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Membrane–cytoskeleton interactions
 

Membrane–cytoskeleton interactions

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    Membrane–cytoskeleton interactions Membrane–cytoskeleton interactions Document Transcript

    • Vincent Das Membrane–cytoskeleton interactions Beatrice Nal ´ during the formation of the Anne Roumier Vannary Meas-Yedid immunological synapse and Christophe Zimmer subsequent T-cell activation Jean-Christophe Olivo-Marin Pascal Roux Pierre Ferrier Alice Dautry-Varsat Andres Alcover ´ Authors’ address Summary: Upon antigen recognition, T cells undergo substantial mem- Vincent Das1, Beatrice Nal1, Anne Roumier1, Vannary ´ brane and cytoskeletal rearrangements that lead to the formation of the Meas-Yedid2, Christophe Zimmer2, Jean-Christophe Olivo- immunological synapse and are necessary for subsequent T-cell activation. Marin2, Pascal Roux3, Pierre Ferrier4, Alice Dautry- However, little is known about how membrane and cytoskeletal mol- Varsat1 and Andres Alcover1, ´ ecules interact during these processes. Here we discuss the involvement 1 Unite de Biologie des Interactions Cellulaires, ´ of the membrane-microfilament linker ezrin. We propose that ezrin is a CNRS URA 1960, 2Unite d’Analyse d’Images ´ component of the cytoskeleton-mediated architecture of the immunolog- Quantitative, CNRS URA 1947, 3Centre ical synapse that plays a role in T-cell receptor clustering, protein kinase d’Imagerie Dynamique, Institut Pasteur, C q translocation and intracellular signaling. Paris, France, 4Centre d’Immunologie de Marseille-Luminy, INSERM-CNRS-Universite ´ de la Mediterranee, Marseilles France ´ ´ Correspondence to: Andres Alcover, ´ Introduction Institut Pasteur Unite de Biologie des Interactions Cellulaires ´ Antigen recognition and subsequent T-cell activation depend 28, rue du Dr Roux on the appropriate interaction between T cells and antigen 75724 Paris Cedex 15 presenting cells (APC). This interaction involves a series of France events that depend on membrane and cytoskeleton dynamics, Tel: π33 1 40 61 30 64 Fax: π33 1 40 61 32 38 namely cell motility, cell–cell adhesion, polarization and re- e-mail: aalcover@pasteur.fr ceptor relocalization. Initial T-cell receptor (TCR) signaling induces actin cytoskeleton rearrangements, which in turn are Acknowledgments The work described here was supported by a necessary for the stability of T cell–APC interactions, for the Programme Transversal de Recherche from the maturation of the immunological synapse and for sustained Institut Pasteur. V. Das is supported by an Allocation de Recherche du Ministere de ` T-cell signaling. During maturation of the immunological l’Education Nationale, de la Recherche et de la synapse, a precise molecular reorganization occurs at the con- Technologie. A. Roumier was supported by a Bourse from La Ligue Contre le Cancer. B. Nal is tact zone between T cells and APCs. Thus, TCR, coreceptors, supported by a Bourse from the Association pour la Recherche sur le Cancer. C. Zimmer is intracellular signaling molecules and adhesion receptors po- supported by the Programme Transversal de larize to the T cell–APC interface and segregate into distinct Recherche from the Institut Pasteur. The expert technical help of Annick Dujeancourt is thankfully supramolecular clusters that redistribute following a precise acknowledged. relative topology. This molecular patterning needs a func- tional actin and myosin cytoskeleton and agonistic TCR stimu- Immunological Reviews 2002 Vol 189: 123–135 lation (reviewed in 1–3). Printed in Denmark. All rights reserved An important question in the field is how plasma mem- brane–cytoskeleton interactions take place and facilitate the Copyright c Blackwell Munksgaard 2002 Immunological Reviews formation of the immunological synapse. The first molecule 0105-2896 found to be likely involved in these membrane–cytoskeleton 123
    • Das et al ¡ Membrane-cytoskeleton links in the IS interactions was talin (4). This protein can interact with ad- cells, which are involved in cell–cell adhesion. The localiz- hesion molecules of the integrin family and with cytoskeletal ation of these transmembrane proteins in the uropod depend components (5), and was found to accumulate in the periph- on their interaction with ERMs and ensure the cell adhesion eral zone of the immunological synapse (6). Moreover, properties of this structure (28–32). CD2AP could also be involved in membrane–cytoskeleton in- The N-terminal domain of ERMs can also interact with teractions by linking the CD2 molecule with the cytoskeleton phosphatidylinositol 