membrane–cytoskeleton interactions

Vincent Das Membrane–cytoskeleton interactionsBeatrice Nal during the formation of theAnne Roumier

immunological synapse andVannary Meas-YedidChristophe Zimmer subsequent T-cell activationJean-Christophe Olivo-MarinPascal RouxPierre FerrierAlice Dautry-VarsatAndres Alcover

Authors’ address

Vincent Das1, Beatrice Nal1, Anne Roumier1, VannaryMeas-Yedid2, Christophe Zimmer2, Jean-Christophe Olivo-Marin2, Pascal Roux3, Pierre Ferrier4, Alice Dautry-Varsat1 and Andres Alcover1,1Unite de Biologie des Interactions Cellulaires,CNRS URA 1960, 2Unite d’Analyse d’ImagesQuantitative, CNRS URA 1947, 3Centred’Imagerie Dynamique, Institut Pasteur,Paris, France, 4Centre d’Immunologie deMarseille-Luminy, INSERM-CNRS-Universitede la Mediterranee, Marseilles France

Correspondence to:

Andres Alcover,Institut PasteurUnite de Biologie des Interactions Cellulaires28, rue du Dr Roux75724 Paris Cedex 15FranceTel: π33 1 40 61 30 64Fax: π33 1 40 61 32 38e-mail: [email protected]

AcknowledgmentsThe work described here was supported by aProgramme Transversal de Recherche from theInstitut Pasteur. V. Das is supported by anAllocation de Recherche du Ministere del’Education Nationale, de la Recherche et de laTechnologie. A. Roumier was supported by aBourse from La Ligue Contre le Cancer. B. Nal issupported by a Bourse from the Associationpour la Recherche sur le Cancer. C. Zimmer issupported by the Programme Transversal deRecherche from the Institut Pasteur. The experttechnical help of Annick Dujeancourt is thankfullyacknowledged.

Immunological Reviews 2002Vol 189: 123–135Printed in Denmark. All rights reserved

Copyright c Blackwell Munksgaard 2002

Immunological Reviews0105-2896

123

Summary: Upon antigen recognition, T cells undergo substantial mem-brane and cytoskeletal rearrangements that lead to the formation of theimmunological synapse and are necessary for subsequent T-cell activation.However, little is known about how membrane and cytoskeletal mol-ecules interact during these processes. Here we discuss the involvementof the membrane-microfilament linker ezrin. We propose that ezrin is acomponent of the cytoskeleton-mediated architecture of the immunolog-ical synapse that plays a role in T-cell receptor clustering, protein kinaseC q translocation and intracellular signaling.

Introduction

Antigen recognition and subsequent T-cell activation depend

on the appropriate interaction between T cells and antigen

presenting cells (APC). This interaction involves a series of

events that depend on membrane and cytoskeleton dynamics,

namely cell motility, cell–cell adhesion, polarization and re-

ceptor relocalization. Initial T-cell receptor (TCR) signaling

induces actin cytoskeleton rearrangements, which in turn are

necessary for the stability of T cell–APC interactions, for the

maturation of the immunological synapse and for sustained

T-cell signaling. During maturation of the immunological

synapse, a precise molecular reorganization occurs at the con-

tact zone between T cells and APCs. Thus, TCR, coreceptors,

intracellular signaling molecules and adhesion receptors po-

larize to the T cell–APC interface and segregate into distinct

supramolecular clusters that redistribute following a precise

relative topology. This molecular patterning needs a func-

tional actin and myosin cytoskeleton and agonistic TCR stimu-

lation (reviewed in 1–3).

An important question in the field is how plasma mem-

brane–cytoskeleton interactions take place and facilitate the

formation of the immunological synapse. The first molecule

found to be likely involved in these membrane–cytoskeleton

Das et al ¡ Membrane-cytoskeleton links in the IS

interactions was talin (4). This protein can interact with ad-

hesion molecules of the integrin family and with cytoskeletal

components (5), and was found to accumulate in the periph-

eral zone of the immunological synapse (6). Moreover,

CD2AP could also be involved in membrane–cytoskeleton in-

teractions by linking the CD2 molecule with the cytoskeleton

(7). More recently, several laboratories including ours showed

that the membrane-microfilament linkers ezrin and moesin

were involved in the organization of membrane components

at the T cell–APC contact zone (8–12). Previously, ezrin and

moesin were shown to be substrates of protein kinases in T

lymphocytes (13–15), suggesting that they are effectors of

TCR signaling. Interestingly, talin, ezrin and moesin belong

to the same large family of proteins, whose prototype is the

band 4.1 of red blood cells.

