Extracellular vesicles such as exosomes are now recognized as key players in intercellular communication. Their role is influenced by the specific repertoires of proteins and lipids, which are enriched when they are generated as intraluminal vesicles (ILVs) in multivesicular endosomes. Here we report that a key component of small extracellular vesicles, the tetraspanin CD63, sorts cholesterol to ILVs, generating a pool that can be mobilized by the NPC1/2 complex, and exported via exosomes to recipient cells. In the absence of CD63, cholesterol is retrieved from the endosomes by actin-dependent vesicular transport, placing CD63 and cholesterol at the centre of a balance between inward and outward budding of endomembranes. These results establish CD63 as a lipid-sorting mechanism within endosomes, and show that ILVs and exosomes are alternative providers of cholesterol. Palmulli, Couty and colleagues show that the tetraspanin CD63 promotes accumulation of cholesterol in intraluminal vesicles (ILVs) at the expense of retrieval from endosomes; cholesterol stored in ILVs and exosomes is recovered in an NPC1-dependent manner.

The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, their impact on EV composition remains under-investigated. In the MCF7 breast cancer cell line CD63 was as expected predominantly intracellular. In contrast CD9 and CD81 strongly colocalized at the plasma membrane, albeit with different ratios at different sites, which may explain a higher enrichment of CD81 in EVs. Absence of these tetraspanins had little impact on the EV protein composition as analysed by quantitative mass spectrometry. We also analysed the effect of concomitant knock-out of CD9 and CD81 because these two tetraspanins play similar roles in several cellular processes and associate directly with two Ig domain proteins, CD9P-1/EWI-F/PTGFRN and EWI-2/IGSF8. These were the sole proteins significantly decreased in the EVs of double CD9- and CD81-deficient cells. In the case of EWI-2, this is primarily a consequence of a decreased cell expression level. In conclusion, this study shows that CD9, CD81 and CD63, commonly used as EV protein markers, play a marginal role in determining the protein composition of EVs released by MCF7 cells and highlights a regulation of the expression level and/or trafficking of CD9P-1 and EWI-2 by CD9 and CD81.

T cells depend on the phosphatase CD45 to initiate T cell receptor signaling. Although the critical role of CD45 in T cells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in T cell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and T cell activation. CD53-negative T cells (Cd53(-/-)) exhibit substantial proliferation defects, and Cd53(-/-) mice show impaired tumor rejection and reduced IFNg-producing T cells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for T cell immunity.

Exosomes are small extracellular vesicles that are enriched in specific proteins and lipids during their generation as intraluminal vesicles in multivesicular endosomes. Contrary to proteins, the endosomal sorting mechanisms of lipids on intraluminal vesicles are still ill-defined. Here, we find that the tetraspanin CD63, highly enriched in ILVs and exosomes, regulates cholesterol sorting on ILVs and exosomes. This process generates a pool of cholesterol that affects exosomes physical properties and can be mobilized by the NPC1/2 complex from ILVs and exosomes. Absence of CD63 redirects cholesterol from endosomes to the Trans-Golgi Network by actin dependent vesicular transport, placing CD63 and cholesterol at the center of a balance between inward and outward budding of endomembranes. These results establish CD63 as cholesterol sorting mechanism within endosomes and place ILVs and exosomes as alternative providers of cholesterol.

CD63, a member of the tetraspanin superfamily, is used as a marker of late endosomes and lysosome-related organelles, as well as a marker of exosomes. Here, we selected rare isotype variants of TS63 by sorting hybridoma cells on the basis of their high expression of surface immunoglobulins of the IgG2a and IgG2b subclass. Pure populations of cells secreting IgG2a and IgG2b variants of TS63 (referred to as TS63a and TS63b) were obtained using two rounds of cell sorting and one limited dilution cloning step. We validate that these new TS63 variants are suitable for co-labeling with mAb of the IgG1 subclass directed to other molecules, using anti mouse subclass antibodies, and for the labeling of exosomes through direct binding to protein A-coated gold particles. These mAbs will be useful to study the intracellular localization of various proteins and facilitate electron microscopy analysis of CD63 localization.

T cells are central to the adaptive immune response, playing a role in both the direct and indirect killing of pathogens and transformed cells. The activation of T cells is the result of a complex signaling cascade, initiated at the T cell receptor (TCR), and ending with the induction of proliferation. CD45, a member of the protein tyrosine phosphatase family, is one of the most abundant membrane proteins on T cells and functions by regulating activation directly downstream of the TCR. As a result of alternative splicing, CD45 can be expressed in multiple isoforms, naive T cells express the CD45RA isoform, while activated T cells gain expression of CD45RO, which has been proposed to increase signaling. Though the importance of CD45 in TCR signaling, proliferation and cytokine production is well established, little is known about the regulation of CD45 activity. We discovered that the immune-specific tetraspanin CD53 directly affects the stability and function of CD45RO in T cells. We have identified CD53 as a T cell co-stimulatory molecule in primary human and murine cells. Furthermore, we have shown that the absence of CD53 leads to an altered CD45 isoform expression as a result of decreased CD45RO stability on the cell surface. This instability was accompanied by increased mobility as measured by FRAP. Together, this indicates that CD53 functions as a stabilizer of CD45RO, and therefore as a positive regulator of TCR signaling at the T cell surface. Our data provides novel insight into the role of tetraspanins in the regulation of immune signaling and may provide a new avenue for the regulation of T cell signaling.

