of leucine

residues was essential for tubulin-binding. Furthermore, ectopic expression of

4.1R in COS-7 cells provoked microtubule disorganization. Our results suggest

an involvement of 4.1R in interphase microtubule architecture and support the

hypothesis that some 4.1R functional activities are cell-type regulated.


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INTRODUCTION

Red blood cell protein 4.1, 4.1R or 4.1R80, was identified as an 80-kDa

multifunctional protein of the membrane skeleton of human erythrocytes. In

these cells, protein 4.1R stabilizes the spectrin-actin network and anchors it to

the overlying lipid bilayer through interactions with cytoplasmic domains of

transmembrane proteins (reviewed in Ref. 1). The formation of the spectrin-

actin-4.1R ternary complex is essential for the maintenance of normal

erythrocyte morphology and membrane mechanical strength as alterations in

the spectrin-actin binding site of 4.1R, located at the C-terminal region of the

molecule (2-5) are associated with congenital hemolytic anemias (6). Protein

4.1R also plays an important role in regulating the glycophorin C-4.1R-p55

ternary complex in the erythrocyte membrane (7).

Many immunological studies have shown that 4.1R protein is more

complex in nonerythroid cells. Thus, 4.1R-immunoreactive polypeptides ranging

in size from 30 to 210 kDa have been detected in different tissue and cell types

(8,9) and 4.1R epitopes have been observed at many different sites, including

stress fibers (10), centrosomes (11) and the nucleus (12-15). The nuclear

localization of specific isoforms of 4.1R has recently been confirmed by

transfection experiments of 4.1R cDNAs isolated from erythroid (16) and

nonerythroid human cells (17-19).

The prototypical erythroid protein 4.1R80 is, therefore, only one of many

isoforms that are generated by a single gene, mainly by extensive alternative

splicing of the 4.1R pre-mRNA (20-23). 4.1R80 protein is produced when 17

nucleotides 5´-upstream from exon 2 are spliced out, and translation is initiated

at the downstream start site present in exon 4 (ATG2). The synthesis of


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isoforms, termed 4.1R135, containing up to 209 amino acids to the N-terminus of

erythroid 4.1R80 occurs when the 17-nucleotide sequence containing the

upstream ATG (ATG1) translation initiation codon is included. These isoforms

are predominantly expressed in nonerythroid cells (20,21). A third type of

isoforms, termed 4.1R60, can be produced in erythroid and nonerythroid cells

when both the 17-nucleotide sequence (containing the ATG1) and exon 4

(containing the ATG2) are spliced out, and translation is initiated from a third

translation initiation site (ATG3) present in exon 8 (16,18).

Although the major functions of 4.1R80 protein have been extensively

characterized in mature erythrocytes, the potential roles of 4.1R isoforms in

nucleated cells have only begun to be characterized. It has been reported that

4.1R protein interacts with various splicing factors (15,24); with pICln (25), an

integral chloride channel component which was recently shown to associate

also with spliceosomal proteins (26); with a novel centrosomal protein, termed

CPAP (27); and with the nuclear mitotic apparatus protein (17). All of these

observations indicate that, in nucleated cells, isoforms of 4.1R protein may play

roles in organizing the nuclear architecture and mitotic spindle poles. These

roles were not suspected from the initial studies, given that they were performed

in anucleate, non-dividing, human red blood cells. Interactions of 4.1R with other

proteins have also been reported (28-34), thus suggesting that 4.1R protein may

be involved in many different events in nucleated cells.

In an attempt to characterize further the functional roles and partners of

nonerythroid 4.1R isoforms, we have analyzed 4.1R distribution in human T

cells and observed that both endogenous and exogenous 4.1R proteins

colocalized with the microtubule skeleton. In vivo and in vitro biochemical


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analyses confirmed an association between 4.1R and microtubules. We have

determined that a region conserved in all 4.1R isoforms, previously designated

by us as the ‘core region’ (18), was involved in tubulin binding and that 22 amino

acids containing leucine residues, organized as heptad repeats, were essential

for the interaction. Our results indicate that both ATG1- and ATG2-translated

4.1R isoforms are able to associate with microtubules in interphase human T

cells, suggesting the involvement of 4.1R in the microtubule architecture. The

finding that ectopic expression of 4.1R in COS-7 cells resulted in disorganization

of the microtubule architecture supports the hypothesis that the functional

activity of protein 4.1R depends on the cell type in which the protein is

expressed.