Ezrin, radixin and moesin are highly homologous polypep-

tides that form, together with merlin/schwannomin, the

ERM (Ezrin/Radixin/Moesin) family of proteins. These pro-

teins control cell shape, cytokinesis and cell adhesion in vari-

ous cell types including lymphocytes. ERMs can mediate the

anchoring of some transmembrane proteins to the actin cyto-

skeleton, either directly or through adapter molecules, such

as EBP50. In addition, ERMs are involved in intracellular sig-

naling (reviewed in 16, 17). As all members of the band 4.1

family, ERMs share a common homologous domain of about

300 amino acids, the FERM domain, which is located at the

N-terminal part of the protein. ERMs interact with the plasma

membrane through their FERM domain, and with the actin

cytoskeleton through their C-terminal domain (18) (Fig. 1A).

These proteins exist under two conformations: a dormant

state, which is folded due to the interaction between the N-

terminal domain and the C-terminal domain (19), and an

active state, which is unfolded and able to interact with the

plasma membrane and the actin-based cytoskeleton (Fig. 1B).

The open conformation is stabilized by the phosphorylation

of a conserved threonine residue in the C-terminal domain

and the presence of phosphatidylinositides (20–25). Protein

kinase C (PKC) q can phosphorylate this threonine residue in

T cells (15), suggesting that ERMs can be activated during T-

cell activation.

The FERM domain can interact with transmembrane pro-

teins, such as intercellular adhesion molecules (ICAMs),

CD43, CD44, or P-selectin glycoprotein ligand 1 (PSGL-1)

in various cell types including lymphocytes (26–30). This is

particularly striking in T cells polarized by chemokines, in

which ERMs and these adhesion receptors strongly accumu-

late in membrane protrusions called uropods. These are

membrane protrusions, budding at the rear end of migrating

124 Immunological Reviews 189/2002

cells, which are involved in cell–cell adhesion. The localiz-

ation of these transmembrane proteins in the uropod depend

on their interaction with ERMs and ensure the cell adhesion

properties of this structure (28–32).

The N-terminal domain of ERMs can also interact with

phosphatidylinositol 4,5-bisphosphate (PIP2). This enhances

the capacity of these proteins to interact with the plasma

membrane, as well as with transmembrane proteins (26–28,

33, 34). Moreover, the N-terminal domain of ERMs can inter-

act with components of various intracellular signaling cas-

cades, such as RhoGDI (35), the regulatory subunit of phos-

phatidylinositol 3-kinase (36), the receptor Fas (37), PKCa

(38), or focal adhesion kinase (FAK) (39). ERM proteins may

also play a role in actin assembly on phagosomal membranes

(40). Therefore, ERMs are multifunctional proteins involved

in cellular architecture, intracellular signaling and membrane

trafficking.

Polarization of the actin cytoskeleton upon T-cell antigenrecognition: involvement of ezrin

Antigen recognition requires an intimate contact between T

cells and APCs. Imaging T-cell antigen recognition in vitro and

in situ revealed that the interaction of T cells with APCs occurs

in a polarized manner. After docking on the APC, T cells ac-

tively crawl on the surface of the APC, and can move from one

APC to another. At the contact site, T cells develop membrane

extensions that contact and scan the APC surface. If a specific

antigen is detected, T cells stop or slow down their move-

ment, trigger intracellular calcium fluxes and progressively

change their shape to increase the area of contact with the

APC. Later on, the cell progressively rounds up again and the

contact surface between both cells diminishes. This coincides

with molecular segregation at the contact zone, which marks

the maturation of the immunological synapse (Fig. 2). Polar-

ization, motility, morphological changes, and molecular seg-

regation taking place in T cells interacting with APCs are sen-

sitive to early signaling and depend on actin and myosin cyto-

skeleton dynamics (6, 7, 41–51).