4,5-bisphosphate (PIP2). This enhances (7). More recently, several laboratories including ours showed the capacity of these proteins to interact with the plasma that the membrane-microfilament linkers ezrin and moesin membrane, as well as with transmembrane proteins (26–28, were involved in the organization of membrane components 33, 34). Moreover, the N-terminal domain of ERMs can inter- at the T cell–APC contact zone (8–12). Previously, ezrin and act with components of various intracellular signaling cas- moesin were shown to be substrates of protein kinases in T cades, such as RhoGDI (35), the regulatory subunit of phos- lymphocytes (13–15), suggesting that they are effectors of phatidylinositol 3-kinase (36), the receptor Fas (37), PKCa TCR signaling. Interestingly, talin, ezrin and moesin belong (38), or focal adhesion kinase (FAK) (39). ERM proteins may to the same large family of proteins, whose prototype is the also play a role in actin assembly on phagosomal membranes band 4.1 of red blood cells. (40). Therefore, ERMs are multifunctional proteins involved Ezrin, radixin and moesin are highly homologous polypep- in cellular architecture, intracellular signaling and membrane tides that form, together with merlin/schwannomin, the trafficking. ERM (Ezrin/Radixin/Moesin) family of proteins. These pro- teins control cell shape, cytokinesis and cell adhesion in vari- Polarization of the actin cytoskeleton upon T-cell antigen ous cell types including lymphocytes. ERMs can mediate the recognition: involvement of ezrin anchoring of some transmembrane proteins to the actin cyto- skeleton, either directly or through adapter molecules, such Antigen recognition requires an intimate contact between T as EBP50. In addition, ERMs are involved in intracellular sig- cells and APCs. Imaging T-cell antigen recognition in vitro and naling (reviewed in 16, 17). As all members of the band 4.1 in situ revealed that the interaction of T cells with APCs occurs family, ERMs share a common homologous domain of about in a polarized manner. After docking on the APC, T cells ac- 300 amino acids, the FERM domain, which is located at the tively crawl on the surface of the APC, and can move from one N-terminal part of the protein. ERMs interact with the plasma APC to another. At the contact site, T cells develop membrane membrane through their FERM domain, and with the actin extensions that contact and scan the APC surface. If a specific cytoskeleton through their C-terminal domain (18) (Fig. 1A). antigen is detected, T cells stop or slow down their move- These proteins exist under two conformations: a dormant ment, trigger intracellular calcium fluxes and progressively state, which is folded due to the interaction between the N- change their shape to increase the area of contact with the terminal domain and the C-terminal domain (19), and an APC. Later on, the cell progressively rounds up again and the active state, which is unfolded and able to interact with the contact surface between both cells diminishes. This coincides plasma membrane and the actin-based cytoskeleton (Fig. 1B). with molecular segregation at the contact zone, which marks The open conformation is stabilized by the phosphorylation the maturation of the immunological synapse (Fig. 2). Polar- of a conserved threonine residue in the C-terminal domain ization, motility, morphological changes, and molecular seg- and the presence of phosphatidylinositides (20–25). Protein regation taking place in T cells interacting with APCs are sen- kinase C (PKC) q can phosphorylate this threonine residue in sitive to early signaling and depend on actin and myosin cyto- T cells (15), suggesting that ERMs can be activated during T- skeleton dynamics (6, 7, 41–51). cell activation. We investigated the involvement of the ERM family of The FERM domain can interact with transmembrane pro- membrane-microfilament linkers in actin cytoskeleton re- teins, such as intercellular adhesion molecules (ICAMs), arrangements occurring at the T cell–APC contact zone CD43, CD44, or P-selectin glycoprotein ligand 1 (PSGL-1) (Fig. 3A). We observed ezrin accumulated at the T cell–APC in various cell types including lymphocytes (26–30). This is contact zone and concentrated in F-actin-rich membrane ex- particularly striking in T cells polarized by chemokines, in tensions that engulfed the APC (Fig. 3B, C) (10). Moesin was which ERMs and these adhesion receptors strongly accumu- also enriched in these membrane extensions (A. Alcover, un- late in membrane protrusions called uropods. These are published data). Between 15 and 30 min of activation, these membrane protrusions, budding at the rear end of migrating membrane extensions retracted and the contact area between 124 Immunological Reviews 189/2002