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EXPERIMENTAL PROCEDURES

Cell Culture and Transfection – The cell lines used in this study were

Human T lymphoid Jurkat and CEM cells and fibroblastic COS-7 cells. Jurkat

and CEM cells were grown in tissue culture flasks in RPMI 1640 medium (Life

Technologies, Inc.). COS-7 cells were grown on culture dishes or on glass

coverslips in Dulbecco´s modified Eagle´s medium (Life Technologies, Inc.).

Both media were supplemented with 1% glutamine, 10% (v/v) fetal calf serum

(Gibco), penicillin (50 units/ml), and streptomycin (50 units/ml). Cultures were

maintained at 37ºC under a 5% CO2/95% air humidified atmosphere.

Transfection experiments were performed by electroporation using the Electro

Cell Manipulator 600 (8BTX, San Diego, Ca). Cells were processed 48h after

transfection. The 4.1R cDNAs used for transfections (pSR 4.1R135 16; pCR3.1-

4.1R135 16,19; pCR3.1-4.1R135 4,5,16; pSR 4.1R80 16 and pCR3.1-

4.1R80 16,19), were isolated from MOLT-4 T cells as previously described

(18,19,35).

Recombinant Proteins - GST-4.1R80 16, GST-Cter and GST-core were

constructed by PCR using pSR 4.1R80 16 as template. For GST-4.1R60 16,18

the template pCR3.1-4.1R60 16,18 was used. Appropriate sense and antisense

primers containing the Bgl II and Xho I restriction sites at the 5´ and 3´ ends,

respectively, were used for the amplification reactions. The amplified cDNAs

were inserted into the BamH I and Xho I sites of pGEX-6P1 vector (Amersham

Pharmacia Biotech) in-frame with the GST coding sequence. The GST-core leu

construct with a 66-nucleotide deletion (1403-1469) in exon 10 (GenBankTM

accession number M61733) (22) was obtained by PCR using the GST-core


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region construct as the template and sense and antisense oligonucleotide

primers annealing to the flanking regions of the sequence to be deleted. All

cDNA constructs were sequenced as previously described (19). The GST-fusion

proteins were overexpressed in E.coli BL21 cells and purified by glutathione

affinity chromatography (Amersham Pharmacia Biotech) using standard

protocols. Subsequently the proteins were dialysed against CSF-XB buffer (10

mM K-Hepes, pH 7.7, 50 mM sucrose, 100 mM KCl, 2 mM MgCl2, 0.1 mM

CaCl2, and 5 mM EGTA) (36), frozen in liquid nitrogen and stored at -70ºC.

Antibodies - Anti-c-Myc monoclonal antibody 9E10 (37) was obtained

from the American Type Culture Collection. Anti-4.1R (10b) antibody was an

affinity-purified polyclonal antibody generated as previously described (2). Anti-

4.1R (762) was a polyclonal antibody raised against a synthetic peptide whose

sequence is encoded by exon 2 (35). The anti-centrosome antibody was a

human autoimmune serum that strongly recognizes centrosomes in mammalian

cells (38). Anti- -tubulin antibody DM1A was a monoclonal antibody obtained

from Sigma. Anti-GST antibody was a polyclonal antibody from Sigma.

Fuorescent- and horseradish peroxidase-labeled antibodies were obtained from

Southern Biotechnology Associates, Inc. (Birmingham, AL, USA).

Immunofluorescence and Confocal Microscopy - Human T cells were

incubated in flat-bottomed, 24-well plates in a final volume of 500 l complete

medium on glass coverslips coated with polylysine at 1 mg/ml. Cells were fixed,

permeabilized and blocked as described (19). Cells were incubated with the

appropriate antibodies and processed as reported (13). As 9E10 and DM1A are

both mouse monoclonal antibodies, in Figs. 3 and 8 antibody 10b was used 25-

fold diluted, relative to concentrations used for detection of endogenous protein,


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in order to detect only exogenous epitope-tagged 4.1R proteins (19). In figure

2B cells were fixed and extracted at the same time with 5% formalin (37%

formaldehyde solution, Sigma) and 0.5% Triton X-100 in PBS (phosphate-

buffered saline) for 3 min at room temperature. Images were obtained using a

Bio-Rad Radiance 2000 Confocal Laser microscope or a Zeiss epifluorescence

microscope.