We investigated the involvement of the ERM family of

membrane-microfilament linkers in actin cytoskeleton re-

arrangements occurring at the T cell–APC contact zone

(Fig. 3A). We observed ezrin accumulated at the T cell–APC

contact zone and concentrated in F-actin-rich membrane ex-

tensions that engulfed the APC (Fig. 3B, C) (10). Moesin was

also enriched in these membrane extensions (A. Alcover, un-

published data). Between 15 and 30min of activation, these

membrane extensions retracted and the contact area between


the T cell and the APC significantly diminished. Ezrin ac-

cumulation at the contact zone became less important, al-

though a local accumulation in a smaller area that overlapped

with the TCR cluster could still be observed at 30 and 60min

of activation (Fig. 3D, E). F-actin followed the distribution of

ezrin during the early stages (5–15min) (10). In contrast, at

later times (30–60min), the accumulation of F-actin ap-

peared relatively less intense than that of ezrin in the same

area, and was located more in the periphery of the TCR clus-

ter central zone (Fig. 3F, G). These data suggest that ezrin is

initially linked to F-actin and occupies the periphery of the T

cell–APC contact zone, whereas at later times it is concen-

trated in the center. How ezrin clustering occurs is not

known. The ability of ezrin to oligomerize could be a possible

mechanism (17), but whether this implies that ezrin detaches

from F-actin to accumulate in a more compact zone will need

further investigation. In addition, the ability of ezrin to as-

sociate with membrane rafts (52, 53 and our unpublished

data) could also favor its clustering in the contact area. In-

deed, an overlap between ezrin and lipid rafts was observed

in the contact zone (53).

We investigated the molecular mechanism responsible for

ezrin relocalization at the T cell–APC contact zone. Two pos-

Fig. 1. A: Domain and functionalorganization of ezrin. The N-terminal 300amino acids of the molecule is the region ofhigher sequence homology between allmembers of the family represented by theband 4.1 of erythrocytes. In particular, ezrin,radixin and moesin (ERMs) are about 87%homologous in this domain. This is afunctional domain where the interactions withmembrane components, phospholipids andvarious signaling molecules occur. The FERMdomain is followed by a less conserved region,the a-domain, predicted to form an a-helicalcoiled-coil. The last 80 amino acids form ahydrophilic domain also highly homologousin ERMs. The C-terminal 34 residues of thisdomain contain a binding site for F-actin. B:Activation of ezrin. ERMs are in anequilibrium between two conformations: aclosed form, which has the F-actin andmembrane-binding sites masked and iscytosolic, and an open form, which can bindto both the membrane and the cytoskeleton.In addition, open conformations can formhomo- or hetero-oligomers, by virtue of theinteraction between N- and C-terminaldomains. The open conformation is stabilizedby phosphorylation of a conserved threonineresidue in the C-terminal domain, and bybinding of phospholipids (16, 17).

125Immunological Reviews 189/2002

sibilities were tested. First, ezrin accumulation could be due to

physical cross-linking of ezrin-associated transmembrane pro-

teins (i.e. ICAMs) by their ligands present on the APC surface.

Alternatively, ezrin relocalization could be due to polarization

of F-actin, which occurs in response to TCR signaling (54, 55).

To test these two possibilities, we replaced the APCs with cell-

size latex beads coated with antibodies (Abs). T cells stimulated

with beads coated with anti-CD3 Abs displayed large mem-

brane extensions engulfing the stimulatory beads, which were

strongly enriched in ezrin and F-actin and were similar to those

observed in cells activated for 15min with Staphylococcus aureus

enterotoxin E (SEE)-pulsed APCs (10). In contrast, beads

coated with Abs directed to transmembrane proteins that inter-

act with ezrin, such as ICAM-2, ICAM-3, or CD43, did not in-

duce morphological changes or ezrin accumulation. This indi-

cated that ezrin relocalization at the T cell–APC contact zone

depends on TCR signaling. Consistently, anti-CD3-induced

ezrin relocalization was impaired in Lck-deficient Jurkat cells

(JCaM-1.6), as well as in wild-type Jurkat cells treated with the

Src inhibitor PP-1 (10). Therefore, these results indicate that

the initial ezrin accumulation at the contact site is linked to po-

larized actin polymerization induced by TCR signaling at the

APC contact site.


Early on, ezrin concentrates in a peripheral zone of the

T cell–APC contact site outside the TCR clustering area, but

coinciding with CD43 (10). CD43 is a highly glycosylated

transmembrane molecule that interacts with the FERM do-

main of ERMs (26, 29). CD43 is excluded from the center of

the immunological synapse upon T-cell activation in an ezrin-

and/or moesin-dependent manner (8, 9, 11, 56). It is worth

noting here that Allespach et al. (8) reported an antipodal

accumulation of ezrin and its associated molecules CD43 in

mouse TCR transgenic T lymphocytes interacting with pep-

tide antigen-pulsed APCs or with anti-TCR-coated beads.