    • Das et al ¡ Membrane-cytoskeleton links in the IS the T cell and the APC significantly diminished. Ezrin ac- sibilities were tested. First, ezrin accumulation could be due to cumulation at the contact zone became less important, al- physical cross-linking of ezrin-associated transmembrane pro- though a local accumulation in a smaller area that overlapped teins (i.e. ICAMs) by their ligands present on the APC surface. with the TCR cluster could still be observed at 30 and 60 min Alternatively, ezrin relocalization could be due to polarization of activation (Fig. 3D, E). F-actin followed the distribution of of F-actin, which occurs in response to TCR signaling (54, 55). ezrin during the early stages (5–15 min) (10). In contrast, at To test these two possibilities, we replaced the APCs with cell- later times (30–60 min), the accumulation of F-actin ap- size latex beads coated with antibodies (Abs). T cells stimulated peared relatively less intense than that of ezrin in the same with beads coated with anti-CD3 Abs displayed large mem- area, and was located more in the periphery of the TCR clus- brane extensions engulfing the stimulatory beads, which were ter central zone (Fig. 3F, G). These data suggest that ezrin is strongly enriched in ezrin and F-actin and were similar to those initially linked to F-actin and occupies the periphery of the T observed in cells activated for 15 min with Staphylococcus aureus cell–APC contact zone, whereas at later times it is concen- enterotoxin E (SEE)-pulsed APCs (10). In contrast, beads trated in the center. How ezrin clustering occurs is not coated with Abs directed to transmembrane proteins that inter- known. The ability of ezrin to oligomerize could be a possible act with ezrin, such as ICAM-2, ICAM-3, or CD43, did not in- mechanism (17), but whether this implies that ezrin detaches duce morphological changes or ezrin accumulation. This indi- from F-actin to accumulate in a more compact zone will need cated that ezrin relocalization at the T cell–APC contact zone further investigation. In addition, the ability of ezrin to as- depends on TCR signaling. Consistently, anti-CD3-induced sociate with membrane rafts (52, 53 and our unpublished ezrin relocalization was impaired in Lck-deficient Jurkat cells data) could also favor its clustering in the contact area. In- (JCaM-1.6), as well as in wild-type Jurkat cells treated with the deed, an overlap between ezrin and lipid rafts was observed Src inhibitor PP-1 (10). Therefore, these results indicate that in the contact zone (53). the initial ezrin accumulation at the contact site is linked to po- We investigated the molecular mechanism responsible for larized actin polymerization induced by TCR signaling at the ezrin relocalization at the T cell–APC contact zone. Two pos- APC contact site. Fig. 1. A: Domain and functional organization of ezrin. The N-terminal 300 amino acids of the molecule is the region of higher sequence homology between all members of the family represented by the band 4.1 of erythrocytes. In particular, ezrin, radixin and moesin (ERMs) are about 87% homologous in this domain. This is a functional domain where the interactions with membrane components, phospholipids and various signaling molecules occur. The FERM domain is followed by a less conserved region, the a-domain, predicted to form an a-helical coiled-coil. The last 80 amino acids form a hydrophilic domain also highly homologous in ERMs. The C-terminal 34 residues of this domain contain a binding site for F-actin. B: Activation of ezrin. ERMs are in an equilibrium between two conformations: a closed form, which has the F-actin and membrane-binding sites masked and is cytosolic, and an open form, which can bind to both the membrane and the cytoskeleton. In addition, open conformations can form homo- or hetero-oligomers, by virtue of the interaction between N- and C-terminal domains. The open conformation is stabilized by phosphorylation of a conserved threonine residue in the C-terminal domain, and by binding of phospholipids (16, 17). Immunological Reviews 189/2002 125