Protein Extractions and Western Blotting - Human T cells were washed

twice with PBS and lysed in Laemmli solubilizing buffer (39). For immunoblot

analysis, protein fractions were separated by SDS/PAGE and transferred to

Immobilon-PVDF (Millipore) in Tris-Borate buffer, pH 8.2. Membranes were

processed and developed as described (13).

Isolation of Human T Cell Microtubules - CEM cells were harvested by

centrifugation and then resuspended in 1/10 of buffer A (0.1 M MES, 0.5 mM

MgCl2, 2 mM EGTA) containing 10 g/ml pepstatin, leupeptin, aprotinin, and 1

mM PMSF. Cells were lysed in 4ºC hypotonic buffer by 25 passages through a

22G1-gauge needle fitted onto a plastic syringe. 10 x buffer A was added to the

extract in order to obtain isotonic buffer A. The lysate was centrifuged at high

speed in a minifuge at 4ºC and processed as described (40). Briefly, the pellet

was discarded and the supernatant was centrifuged at 100,000 x g for 60 min at

4ºC in a Beckman TL-100 Tabletop Ultracentrifuge using a TLA-100.1 fixed-

angle rotor. The pellet, corresponding to the membrane-containing fraction, was

discarded. The supernatant was supplemented with 10 M Taxol and 0.1 mM

GTP and incubated for 35 min at 37ºC. Microtubules were centrifuged through a

15 % sucrose cushion in buffer A containing 5 M Taxol (30,000 x g, 35 min,


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37ºC). The microtubule pellets and supernantants were boiled in Laemmli buffer

and analyzed by Western blotting with either 9E10, 10b or 762 antibodies.

In Vitro Microtubule Binding - PC-Tubulin from bovine brain (41) was

polymerized as described (36), mixed with recombinant proteins in buffer

BRB80 (80 mM KPipes pH 6.8, 1 mM EGTA, 1 mM MgCl2) containing 10 M

Taxol and incubated for 15 min at room temperature. The samples were

centrifuged through a 15 % sucrose cushion in BRB80, as described previously

(36). Equivalent aliquots of supernatants and pellets were analyzed on

Coomassie-stained gels and Western blots probed with anti-GST antibody.


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RESULTS

Immunolocalization of Endogenous 4.1R Proteins in Human T Cells- To

understand how 4.1R is distributed in human T cells we analyzed CEM cells,

fixed in the absence (Fig. 2A) or in the presence (Fig. 2B) of TX-100, by

confocal microscopy. Fig. 2A,a shows a representative image of cells stained

with the anti-4.1R 10b antibody. Diffuse cytoplasmic staining, which was more

concentrated at the pericentrosomal region, and staining of both discrete

cytoplasmic filaments and the plasma membrane was observed. Nuclear

staining (not shown in this confocal plane) was also detected. The cytoplasmic

filaments decorated with antibody 10b corresponded to microtubules, as

revealed by the anti-tubulin DM1A antibody (Fig. 2A, b). Microtubules were also

decorated with the anti-4.1R 762 antibody, which recognizes the extra amino-

terminal region of 4.1R135 isoforms (35) (Fig. 2A, c and d). These results

indicated that the microtubule network of human T cells contains 4.1R epitopes.

The 4.1R staining pattern of CEM cells fixed in the presence of Triton X-

100 is shown in Fig. 2B. The Triton X-100 treatment resulted in the extraction of

most of the 4.1R immunoreactivity; however, nuclear speckles and a bright spot

in the pericentrosomal region were clearly observed in cells stained with the 10b

antibody (Fig. 2B, a). The bright spot was stained with antibodies that recognize

the centrosome (Fig. 2B, b). Antibody 762 did not stain the centrosome (Fig. 2B,

c). These results show that centrosomal 4.1R immunoreactivity is better

detected when cells are fixed in the presence of Triton X-100.