However, antipodal translocation of ezrin was not observed

by us, either in Jurkat or in human peripheral blood T cells,

or reported by others (9, 11, 53), suggesting differences be-

tween the various experimental systems. Interestingly, Mon-

toya et al. (57) reported that ICAM-3 and P-selectin glyco-

protein ligand (PSGL)-1, which also interact with ezrin and

moesin (30), accumulated at opposite poles of the T cell dur-

ing APC interactions. Thus, ICAM-3 was relocated to the APC

contact site in an antigen-independent manner, whereas

PSGL-1 relocated to the pole away from the APC contact site

Fig. 2. Early stages of T lymphocyteinteraction with stimulatory APCs:morphological changes and actincytoskeleton rearrangements. T cells dockon APCs and scan their surface. If the specificantigen-MHC complexes are detected, the T cellstops or slows down its movement andtriggers early signaling, such as calciumfluxes. This is followed by strongmorphological changes, which depend onactin cytoskeleton dynamics. At this time,active actin polymerization takes place at thesite of contact with the APC, which results inan enlargement of the area of contact of theT cell on the APC. Finally, the T cell rounds upand the area of contact again becomes morereduced. This coincides with the coalescenceand segregation of supramolecular clusters atthe contact site, a phenomenon that marks thematuration of the immunological synapse.


only in activated T cells (57). This indicates that a complex

set of phenomena involving interactions between ERMs and

various adhesion molecules takes place during T cell–APC in-

teractions. This may be important for setting up the appropri-

ate membrane–cytoskeleton interactions necessary for T-cell

stimulation by APCs. In summary, our data indicate that ezrin

is part of the actin cytoskeleton-mediated scaffold involved in

the molecular architecture of the immunological synapse.

A method of quantitative image analysis forevaluating morphological changes and molecularpolarization at the T cell–APC contact zone

In order to have a more accurate measurement of cytoskeletal

modifications during early stages of T lymphocyte responses,

we developed a method of quantitative image analysis capable

of evaluating morphological changes, as well as the molecular

accumulation of various molecules at the T cell–APC contact

zone. This method obtains complementary information from


multimodal imaging and utilizes active contours to define the

cell borders in a semiautomatic manner (Fig. 4).

Three types of images were acquired using a CCD camera,

nuclear staining, phase contrast and cytoskeleton staining.

Nuclear staining allows individualization of each cell and pro-

vides the initial contours (Fig. 4A). These initial contours are

then projected on the phase contrast image of the same field

of cells (Fig. 4B). Initial contours then evolve on the phase

contrast image to fit the cell perimeter, thus providing cell

profiles in two dimensions (Fig. 4C). The sites where the con-

tours of two different cells encounter each other define the

cell–cell contacts (Fig. 4C, D, arrows). Finally, the extracted con-

tours are projected on the fluorescence image, allowing analy-

sis of the ratio of fluorescence between the cell–cell contact

area and the rest of the cell (Fig. 4D, F).

A difficulty may be encountered when trying to distinguish

cell–cell contacts occurring between a T cell and an APC from

Fig. 3. Relocalization of ezrin to the T cell–APC contact site. Jurkat T cells interactingwith Raji B cells in the absence (A) or in thepresence of Staphylococcus aureus enterotoxin E(SEE) superantigen. Cells were activated, fixed,stained with anti-TCR-CD3 and anti-ezrinAbs (A-E), or anti-TCR-CD3 Abs andphalloidin (F, G), and analyzed by confocalmicroscopy as previously described (10). Amedial optical section is shown in each image.At 15min, ezrin accumulates in membraneextensions that engulf the APC (B) and ismore concentrated in the peripheral area ofthe immunological synapse (C, red),surrounding the more central TCR clusters (C,green). At 30min, the area of contact appearedmore reduced, ezrin was still concentrated inthe contact zone but in an area that overlappedwith the TCR cluster (D, E), whereas F-actinappeared less concentrated and remained in theperiphery of the contact zone (F, G). BarΩ5 mm.


those taking place between two T cells or two APCs. One

should therefore use criteria to distinguish the two cell types.

In our experimental system, we took advantage of the fact

that APCs (Raji cells) displayed a nuclear morphology differ-

ent from that of T cells (Jurkat T cells). Thus, Raji nuclei have

a more irregular shape than those of Jurkat T cells (Fig. 4A,

asterisks).