    • Das et al ¡ Membrane-cytoskeleton links in the IS Early on, ezrin concentrates in a peripheral zone of the only in activated T cells (57). This indicates that a complex T cell–APC contact site outside the TCR clustering area, but set of phenomena involving interactions between ERMs and coinciding with CD43 (10). CD43 is a highly glycosylated various adhesion molecules takes place during T cell–APC in- transmembrane molecule that interacts with the FERM do- teractions. This may be important for setting up the appropri- main of ERMs (26, 29). CD43 is excluded from the center of ate membrane–cytoskeleton interactions necessary for T-cell the immunological synapse upon T-cell activation in an ezrin- stimulation by APCs. In summary, our data indicate that ezrin and/or moesin-dependent manner (8, 9, 11, 56). It is worth is part of the actin cytoskeleton-mediated scaffold involved in noting here that Allespach et al. (8) reported an antipodal the molecular architecture of the immunological synapse. 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 A method of quantitative image analysis for by us, either in Jurkat or in human peripheral blood T cells, evaluating morphological changes and molecular or reported by others (9, 11, 53), suggesting differences be- polarization at the T cell–APC contact zone tween the various experimental systems. Interestingly, Mon- toya et al. (57) reported that ICAM-3 and P-selectin glyco- In order to have a more accurate measurement of cytoskeletal protein ligand (PSGL)-1, which also interact with ezrin and modifications during early stages of T lymphocyte responses, moesin (30), accumulated at opposite poles of the T cell dur- we developed a method of quantitative image analysis capable ing APC interactions. Thus, ICAM-3 was relocated to the APC of evaluating morphological changes, as well as the molecular contact site in an antigen-independent manner, whereas accumulation of various molecules at the T cell–APC contact PSGL-1 relocated to the pole away from the APC contact site zone. This method obtains complementary information from Fig. 2. Early stages of T lymphocyte interaction with stimulatory APCs: morphological changes and actin cytoskeleton rearrangements. T cells dock on APCs and scan their surface. If the specific antigen-MHC complexes are detected, the T cell stops or slows down its movement and triggers early signaling, such as calcium fluxes. This is followed by strong morphological changes, which depend on actin cytoskeleton dynamics. At this time, active actin polymerization takes place at the site of contact with the APC, which results in an enlargement of the area of contact of the T cell on the APC. Finally, the T cell rounds up and the area of contact again becomes more reduced. This coincides with the coalescence and segregation of supramolecular clusters at the contact site, a phenomenon that marks the maturation of the immunological synapse. 126 Immunological Reviews 189/2002
    • Das et al ¡ Membrane-cytoskeleton links in the IS multimodal imaging and utilizes active contours to define the those taking place between two T cells or two APCs. One cell borders in a semiautomatic manner (Fig. 4). should therefore use criteria to distinguish the two cell types. Three types of images were acquired using a CCD camera, In our experimental system, we took advantage of the fact nuclear staining, phase contrast and cytoskeleton staining. that APCs (Raji cells) displayed a nuclear morphology differ- Nuclear staining allows individualization of each cell and pro- ent from that of T cells (Jurkat T cells). Thus, Raji nuclei have vides the initial contours (Fig. 4A). These initial contours are a more irregular shape than those of Jurkat T cells (Fig. 4A, then projected on the phase contrast image of the same field asterisks). of cells (Fig. 4B). Initial contours then evolve on the phase Once the cell perimeters are defined, the shape of the cells contrast image to fit the cell perimeter, thus providing cell can be estimated by calculating the normalized compactness profiles in two dimensions (Fig. 4C). The sites where the con- (Cn), which has a value of 0 in the case of a perfect circle, tours of two different cells encounter each other define the and a value of 1 in the case of an elongated shape. By this cell–cell contacts (Fig. 4C, D, arrows). Finally, the extracted con- means, morphological changes can be quantified (Fig. 4E). For tours are projected on the fluorescence image, allowing analy- instance, we determined that the compactness of T cells in sis of the ratio of fluorescence between the cell–cell contact conjugates formed in the absence of superantigen stimulation area and the rest of the cell (Fig. 4D, F). was similar in Jurkat T cells and in APCs. In contrast, the A difficulty may be encountered when trying to distinguish coefficient of compactness of activated T cells at 15 min was cell–cell contacts occurring between a T cell and an APC from higher (V. Das et al., in preparation). Fig. 3. Relocalization of ezrin to the T cell– APC contact site. Jurkat T cells