Colocalization of Exogenously Expressed 4.1R Isoforms with

Microtubules of Human T Cells- To identify specific 4.1R isoforms that

colocalize with microtubules, we transfected T cells with different 4.1R cDNAs


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previously isolated by us from human T cells (18,35) and compared the

intracellular distribution of the expressed 4.1R proteins and that of tubulin by

confocal microscopy. Two of the 4.1R cDNAs (4.1R80 16 and 4.1R80 16,19)

encode 4.1R isoforms that are translated from the ATG-2 translation initiation

codon present in exon 4 (Fig. 1B), whereas the other three 4.1R cDNAs

(4.1R135 16; 4.1R135 16,19 and 4.1R135 4,5,16) encode 4.1R isoforms that are

translated from the 5’ upstream-ATG-1 translation initiation site (Fig. 1B). The

only difference between the isoforms 4.1R135 16 and 4.1R135 16,19 and their

respective counterparts 4.1R80 16 and 4.1R80 16,19 is that the first two ones

have the 209-amino acid N-terminal extension. Figure 3 shows confocal

microscopy images of Jurkat T cells transiently expressing the 4.1R isoforms

and double-stained with 10b (10b) and DM1A (DM1A) antibodies. Antibody 10b

was highly diluted to react only with the exogenously expressed 4.1R isoforms

but not with the endogenous 4.1R proteins. All 4.1R isoforms localized to

cytoplasmic filaments (Fig. 3A, a, c and e and Fig. 3B, a and c) that were also

stained by the tubulin-recognizing monoclonal antibody DM1A (Fig. 3A, b, d and

f and Fig. 3B, b and d). The distribution of the exogenously expressed 4.1R

isoforms on the microtubule network resembled that of the endogenous 4.1R

proteins (compare Figs. 2 and 3).

Endogenous and Exogenous 4.1R Proteins Cosedimented with Taxol-

Polymerized Microtubules Isolated from Human T Cells- To determine whether

4.1R and microtubules interacted in vivo we performed biochemical assays

using T cells stably transfected with either 4.1R135 16 or 4.1R80 16 cDNAs.

Taxol-polymerized microtubules were isolated from these cells by centrifugation

through a sucrose cushion and the presence or absence of the expressed 4.1R


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proteins in the pellet fractions was analyzed by Western blot. Control cells,

transfected with an empty plasmid, were processed in parallel.

Expression of 4.1R135 16 and 4.1R80 16 in CEM cells was confirmed by

Western blot analysis revealed with antibody 9E10 (Fig. 4A), as the exogenous

4.1R isoforms were tagged at their C-terminal region with the 9E10 c-Myc

epitope to distinguish them from the endogenous 4.1R proteins (19). Western

blots of equivalent aliquots of microtubule pellets and supernatants isolated from

control and transfected cells were revealed with 9E10 antibody (Fig. 4B).

Isoforms 4.1R135 16 and 4.1R80 16 were present in the microtubule pellets

(Fig. 4B, lanes 3 and 5) suggesting their in vivo association with the tubulin

skeleton.

Duplicates of the microtubule pellet shown in Fig. 4B, lane 1, were

revealed with anti-4.1 antibodies to analyze endogenous 4.1R proteins

cosedimenting in the microtubule pellet (Fig. 4C). Antibody 10b (Fig. 4C, 10b)

detected major bands of approximately 80 kDa and 145 kDa. Antibody 762 (Fig.

4C, 762) reacted with a 145-kDa band. These results and those obtained for

the exogenously expressed 4.1R isoforms indicate that both ATG1- and ATG2-

translated 4.1R isoforms cosediment with microtubules.

The Core Region of Protein 4.1R is Responsible for Tubulin-Binding- To

investigate whether 4.1R80 16 could bind directly to tubulin, the major

component of microtubules, we prepared tubulin depleted in microtubule-

associated proteins (PC-tubulin) and performed in vitro binding assays. Taxol-

polymerized tubulin was incubated with a GST-fusion protein containing

4.1R80 16 (GST-4.1R80 16) and processed as indicated in Experimental

Procedures. Protein GST-4.1R80 16 was detected in the pellet fraction with


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polymerized tubulin (Fig. 5A, lane 1) but not in the supernatant fraction (Fig. 5A,

lane 5). Similar results were confirmed by the blot analysis (Fig. 5B, lanes 1 and

5).