Once the cell perimeters are defined, the shape of the cells

can be estimated by calculating the normalized compactness

(Cn), which has a value of 0 in the case of a perfect circle,

and a value of 1 in the case of an elongated shape. By this

means, morphological changes can be quantified (Fig. 4E). For

instance, we determined that the compactness of T cells in

conjugates formed in the absence of superantigen stimulation

was similar in Jurkat T cells and in APCs. In contrast, the

coefficient of compactness of activated T cells at 15min was

higher (V. Das et al., in preparation).


After determining the intersection of cell contours, as de-

scribed above, we quantified the number of pixels having

values above background levels within the contact zone, or

outside this area. We could then calculate a ‘relocalization

index’ R (Fig. 4F). For instance, when we measured the ac-

cumulation of F-actin at the T cell–APC junctions, we calcu-

lated that activated T cells at 15min had a value of R 0.9,

whereas for non-activated cells, its value was R 0.4 (V. Das

et al., in preparation). Although this method still requires sys-

tematization to analyze large numbers of samples, it already

provides more precise data on cell shape and cytoskeletal re-

modeling during T cell–APC interactions.

Fig. 4. New method for quantitative analysisof morphological changes and molecularpolarization at the T cell–APC contact zone.This method obtains complementaryinformation from multimodal imaging andutilizes active contours to define the cellborders in a semiautomatic manner. Threetypes of images acquired using a CCD camerawere used in this example: nuclear staining,phase contrast and cytoskeleton staining.Nuclear staining allows individualization ofeach cell and provides the initial contours (A),which are then projected on the phase contrastimage of the same field of cells (B). Initialcontours then evolve on the phase contrastimage to fit the cell perimeter, thus providingcell profiles in two dimensions (C). The siteswhere the contours of two different cellsencounter each other define the cell–cellcontacts (C, D, arrows). Finally, the extractedcontours are projected on the fluorescenceimage (D, F). From the contours, the shapeof the cell can be estimated by calculating thenormalized compactness (Cn), according to theformula, where A is the area and P theperimeter. Cn has a value of 0 in the case ofa perfect circle, and a value of 1 in the case ofan elongated shape. By these means,morphological changes can be quantified. Anexample of Cn calculated for an activated T cell(E, left), vs. a control T cell (E, right) isshown. After determining the intersection ofcell contours, the molecular polarization couldbe estimated. The number of pixels havingvalues above background were quantifiedwithin the contact zone (Njunction), or outsidethis area (NC

junction). A ‘relocalization index’ Rwas calculated according to the formula (F,bottom).


A role for ezrin in TCR-CD3 clustering at theimmunological synapse: quantitative imaging analysis

Molecular polarization and subsequent segregation into dis-

tinct clusters in the immunological synapse require an intact

actin-myosin cytoskeleton (6, 45, 46). Ezrin is known to link

the membrane with the actin cytoskeleton and could there-

fore facilitate molecular polarization and/or clustering in the

T cell–APC contact zone. To investigate this possibility, we

studied the effect of a dominant negative mutant of ezrin.

This mutant contains only the FERM domain, which interacts

with the membrane but lacks the main F-actin-binding re-


gion (18). It has previously been shown to inhibit morpho-

genesis and migration of epithelial cells (58). We observed

that overexpression of this ezrin mutant inhibited the coalesc-

ence of dispersed TCR clusters into more compact ones, a

characteristic of the immunological synapse in mature

lymphocytes (6, 46).

As shown in Fig. 5, Jurkat T cells activated with bacterial

superantigen-pulsed Raji B cells formed immunological

synapses similar to those previously described in mouse T

cells activated with peptide antigen-bearing B cells, or with

artificial lipid bilayers presenting major histocompatibility

complex (MHC)-peptide complexes (6, 46). Thus, we ob-

served a compact cluster of TCRs embedded into a larger ac-

cumulation of adhesion molecules, whose density was higher

in the peripheral contact zone. This indicates that superanti-

gen activation of Jurkat cells triggers molecular polarization

and segregation, forming a canonical immunological synapse.