interacting with Raji B cells in the absence (A) or in the presence of Staphylococcus aureus enterotoxin E (SEE) superantigen. Cells were activated, fixed, stained with anti-TCR-CD3 and anti-ezrin Abs (A-E), or anti-TCR-CD3 Abs and phalloidin (F, G), and analyzed by confocal microscopy as previously described (10). A medial optical section is shown in each image. At 15 min, ezrin accumulates in membrane extensions that engulf the APC (B) and is more concentrated in the peripheral area of the immunological synapse (C, red), surrounding the more central TCR clusters (C, green). At 30 min, the area of contact appeared more reduced, ezrin was still concentrated in the contact zone but in an area that overlapped with the TCR cluster (D, E), whereas F-actin appeared less concentrated and remained in the periphery of the contact zone (F, G). Bar Ω 5 mm. Immunological Reviews 189/2002 127
    • Das et al ¡ Membrane-cytoskeleton links in the IS After determining the intersection of cell contours, as de- A role for ezrin in TCR-CD3 clustering at the scribed above, we quantified the number of pixels having immunological synapse: quantitative imaging analysis values above background levels within the contact zone, or outside this area. We could then calculate a ‘relocalization Molecular polarization and subsequent segregation into dis- index’ R (Fig. 4F). For instance, when we measured the ac- tinct clusters in the immunological synapse require an intact cumulation of F-actin at the T cell–APC junctions, we calcu- actin-myosin cytoskeleton (6, 45, 46). Ezrin is known to link lated that activated T cells at 15 min had a value of R 0.9, the membrane with the actin cytoskeleton and could there- whereas for non-activated cells, its value was R 0.4 (V. Das fore facilitate molecular polarization and/or clustering in the et al., in preparation). Although this method still requires sys- T cell–APC contact zone. To investigate this possibility, we tematization to analyze large numbers of samples, it already studied the effect of a dominant negative mutant of ezrin. provides more precise data on cell shape and cytoskeletal re- This mutant contains only the FERM domain, which interacts modeling during T cell–APC interactions. with the membrane but lacks the main F-actin-binding re- Fig. 4. New method for quantitative analysis of morphological changes and molecular polarization 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. Three types of images acquired using a CCD camera were used in this example: nuclear staining, phase contrast and cytoskeleton staining. Nuclear staining allows individualization of each cell and provides the initial contours (A), which are then projected on the phase contrast image of the same field of cells (B). Initial contours then evolve on the phase contrast image to fit the cell perimeter, thus providing cell profiles in two dimensions (C). The sites where the contours of two different cells encounter each other define the cell–cell contacts (C, D, arrows). Finally, the extracted contours are projected on the fluorescence image (D, F). From the contours, the shape of the cell can be estimated by calculating the normalized compactness (Cn), according to the formula, where A is the area and P the perimeter. Cn 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 these means, morphological changes can be quantified. An example of Cn calculated for an activated T cell (E, left), vs. a control T cell (E, right) is shown. After determining the intersection of cell contours, the molecular polarization could be estimated. The number of pixels having values above background were quantified within the contact zone (Njunction), or outside this area (NCjunction). A ‘relocalization index’ R was calculated according to the formula (F, bottom). 128 Immunological Reviews 189/2002
    • Das et al ¡ Membrane-cytoskeleton links in the IS gion (18). It has previously been shown to inhibit morpho- this phenomenon, we performed quantitative image analysis genesis and migration of epithelial cells (58). We observed of TCR clustering in cells overexpressing dominant negative that overexpression of this ezrin mutant inhibited the coalesc- ezrin, wild type ezrin, or an irrelevant protein (i.e. GFP). To ence of dispersed TCR clusters into more compact ones, a this end, confocal images were utilized. A z-series of 20 ‘xy’ characteristic of the immunological synapse in mature optical sections was acquired. The region corresponding to lymphocytes (6, 46). the contact zone was then extracted over the whole z-series, As shown in Fig. 5, Jurkat T cells activated with bacterial and an ‘xz’-projection of