To identify the 4.1R80 16 region interacting with tubulin we fused various

4.1 fragments to GST (Fig. 1C) and assayed in vitro their ability to associate

with PC-tubulin. Figure 5C shows a Coomassie-stained gel of the different GST-

fusion proteins used for the study. Protein GST-Cter (Fig. 5C, lane 3) contained

the carboxy-terminal region of protein 4.1R80 16 (see Fig. 1C) and the GST-

4.1R60 16,18 protein (Fig. 5C, lane 2) contained a short 4.1R isoform translated

from the ATG3 triplet (18) whose sequence is comprised in 4.1R80 16 (see Fig.

1C). Fig. 5A (Coomassie-stained gels) and Fig. 5B (Western blots revealed with

anti-GST) show that GST-4.1R60 16,18 protein was detected in the tubulin

pellets (Fig. 5A,B lanes 2) but not in the supernatants (Fig. 5A,B lanes 6). By

contrast, protein GST-Cter, assayed at two different concentrations, was

observed in the supernatant fractions (Fig. 5A,B lanes 7 and 8). The absence of

GST-Cter protein from the tubulin pellets was confirmed by Western blot

analysis (Fig. 5B, lanes 3 and 4). The fact that GST-Cter did not bind to tubulin

whereas the GST-4.1R60 16,18 and GST-4.1R80 16 proteins did, suggested

that the common central region of the latter proteins, previously designated by

us as ‘the core region’ (18), was responsible for their association with tubulin.

A GST-fusion protein containing the core region (GST-core) was assayed

for its tubulin binding ability. Fig. 6A shows the Coomassie-stained gel of the

pellet and the supernatant fractions and Fig. 6B the Western blot revealed with

anti-GST antibody. Protein GST-core was present in the pellet with tubulin (Fig.

6A,B, lanes 3) and absent in the supernatant (Fig. 6A,B, lanes 6). The fusion


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proteins GST-4.1R80 16 (Fig. 6A,B lanes 1 and 4) and GST-Cter (Fig. 6A,B

lanes 2 and 5) were used as positive and negative controls, respectively. These

results confirmed that the core region, present in all 4.1R isoforms, is

responsible for tubulin binding.

Twenty-two Amino Acids in the 4.1R Core Region are Required for

Tubulin Interaction- Primary sequence analysis of the core region revealed that

the N-terminal region of exon-10-encoded sequences comprised heptad repeats

of leucine residues resembling a putative leucine-zipper motif (Fig. 7A). To

investigate whether this characteristic sequence was involved in tubulin

association, we created a deletion mutant, termed core- leu, that lacked 22

amino acids from the leucine zipper-resembling motif (amino acids boxed in Fig.

7A), fused it to GST (GST-core- leu) (see Fig. 1C) and determined its tubulin-

binding capacity. As expected, GST-core was detected in the pellet fraction (Fig.

7B,C lanes 1) whereas GST-core- leu remained in the supernatant (Fig. 7B,C

lanes 4), indicating that the 22-amino acid stretch containing the heptad repeats

of leucine is essential for tubulin binding.

Exogenous Expression of 4.1R80 16 in COS-7 Cells Leads to Disruption

of the Microtubule Architecture- COS-7 cells were transfected with 4.1R80 16

cDNA to compare the distribution pattern of the expressed 4.1R isoform with

that of tubulin by double-immunofluorescence microscopy (Fig. 8). Isoform

4.1R80 16 did not result to colocalize with microtubules as it did in T cells but

instead led to disorganization of the microtubule network (compare Fig. 8 and

Fig. 3B, a and b). Similar disorganization of the microtubule architecture was

also observed with other 4.1R cDNAs assayed (not shown). A representative


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image of the altered microtubule network showing microtubules that no longer

radiate from a single perinuclear focus is represented in Fig. 8.