We observed that immunological synapses formed by Jurkat

T cells transfected with dominant negative ezrin displayed a

TCR clustering pattern often more dispersed than that shown

by untransfected cells. This suggested that dominant negative

ezrin perturbs TCR clustering during maturation of the im-

munological synapse. To have a more accurate estimation of

Fig. 5. Immunological synapses formed between Jurkat T cells and superantigen-pulsed Raji B cells. Cells were activated for 30min, thenfixed, stained with anti-TCR-CD3, anti-ICMA-1 Abs and analyzed by confocal microscopy as previously described (10). A: TCR and ICAM-1concentrate in the contact zone. The TCR cluster occupies a restricted central zone, which coincides with the accumulation of the adhesionmolecule ICAM-1 from the APC. The TCR cluster appears embedded in the larger cluster of ICAM-1, as observed in the 3 D reconstruction of thecontact zone (A, right panel), carried out using Imaris software (Bitplane, Switzerland). Note that ICAM-1 is expressed only in the APC and likelyreflects the accumulation of its counter adhesion receptor LFA-1 expressed on the T cell. The staining of surface molecules from the T cell and theAPC gives a view of the tightness of the synapse. B: TCRs and membrane rafts accumulate in the center of the contact zone. TCRs and membranerafts were stained with anti-TCR-CD3 Abs and cholera toxin B fragment, respectively. Both TCRs and membrane rafts cluster in the center of the Tcell–APC contact zone, as observed in the zx-projection of the contact zone, carried out using Zeiss LSM-510 software (right panel).


this phenomenon, we performed quantitative image analysis

of TCR clustering in cells overexpressing dominant negative

ezrin, wild type ezrin, or an irrelevant protein (i.e. GFP). To

this end, confocal images were utilized. A z-series of 20 ‘xy’

optical sections was acquired. The region corresponding to

the contact zone was then extracted over the whole z-series,

and an ‘xz’-projection of this region was obtained, using

Zeiss LSM-510 confocal microscope and software.

A computer program was developed that detects, counts

and measures clusters in these projection images. This pro-

gram is based on the multiscale product of subband images

resulting from an undecimated wavelet transform decompo-

sition of the original image, after thresholding of nonsignifi-

cant coefficients (59). To characterize the spatial distribution

of clusters, the algorithm then computes the surface of

bounding area that includes all the detected clusters. By these

means, TCR clusters could be individualized and quantified

with respect to their number, individual surface and form,

and spatial distribution (Fig. 6). This method provides a meas-

urement of the phenomenon of coalescence of small TCR

clusters into one or few more compact ones, which is a fea-

ture of immunological synapse maturation (6, 46, 48).

This quantitative image analysis revealed that immunolog-


ical synapses formed by T cells overexpressing dominant

negative ezrin displayed significant higher numbers of TCR

clusters, which had smaller individual surface and were

spread on a larger area, than cells overexpressing wild type

ezrin or the irrelevant GFP protein (10 and our unpublished

data). This finding suggests that ezrin provides a link between

membrane components and the actin cytoskeleton that facili-

tates the coalescence of TCR clusters.

The effect of the dominant negative mutant of ezrin on

molecular rearrangements at the T cell–APC contact zone was

not restricted to the TCR. It also affected a key signaling mol-

ecule that accumulates at the immunological synapse, PKCq

(60). Thus, we observed that the number of T cell–APC con-

jugates displaying an accumulation of PKCq was significantly

diminished in cells overexpressing dominant negative ezrin

with respect to controls (10). In contrast, Allenspach et al.

(8) did not report this type of inhibition. The reasons for this

discrepancy are not clear at present. Our data suggest that

ezrin helps the targeting (or the retention) of signaling com-

plexes to the T cell–APC contact zone as well as the coalesc-

ence of clusters in this area, phenomena which are likely

needed for T-cell activation.

Fig. 6. A method for quantitative image analysis of TCR clustering at the T cell–APC contact zone. A z-series of 20 confocal ‘xy’ opticalsections was acquired. Then, a region corresponding to the contact zone (A, B, square inset) was extracted over the whole z-series and an ‘xz’projection performed using Zeiss LSM-510 software (A, B, right lower panels). Panels A and B represent examples of immature (A: TCR clustersare smaller and more dispersed) and mature (B: TCR cluster is condensed), respectively. XZ-projections were then transformed (C) to obtain animage in which the spots corresponding to TCR clusters could be easily quantified. The computer then provides the coordinates of each object,its surface, its roundness and its average pixel intensity. From this, we can calculate the average number of clusters per synapse, their average surfaceand the surface they occupy (bounding area). Panel C shows three examples of these transformed images, corresponding to an immature synapse(upper panel, where TCR clusters were very small and dispersed), intermediate synapse (middle panel, where TCR clusters were fewer, larger andoccupied a smaller surface than in the upper panel), and mature synapse (lower panel, where TCR clusters appeared condensed in a continuouslarge cluster). The square in the transformed images represents the bounding area occupied by the clusters).