this region was obtained, using superantigen-pulsed Raji B cells formed immunological Zeiss LSM-510 confocal microscope and software. synapses similar to those previously described in mouse T A computer program was developed that detects, counts cells activated with peptide antigen-bearing B cells, or with and measures clusters in these projection images. This pro- artificial lipid bilayers presenting major histocompatibility gram is based on the multiscale product of subband images complex (MHC)-peptide complexes (6, 46). Thus, we ob- resulting from an undecimated wavelet transform decompo- served a compact cluster of TCRs embedded into a larger ac- sition of the original image, after thresholding of nonsignifi- cumulation of adhesion molecules, whose density was higher cant coefficients (59). To characterize the spatial distribution in the peripheral contact zone. This indicates that superanti- of clusters, the algorithm then computes the surface of gen activation of Jurkat cells triggers molecular polarization bounding area that includes all the detected clusters. By these and segregation, forming a canonical immunological synapse. means, TCR clusters could be individualized and quantified We observed that immunological synapses formed by Jurkat with respect to their number, individual surface and form, T cells transfected with dominant negative ezrin displayed a and spatial distribution (Fig. 6). This method provides a meas- TCR clustering pattern often more dispersed than that shown urement of the phenomenon of coalescence of small TCR by untransfected cells. This suggested that dominant negative clusters into one or few more compact ones, which is a fea- ezrin perturbs TCR clustering during maturation of the im- ture of immunological synapse maturation (6, 46, 48). munological synapse. To have a more accurate estimation of This quantitative image analysis revealed that immunolog- Fig. 5. Immunological synapses formed between Jurkat T cells and superantigen-pulsed Raji B cells. Cells were activated for 30 min, then fixed, stained with anti-TCR-CD3, anti-ICMA-1 Abs and analyzed by confocal microscopy as previously described (10). A: TCR and ICAM-1 concentrate in the contact zone. The TCR cluster occupies a restricted central zone, which coincides with the accumulation of the adhesion molecule 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 the contact zone (A, right panel), carried out using Imaris software (Bitplane, Switzerland). Note that ICAM-1 is expressed only in the APC and likely reflects the accumulation of its counter adhesion receptor LFA-1 expressed on the T cell. The staining of surface molecules from the T cell and the APC gives a view of the tightness of the synapse. B: TCRs and membrane rafts accumulate in the center of the contact zone. TCRs and membrane rafts were stained with anti-TCR-CD3 Abs and cholera toxin B fragment, respectively. Both TCRs and membrane rafts cluster in the center of the T cell–APC contact zone, as observed in the zx-projection of the contact zone, carried out using Zeiss LSM-510 software (right panel). Immunological Reviews 189/2002 129
    • Das et al ¡ Membrane-cytoskeleton links in the IS ical synapses formed by T cells overexpressing dominant Role of F-actin-ezrin complexes in molecular clustering at negative ezrin displayed significant higher numbers of TCR the immunological synapse: a working model clusters, which had smaller individual surface and were spread on a larger area, than cells overexpressing wild type A possible model to explain ezrin’s involvement in TCR clus- ezrin or the irrelevant GFP protein (10 and our unpublished tering could be the following (Fig. 7): TCR triggering induces data). This finding suggests that ezrin provides a link between the polarization of F-actin and ezrin to the APC contact zone, membrane components and the actin cytoskeleton that facili- producing membrane extensions that cover a large surface of tates the coalescence of TCR clusters. the APC (Fig. 3B). Ezrin-binding proteins, such as CD43, The effect of the dominant negative mutant of ezrin on would be anchored, creating a ‘collar’ of cytoskeleton-an- molecular rearrangements at the T cell–APC contact zone was chored transmembrane proteins at the periphery of the con- not restricted to the TCR. It also affected a key signaling mol- tact zone (10). This collar would function as a transmem- ecule that accumulates at the immunological synapse, PKCq brane ‘fence’ to reduce the diffusion of TCRs out of the con- (60). Thus, we observed that the number of T cell–APC con- tact zone. This process would occur early during T cell–APC jugates displaying