ADAM10 is a transmembrane metalloprotease that is essential for development and tissue homeostasis. It cleaves the ectodomain of many proteins, including amyloid precursor protein, and plays an essential role in Notch signaling. ADAM10 associates with six members of the tetraspanin superfamily referred to as TspanC8 (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33), which regulate its exit from the endoplasmic reticulum and its substrate selectivity. We now show that ADAM10, Tspan5, and Tspan15 influence each other's expression level. Notably, ADAM10 undergoes faster endocytosis in the presence of Tspan5 than in the presence of Tspan15, and Tspan15 stabilizes ADAM10 at the cell surface yielding high expression levels. Reciprocally, ADAM10 stabilizes Tspan15 at the cell surface, indicating that it is the Tspan15/ADAM10 complex that is retained at the plasma membrane. Chimeric molecules indicate that the cytoplasmic domains of these tetraspanins contribute to their opposite action on ADAM10 trafficking and Notch signaling. In contrast, an unusual palmitoylation site at the end of Tspan15 C-terminus is dispensable. Together, these findings uncover a new level of ADAM10 regulation by TspanC8 tetraspanins.

Tspan5 is a member of a subgroup of tetraspanins referred to as TspanC8. These tetraspanins directly interact with the metalloprotease ADAM10, regulate its exit from the endoplasmic reticulum and subsequent trafficking, and differentially regulate its ability to cleave various substrates and activate Notch signaling. The study of Tspan5 has been limited by the lack of good antibodies. This study provides new insights into Tspan5 using new monoclonal antibodies (mAbs), including two mAbs recognizing both Tspan5 and the highly similar tetraspanin Tspan17. Using these mAbs, we show that endogenous Tspan5 associates with ADAM10 in human cell lines and in mouse tissues where it is the most abundant, such as the brain, the lung, the kidney, or the intestine. We also uncover two TspanC8-specific motifs in the large extracellular domain of Tspan5 that are important for ADAM10 interaction and exit from the endoplasmic reticulum. One of the anti-Tspan5 mAbs does not recognize Tspan5 associated with ADAM10, providing a convenient way to measure the fraction of Tspan5 not associated with ADAM10. This fraction is minor in the cell lines tested, and it increases upon transfection of cells with TspanC8 tetraspanins such as Tspan15 or Tspan33 that inhibit Notch signaling. Finally, two antibodies inhibit ligand-induced Notch signaling, and this effect is stronger in cells depleted of the TspanC8 tetraspanin Tspan14, further indicating that Tspan5 and Tspan14 can compensate for each other in Notch signaling.

By interacting directly with partner proteins and with one another, tetraspanins organize a network of interactions referred to as the tetraspanin web. ADAM10 (A Disintegrin And Metalloprotease 10), an essential membrane-anchored metalloprotease that cleaves off the ectodomain of a large variety of cell surface proteins including cytokines, adhesion molecules, the precursor of the β-amyloid peptide APP or Notch, has emerged as a major component of the tetraspanin web. Recent studies have shown that ADAM10 associates directly with all members (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33) of a subgroup of tetraspanins having eight cysteines in the large extracellular domain and referred to as TspanC8. All TspanC8 regulate ADAM10 exit from the endoplasmic reticulum, but differentially regulate its subsequent trafficking and its function, and have notably a different impact on Notch signaling. TspanC8 orthologs in invertebrates also regulate ADAM10 trafficking and Notch signaling. It may be possible to target TspanC8 tetraspanins to modulate in a tissue- or substrate-restricted manner ADAM10 function in pathologies such as cardiovascular diseases, cancer or Alzheimer's disease.