Role of F-actin-ezrin complexes in molecular clustering atthe immunological synapse: a working model

A possible model to explain ezrin’s involvement in TCR clus-

tering could be the following (Fig. 7): TCR triggering induces

the polarization of F-actin and ezrin to the APC contact zone,

producing membrane extensions that cover a large surface of

the APC (Fig. 3B). Ezrin-binding proteins, such as CD43,

would be anchored, creating a ‘collar’ of cytoskeleton-an-

chored transmembrane proteins at the periphery of the con-

tact zone (10). This collar would function as a transmem-

brane ‘fence’ to reduce the diffusion of TCRs out of the con-

tact zone. This process would occur early during T cell–APC

interactions (5–15min under our experimental conditions,

Fig. 3B, C) and would facilitate initial concentration of TCRs

into small clusters. Later, membrane extensions retract, al-

though ezrin remains accumulated in this area. At this time a

constriction of the F-actin–ezrin collar occurs concomitantly

with the reduction of the cell–cell contact area (Fig. 3D, E,

30min). This constriction of the cytoskeleton collar could

compress dispersed small TCR clusters, facilitating their co-

alescence.


The TCR clustering observed may be part of a more general

phenomenon involving cholesterol and sphingolipid-enrich-

ed membrane microdomains, also called membrane rafts

(61). In T cells, membrane rafts contain TCRs and signaling

molecules (62, 63), and concentrate at the T cell–APC contact

zone (64) (Fig. 5B) upon activation. Interestingly, ERMs can

interact with membrane rafts, as assessed by sucrose gradient

buoyancy (53 and our unpublished data). This may facilitate

raft coalescence in this zone.

According to this model, a crucial step for cluster coalescence

would be the constriction of the F-actin–ezrin network and the

concomitant reduction of the T cell–APC contact area, which

we observed between 15 and 30min (Figs 3 and 7). This con-

striction could occur by shifting from a state of high actin poly-

merization dynamics, which generates the membrane exten-

sions observed at early times, to a state of lower actin poly-

merization and higher cross-linking of actin fibers. In addition,

ezrin could also oligomerize in this area. The cytoskeleton col-

lar would then become a more compact and stable structure,

which could restrict molecular diffusion and stabilize supra-

molecular clusters. These phenomena would be perturbed by

the overexpression of the dominant negative mutant of ezrin,

Fig. 7. Working model to explain the role of F-actin-ezrincomplexes in molecular clustering at the immunologicalsynapse. TCR triggering induces the polarization of F-actin andezrin to the APC contact zone producing membrane extensionsthat cover a large surface of the APC. We propose that this anchorsezrin-binding proteins, such as CD43, creating a ‘collar’ ofcytoskeleton-anchored transmembrane proteins at the periphery ofthe contact zone. This collar would function as a transmembrane‘fence’ reducing the diffusion of TCRs out of the contact zone andfacilitating the initial concentration of TCRs into small clusters (2).Later on, membrane extensions retract although ezrin remainsaccumulated in this area. At this time a constriction of the F-actin-ezrin collar occurs which is concomitant to the reduction ofthe cell–cell contact area. This constriction of the cytoskeletoncollar could compress dispersed small TCR clusters facilitating theircoalescence into a single cluster (3).


which lacks the C-terminal half of the molecule, and is there-

fore unable to bind to actin fibers and to oligomerize.

The actin-cytoskeleton associated protein coronin-1 istransiently recruited to the T cell–APC contact area

First described in Distyostelium discoideum, coronin and coronin-

like proteins are expressed in many eukaryotic species from

yeast to mammals. They have been described as actin-binding

proteins implicated in cellular processes involving actin cyto-

skeleton dynamics, such as cell migration, cytokinesis, phago-

cytosis, or morphogenesis (65). Coronins from different spe-

cies display a conserved structural organization. Common fea-

tures include 5 WD repeats, a coiled-coil domain at the C-

terminus, and a linker sequence in between of variable length

referred to as the unique region. Similar to WD-containing Gb

proteins (66), WD repeats of coronins are thought to be organ-

ized in a b-propeller domain, likely involved in protein interac-

tions. Coronin-1 is the mammalian paralogue specifically en-

riched in hematopoietic tissues. Depending on the cell lineage,

coronin-1 appears to have different molecular interactions and,

presumably, different functional activities. For instance, in neu-

trophils, coronin-1 was found associated with components of

the NADPH oxidase complex, which produces superoxide in

phagocytic cells (67). Moreover, it was found as a component

of the phagosome coat in macrophages (68, 69).