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DISCUSSION

The great diversity of 4.1R isoforms present in nonerythroid cells makes

it necessary to assay individual isoforms to specifically assign them cellular

functions. In recent years, much effort has been concentrated on isolating 4.1R

cDNAs from different cell sources for use in the assignment of roles and

partners for specific 4.1R isoforms. In this study we show that naturally

occurring exogenously expressed ATG1- and ATG2-translated 4.1R isoforms,

and also endogenous 4.1R proteins, colocalize with the tubulin cytoskeleton of

interphase human T cells. Cosedimentation of 4.1R proteins with microtubules

of human T cells and direct in vitro binding to tubulin revealed that a common

region present in all 4.1R isoforms, the ‘core region’, is responsible for the

association of 4.1R with tubulin. This is the first demonstration of an association

between 4.1R and interphase microtubules.

It is of particular note that the 22-amino acid sequence required for 4.1R-

tubulin interaction contains heptad repeats of leucine resembling leucine zippers

(see Fig. 7A) but, unlike the latter, it does not adopt an -helix conformation

(42). The crystal structure of the N-terminal 30-kDa domain of 4.1R has been

determined and has the form of a three-lobed cloverleaf (43). The C-terminal

lobe contains two -sheets and ends in an -helix; one of the -sheets contains

the binding site for p55, a palmitoylated peripheral membrane protein belonging

to a membrane-associated guanylate kinase homologue (MAGUK) family of

signaling and cytoskeletal proteins (7,44). The 22 amino acids identified in this

study as being required for 4.1R-tubulin associations would be located on the


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other -sheet of the C-terminal lobe. Removal of the 22 amino acids would

result in an almost complete absence of this -sheet structure.

ERM (ezrin-radixin-moesin) proteins belong to ‘the 4.1R protein family’

and are thought to link actin filaments to the plasma membrane at cortical foci

(45). Ezrin has also been isolated from microtubule-associated protein

preparations from MDCK and A72 cells and the ERM protein merlin contains a

‘cryptic’ microtubule binding site exposed specifically in the activated or ‘open’

ERM conformation (reviewed in Ref. 45) . Thus, it has been suggested that

some ERM proteins may play an additional role in linking microtubules to the

cell cortex, thus having the capacity to associate with the microtubule and actin

cytoskeletons. One of the major functions of erythroid 4.1R is the stabilization of

the spectrin-actin complex through the 10-kDa domain (2-5) encoded by exons

16 and 17. We may speculate that, in nonerythroid cells, 4.1R isoforms

expressing the alternative exon 16 would have the capacity to bind to the actin

and the microtubule cytoskeletons, whereas those 4.1R isoforms lacking exon

16 expression would only retain the ability to bind to the microtubule

cytoskeleton.

It has been indicated that non-erythroid 4.1R isoforms may have different

functional activities, depending on the cell type in which they are expressed

(31). Thus, 4.1R interacts through its spectrin-actin binding domain with a

protein-complex in skeletal muscle (34) that differs from that described in PC12

cells (31), even though the latter type of cell also contains the proteins to which

4.1R binds in skeletal muscle. Concomitantly, we show in this study that

transfection of T cells with T cell-4.1R cDNAs resulted in overexpression of 4.1R

isoforms colocalizing with the microtubule network, whereas ectopic expression


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of 4.1R isoforms in COS-7 cells did not result in 4.1R binding to the

microtubules but instead in the disruption of the microtubule architecture. It is

evident, therefore, that different cell types respond in distinct manners to the

expression of 4.1R, supporting the hypothesis that some functional activities of

protein 4.1R are cell-type regulated. A major difference between T and COS-7

cells is that T cells experiment internal protein rearrangements during

polarization in response to many stimuli (see below). Whether some cell type-

specific partners of 4.1R are required for specialized 4.1R roles remains to be

established.

Polarization is a key feature in the biology of T cells as T lymphocytes

acquire a polarized phenotype after activation, upon interaction with antigen-

presenting cells and during transendothelial migration. To extravasate,

circulating lymphocytes must adopt a polarized flexible form suitable for tissue

invasion. The anterior region of the polarized lymphocyte bears multiple, highly

labile lamellipodia, whereas the posterior part is drawn out into a single slender

appendage called the uropod (reviewed in Ref. 46). Polarization involves a

reorganization of the cytoskeleton, including polymerization and redistribution of

actin and a drastic reconfiguration of the tubulin cytoskeleton which, in

conjunction with the microtubule organizing center, retract into the uropod

lumen, collapsing into a thin, compact sheaf. Microtubule retraction has been

suggested as being a strategy for accelerating extravasation without

disassembling the microtubule-based transport system (47).