an accumulation of PKCq was significantly interactions (5–15 min under our experimental conditions, diminished in cells overexpressing dominant negative ezrin Fig. 3B, C) and would facilitate initial concentration of TCRs with respect to controls (10). In contrast, Allenspach et al. into small clusters. Later, membrane extensions retract, al- (8) did not report this type of inhibition. The reasons for this though ezrin remains accumulated in this area. At this time a discrepancy are not clear at present. Our data suggest that constriction of the F-actin–ezrin collar occurs concomitantly ezrin helps the targeting (or the retention) of signaling com- with the reduction of the cell–cell contact area (Fig. 3D, E, plexes to the T cell–APC contact zone as well as the coalesc- 30 min). This constriction of the cytoskeleton collar could ence of clusters in this area, phenomena which are likely compress dispersed small TCR clusters, facilitating their co- needed for T-cell activation. alescence. 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’ optical sections 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 clusters are smaller and more dispersed) and mature (B: TCR cluster is condensed), respectively. XZ-projections were then transformed (C) to obtain an image 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 surface and 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 and occupied a smaller surface than in the upper panel), and mature synapse (lower panel, where TCR clusters appeared condensed in a continuous large cluster). The square in the transformed images represents the bounding area occupied by the clusters). 130 Immunological Reviews 189/2002
    • Das et al ¡ Membrane-cytoskeleton links in the IS The TCR clustering observed may be part of a more general which lacks the C-terminal half of the molecule, and is there- phenomenon involving cholesterol and sphingolipid-enrich- fore unable to bind to actin fibers and to oligomerize. ed membrane microdomains, also called membrane rafts (61). In T cells, membrane rafts contain TCRs and signaling The actin-cytoskeleton associated protein coronin-1 is molecules (62, 63), and concentrate at the T cell–APC contact transiently recruited to the T cell–APC contact area zone (64) (Fig. 5B) upon activation. Interestingly, ERMs can interact with membrane rafts, as assessed by sucrose gradient First described in Distyostelium discoideum, coronin and coronin- buoyancy (53 and our unpublished data). This may facilitate like proteins are expressed in many eukaryotic species from raft coalescence in this zone. yeast to mammals. They have been described as actin-binding According to this model, a crucial step for cluster coalescence proteins implicated in cellular processes involving actin cyto- would be the constriction of the F-actin–ezrin network and the skeleton dynamics, such as cell migration, cytokinesis, phago- concomitant reduction of the T cell–APC contact area, which cytosis, or morphogenesis (65). Coronins from different spe- we observed between 15 and 30 min (Figs 3 and 7). This con- cies display a conserved structural organization. Common fea- striction could occur by shifting from a state of high actin poly- tures include 5 WD repeats, a coiled-coil domain at the C- merization dynamics, which generates the membrane exten- terminus, and a linker sequence in between of variable length sions observed at early times, to a state of lower actin poly- referred to as the unique region. Similar to WD-containing Gb merization and higher cross-linking of actin fibers. In addition, proteins (66), WD repeats of coronins are thought to be organ- ezrin could also oligomerize in this area. The cytoskeleton col- ized in a b-propeller domain, likely involved in protein interac- lar would then become a more compact and stable structure, tions. Coronin-1 is the mammalian paralogue specifically en- which could restrict molecular diffusion and stabilize supra- riched in hematopoietic tissues. Depending on the cell lineage, molecular clusters. These phenomena would be perturbed by coronin-1 appears to have different molecular interactions and, the overexpression of the dominant negative mutant of ezrin, 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 Fig. 7. Working model to explain the role of F-actin-ezrin complexes in molecular clustering at the immunological and ezrin are enriched in these membrane structures; they synapse. TCR triggering induces the polarization of F-actin and partially overlap, although they have distinct relative spatial ezrin to the APC contact zone producing membrane extensions localizations. Coronin-1 is