We found that the coronin-1 gene is expressed in lymphoid

precursor cells within mice embryos as well as in differentiat-

ing thymocytes. Coronin-1 is enriched in subcellular struc-

tures rich in F-actin, such as lamellipodia and filopodia, in

both mouse and human T lymphocytes (Nal et al., submit-

ted). In T cells encountering superantigen-pulsed APCs, co-

ronin-1 transiently accumulates at the cell–cell contact area

(Fig. 8). The maximal accumulation occurs within the large

membrane protrusions that engulf the APC at early stages of

the interaction between T cells and APCs. Coronin-1, F-actin

and ezrin are enriched in these membrane structures; they

partially overlap, although they have distinct relative spatial

localizations. Coronin-1 is more enriched in the area closer

to the cell body, F-actin in the middle and ezrin in the more

cortical area (10) (Fig. 8B). In contrast, at later times, coronin-

1 accumulation in the contact area mostly disappeared,

whereas ezrin remained accumulated (Figs. 3D, E, 8F, G).

Interestingly, coronins were shown to be related to actin

filament assembly rather than to stability. For instance, co-

ronins were found enriched in actin tails induced by Listeria

monocytogenes in infected cells, at phagocytic cups, in lamellipo-

dia and in membrane ruffles, but less in stress fibers (68, 70–


72 and our unpublished data). Coronin-1 could therefore be

considered a marker for local active actin polymerization. We

suggest that the transient accumulation of coronin-1 at the

T cell–APC contact area reflects distinct actin polymerization

dynamics along the maturation of the immunological syn-

apse, very intense at early times and much lower at later

times.

Ezrin is necessary for T-cell activation leading to IL2 genetranscription

Definite evidence for the functional importance of ezrin in T

lymphocyte function came from the observation that overex-

pression of the dominant negative mutant of ezrin in Jurkat

Fig. 8. Coronin-1 is transiently recruited tothe T cell–APC contact zone. Jurkat T cellsinteracting with Raji B cells in the absence (A,C), or in the presence of SEE superantigen (B,D-G). Cells were activated, fixed, stained withanti-coronin-1 Abs and phalloidin (A, B), oranti-coronin-1 and anti-TCR-CD3 (C-G), andanalyzed by confocal microscopy as previouslydescribed (10). At 15min, coronin-1 wasaccumulated in the membrane extensions thatcontact the APC, partially overlapping with F-actin, but more concentrated in an area closerto the cell body (B). When localized withrespect to the TCR, coronin-1 occupied theperiphery of the T cell–APC contact zone (D,E). At later times, coronin-1 accumulation wasstrongly reduced (F). When compared withthe TCR, it appeared staining the peripheralcontact area, being rather excluded from thearea where the TCR cluster is located (G).BarΩ5 mm.


cells inhibited the activation of nuclear factor for activated T

cells in response to stimulation to peptide antigen or super-

antigen (10). Moreover, Allenspach et al. (8) also reported

that T cells from TCR transgenic mice overexpressing a similar

dominant negative mutant of ezrin had a reduced capacity to

produce various cytokines in response to antigenic stimula-

tion.

How ezrin and moesin are involved in T-cell activation is

not clear at present. As described above, ezrin may help mol-

ecular reorganization at the immunological synapse and, as a

consequence, help signaling. Ezrin and moesin could inter-

vene in the clustering of receptors and signaling molecules

(10). This could occur either directly, via interaction with

lipid rafts (12, 53), or by actively removing highly glycosylat-


ed molecules, such as CD43 (8, 9, 11), or PSGL-1 (57),

whose presence could destabilize T cell–APC interactions and

therefore activation. It might also act by facilitating initial in-

teractions between T cells and APC (57). Ezrin and moesin

might also intervene in one or various T-cell signaling path-

ways, by directly interacting with signal transduction mol-

ecules, as shown in other experimental systems (17). An in-

volvement of ERM molecules at various stages of the T-cell

activation process could explain the strong effect of dominant

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the molecular reorganization of the T cell–APC contact zone

and in promoting T-cell activation will be of particular inter-

est.


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membrane–cytoskeleton interactions

Technology

t cells

t cades

tcell receptor clustering

initial tcell receptor

t cellapc interface

t cellapc contact zone

cell motility

membranecytoskeleton