The distribution of spectrin has been analyzed during lymphocyte

activation, revealing a rapid reorganization of the protein, whereby the initial

aggregated cytoplasmic structures are translocated to specific areas of the


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plasma membrane (48). Different distributions are observed for proteins of the

ERM family in T lymphocytes induced to polarize by chemokines. Thus, radixin

colocalizes with myosin II in the neck of the uropod, while moesin is

preferentially located at the uropod tip, interacting with the cytoplasmic tail of

ICAM-3, CD43 and CD44 (49). Interactions between 4.1R and CD44 have also

been reported (28). Further studies will be conducted to determine the

behaviour of 4.1R during T cell polarization, which may provide new clues for

understanding the complex cytoskeletal reorganization experienced by this cell

type.

ACKNOWLEDGEMENTS

We are very grateful to Dr. Isabelle Vernos for invaluable help. We also

thank Drs. Jesús Avila and Jorge Domínguez for providing us with PC-tubulin

and anti-centrosome antibody, and Drs. Miguel A. Alonso, Jaime Millán and

Antonio Rodríguez for generous comments. We acknowledge Dr. Carlos

Sánchez for his assistance with confocal microscopy techniques.


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FOOTNOTES

*This work was supported by grant no. PM98-0002 from the Ministerio de Ciencia y

Tecnología, Spain.

§C. M. Pérez-Ferreiro is a Predoctoral Fellow of the Ministerio de Educación y

Cultura, Spain.

+Present address: European Molecular Biology Laboratory, Meyerhoffstrasse 1, D-

69117 Heidelberg, Germany

Abbreviations: GST, glutathione-S-transferase; PBS, phosphate-buffered saline

Key words: protein 4.1R, T cells, microtubules


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FIGURE LEGENDS

FIG. 1. Schematic representation of the exon map for the 4.1R protein and

the cDNA constructs used in this study. (A), schematic representation for

the protein 4.1R. Exons are coded as follows: shaded, alternative; white,

constitutive; black, noncoding. The number of each individual exon is

represented at the bottom. Three translation initiation sites at exons 2’ (ATG-1),

4 (ATG-2) and 8 (ATG-3) are indicated, and the stop codon (TGA) at exon 21.

These data have been taken from Refs. (16,18,22,50-52). (B), exon composition

of the T cell-4.1R cDNAs used for the T cell-transfection experiments. The

nucleotide sequence for c-Myc-epitope-tagging (myc) was added at the 3´ end

of all cDNAs. (C), GST-fusion proteins used for the identification of the 4.1R

region involved in tubulin association.

FIG. 2. Colocalization of endogenous 4.1R with the tubulin skeleton of T

cells. (A), CEM cells fixed with formalin in the absence of Triton X-100 were

double-labeled with the anti-tubulin antibody DM1A (b and d) and the anti-4.1R

antibodies 10b (a) or 762 (c). Areas in lower right (a-d) of each panel show

enlargements of indicated areas. (B), CEM cells fixed with formalin containing

Triton X-100 were double-labeled with a human anti-centrosome antibody (b

and d) and the anti-4.1R antibodies 10b (a) or 762 (c). The samples were

analyzed by confocal microcopy.

FIG. 3. Exogenously expressed 4.1R isoforms colocalize with

microtubules of T cells. Jurkat cells were transfected with 4.1R cDNAs

encoding either (A), ATG1-translated 4.1R isoforms: 4.1R135 16 (a, b),


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4.1R135 16,19 (c, d) and 4.1R135 4,5,16 (e, f) or (B), ATG2-translated 4.1R

isoforms: 4.1R80 16 (a, b) and 4.1R80 16,19 (c, d). Cells were double-labeled

with antibodies 10b (10b) and DM1A (DM1A) 48h after transfection and

examined by confocal microscopy. Areas in lower right (a and b) of each panel

show enlargements of indicated areas. Antibody 10b was used at dilutions that

only detect exogenous epitope-tagged 4.1R proteins.