more enriched in the area closer that cover a large surface of the APC. We propose that this anchors ezrin-binding proteins, such as CD43, creating a ‘collar’ of to the cell body, F-actin in the middle and ezrin in the more cytoskeleton-anchored transmembrane proteins at the periphery of cortical area (10) (Fig. 8B). In contrast, at later times, coronin- the contact zone. This collar would function as a transmembrane 1 accumulation in the contact area mostly disappeared, ‘fence’ reducing the diffusion of TCRs out of the contact zone and whereas ezrin remained accumulated (Figs. 3D, E, 8F, G). facilitating the initial concentration of TCRs into small clusters (2). Later on, membrane extensions retract although ezrin remains Interestingly, coronins were shown to be related to actin accumulated in this area. At this time a constriction of the F- filament assembly rather than to stability. For instance, co- actin-ezrin collar occurs which is concomitant to the reduction of ronins were found enriched in actin tails induced by Listeria the cell–cell contact area. This constriction of the cytoskeleton collar could compress dispersed small TCR clusters facilitating their monocytogenes in infected cells, at phagocytic cups, in lamellipo- coalescence into a single cluster (3). dia and in membrane ruffles, but less in stress fibers (68, 70– Immunological Reviews 189/2002 131
    • Das et al ¡ Membrane-cytoskeleton links in the IS 72 and our unpublished data). Coronin-1 could therefore be cells inhibited the activation of nuclear factor for activated T considered a marker for local active actin polymerization. We cells in response to stimulation to peptide antigen or super- suggest that the transient accumulation of coronin-1 at the antigen (10). Moreover, Allenspach et al. (8) also reported T cell–APC contact area reflects distinct actin polymerization that T cells from TCR transgenic mice overexpressing a similar dynamics along the maturation of the immunological syn- dominant negative mutant of ezrin had a reduced capacity to apse, very intense at early times and much lower at later produce various cytokines in response to antigenic stimula- times. tion. How ezrin and moesin are involved in T-cell activation is not clear at present. As described above, ezrin may help mol- Ezrin is necessary for T-cell activation leading to IL2 gene ecular reorganization at the immunological synapse and, as a transcription consequence, help signaling. Ezrin and moesin could inter- Definite evidence for the functional importance of ezrin in T vene in the clustering of receptors and signaling molecules lymphocyte function came from the observation that overex- (10). This could occur either directly, via interaction with pression of the dominant negative mutant of ezrin in Jurkat lipid rafts (12, 53), or by actively removing highly glycosylat- Fig. 8. Coronin-1 is transiently recruited to the T cell–APC contact zone. Jurkat T cells interacting with Raji B cells in the absence (A, C), or in the presence of SEE superantigen (B, D-G). Cells were activated, fixed, stained with anti-coronin-1 Abs and phalloidin (A, B), or anti-coronin-1 and anti-TCR-CD3 (C-G), and analyzed by confocal microscopy as previously described (10). At 15 min, coronin-1 was accumulated in the membrane extensions that contact the APC, partially overlapping with F- actin, but more concentrated in an area closer to the cell body (B). When localized with respect to the TCR, coronin-1 occupied the periphery of the T cell–APC contact zone (D, E). At later times, coronin-1 accumulation was strongly reduced (F). When compared with the TCR, it appeared staining the peripheral contact area, being rather excluded from the area where the TCR cluster is located (G). Bar Ω 5 mm. 132 Immunological Reviews 189/2002
    • Das et al ¡ Membrane-cytoskeleton links in the IS ed molecules, such as CD43 (8, 9, 11), or PSGL-1 (57), negative ezrin in late activation events, such as cytokine gene whose presence could destabilize T cell–APC interactions and activation (10). therefore activation. It might also act by facilitating initial in- Clearly, more work is necessary to elucidate the role of the teractions between T cells and APC (57). Ezrin and moesin actin cytoskeleton, and in particular the role of ezrin and co- might also intervene in one or various T-cell signaling path- ronin-1 in early T-cell functions. The role of these proteins in ways, by directly interacting with signal transduction mol- the molecular reorganization of the T cell–APC contact zone ecules, as shown in other experimental systems (17). 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