FIG. 4. Endogenous and exogenous 4.1R proteins cosediment with taxol-

polymerized microtubules of T cells. (A), Western blot of total lysates of CEM

cells transfected with an empty plasmid (control) or CEM cells stably expressing

isoforms 4.1R80 16 (4.1R80 16) or 4.1R135 16 (4.1R135 16), revealed with the

9E10 antibody, which recognizes exogenous 4.1R isoforms. (B), sedimentation

of exogenous 4.1R proteins. Control CEM cells (control), CEM cells stably

expressing the ATG2-translated isoform 4.1R80 16 (4.1R80 16) or the ATG1-

translated isoform 4.1R135 16 (4.1R135 16) were homogenized and processed

to isolate the taxol-polymerized microtubule fraction, as indicated in

Experimental Procedures. MT, microtubule pellet obtained by centrifugation at

30,000 x g after taxol addition; S, 30,000 x g supernatant. (C), sedimentation of

endogenous 4.1R proteins. Replicas of the microtubule pellet shown in lane 1

revealed with anti-4.1R 10b (10b) or 762 (762) antibodies to detect endogenous

4.1R immunoreactive proteins cosedimenting with the microtubules.

FIG. 5. In vitro association of protein 4.1R isoforms with tubulin. (A),

Coomassie blue-stained SDS-gels and (B), Western blots revealed with anti-

GST antibody of equivalent aliquots of taxol-polymerized tubulin (pellets) and


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supernatant fractions (supernatants) obtained in in vitro binding assays

performed with PC-tubulin and the indicated GST-fusion proteins. (C),

Coomassie blue-stained SDS-gel of all purified GST-fusion proteins used in this

study for the in vitro binding assays. The composition of the GST-fusion proteins

is shown in Fig. 1C. Note that the fusion protein GST-Cter remains unbound to

tubulin (lanes 7 and 8 in A and B) whereas GST-4.1R80 16 and GST-

4.1R60 16,18 remain in the pellet associated with PC-tubulin (lanes 1 and 2,

respectively, in A and B).

FIG. 6. Identification of the 4.1R region involved in tubulin association.

Coomassie blue-stained SDS-gel (A) and Western blots revealed with anti-GST

(B) of equivalent aliquots of taxol-polymerized tubulin (pellets) and supernatant

fractions (supernatants) from in vitro binding assays. Note that the ‘core region’

common to all protein 4.1R isoforms is involved in tubulin association (lane 3 in

A and B). GST-4.1R80 16 and GST-Cter were used in the assay as positive and

negative controls, respectively.

FIG. 7. Twenty-two amino acids in the 4.1R core region are required for

tubulin interaction. (A), amino acid sequence containing four heptad repeats,

resembling a leucine zipper motif, in the core region of 4.1R. The leucine

residues are highlighted. The 22 boxed amino acids are deleted in GST-

core leu. (B), Coomassie blue-stained SDS-gel and (C), Western blot revealed

with anti-GST of equivalent aliquots of tubulin pellets (P) and supernatants (S)

from the in vitro binding assays. Note that GST-core binds to tubulin (Fig. 7B, C

lane 1) whereas removal of the heptad repeats of leucine (GST-core leu)

abolishes the association (Fig. 7B, C lane 4).


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FIG. 8. Exogenous expression of 4.1R induces microtubule

disorganization in COS-7 cells. COS-7 cells were transfected with a 4.1R

cDNA encoding isoform 4.1R80 16 and processed for double

immunofluorescence with antibodies 10b (10b) and DM1A (DM1A) 48 h after

transfection. Cells were analyzed by epifluorescence microscopy. The white

arrow indicates a representative transfected cell presenting a disorganized

microtubule network which is no longer well-focused at the centrosome.

Untransfected cells show typical microtubules radiating from the centrosome.


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Carmen M Pérez-Ferreiro, Carlos M Luque and Isabel Correasconstitutive region is involved in tubulin-binding

4.1R proteins associate with interphase microtubules in human T cells. A 4.1R

published online September 28, 2001J. Biol. Chem.

10.1074/jbc.M107369200Access the most updated version of this article at doi:

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Documents

1 Carmen M. Pérez-Ferreiro§, Carlos M. Luque+ and Isabel Correas