Tspan5 is a member of a subgroup of tetraspanins referred to as TspanC8. These tetraspanins directly interact with the metalloprotease ADAM10, regulate its exit from the endoplasmic reticulum and subsequent trafficking, and differentially regulate its ability to cleave various substrates and activate Notch signaling. The study of Tspan5 has been limited by the lack of good antibodies. This study provides new insights into Tspan5 using new monoclonal antibodies (mAbs), including two mAbs recognizing both Tspan5 and the highly similar tetraspanin Tspan17. Using these mAbs, we show that endogenous Tspan5 associates with ADAM10 in human cell lines and in mouse tissues where it is most abundant such as the brain, the lung, the kidney or the intestine. We also uncover two TspanC8-specific motifs in the large extracellular domain of Tspan5 that are important for ADAM10 interaction and exit from the endoplasmic reticulum. One of the antiTspan5 mAb does not recognize Tspan5 associated with ADAM10, providing a convenient way to measure the fraction of Tspan5 not associated with ADAM10. This fraction is minor in the cell lines tested, and increases upon transfection of cells with TspanC8 tetraspanins such as Tspan15 or Tspan33 that inhibit Notch signalling. Finally, two antibodies inhibit ligand-induced Notch signalling, and this effect is stronger in cells depleted of the TspanC8 Tspan14, further indicating that Tspan5 and Tspan14 can compensate for each other in Notch signalling. Tetraspanins form a family of proteins with 4 transmembrane domains expressed by all metazoans. These proteins possess a number of specific features, including conserved residues and a specific fold in the largest of the two extracellular domains (the Large Extracellular Loop or LEL) that differentiate them from other proteins with 4 transmembrane domains (1-4). Identification of pathological mutations in humans, as well as genetic approaches in the mouse or invertebrates have shown the importance of these molecules. In mammals, particular tetraspanins have for example been shown to play a key role in sperm-egg fusion, vision, kidney function, immunity or muscle regeneration (1-4). A remarkable property of the most characterized tetraspanins is to associate at the cell surface with one another and with nontetraspanin integral proteins to organize a network of interaction referred to as the “tetraspanin web” or tetraspanin-enriched microdomains. In this network, tetraspanins associate directly and specifically with a limited number of molecular partners that they connect to other tetraspanins. Well characterized primary complexes include the complexes formed by CD151 (Tspan24) and the laminin-binding integrins α3β1 and α6β1 as well as the complex formed by CD81 (Tspan28) and CD19, a costimulatory molecule of B lymphocytes. In addition, CD81 shares with CD9 (Tspan29) two common partners, CD9P-1/EWI-F and EWI-2 which are Ig domain proteins of unknown function (1-4). Tspan5 is a highly conserved tetraspanin: the human, mouse and rat proteins are completely identical, and share 91%, 44% and http://www.jbc.org/cgi/doi/10.1074/jbc.M116.765669 The latest version is at JBC Papers in Press. Published on April 20, 2017 as Manuscript M116.765669 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. at ST A T E U N IV O F N E W Y O R K on A ril 2, 2017 hp://w w w .jb.org/ D ow nladed from Characterization of the tetraspanin Tspan5 2 38% identity with the closest orthologs found in D. rerio, D. melanogaster and C. elegans respectively (5-8). Tspan5 is a member of a subgroup of tetraspanins that have 8 cysteines in the LEL (others have 6 or 4 cysteines), and are consequently referred to as TspanC8 (7-10). Mammals express 6 of these TspanC8 tetraspanins which share a common partner, the metalloprotease ADAM10, a member of the ADAM (A Disintegrin And Metalloprotease domain) family of metalloproteases (8,10,11). These membrane-anchored enzymes mediate a proteolytic cleavage of various transmembrane proteins within their extracellular region, a process referred to as ectodomain shedding (12,13). ADAM10 cleaves off the ectodomain of more than 40 transmembrane proteins, including cytokine and growth factor precursors, as well as adhesion proteins such as E and N-cadherins (13). Notably, ADAM10-mediated cleavage of the amyloid precursor protein (APP) prevents the formation of the amyloid peptide A β, a major component of amyloid plaques observed in Alzheimer’s disease (14). ADAM10 plays also an essential role in Notch signalling: Notch ectodomain cleavage by ADAM10 allows a second cleavage by the γ-secretase complex that results in the release of Notch intracellular domain and its translocation to the nucleus where it acts as a transcriptional cofactor. (15-18). TspanC8 tetraspanins regulate several aspects of ADAM10. They all regulate the exit of ADAM10 from the ER, and target it either to late endosomes (Tspan10, 17) or the plasma membrane (Tspan5, 14, 15, 33) (8,10,11). In addition, TspanC8 tetraspanins modulate the substrate specificity of ADAM10 (19,20). In particular, Tspan5 and Tspan14 are positive and Tspan15 and Tspan33 negative regulators of Notch signalling (8,19). Also, of all TspanC8 tetraspanins tested, only Tspan15 was shown to regulate ADAM10-mediated cleavage of Ncadherin (11,19,20). These functional differences may be the result of a different action of TspanC8 on ADAM10 membrane compartmentalization (19). Alternatively, TspanC8 might direct substrate specificity by constraining ADAM10 into defined conformations (20) In the absence of good antibodies, the study of Tspan5 and other TspanC8 has relied on the transfection of tagged molecules, with potential pitfalls arising from over-expression or the addition of a tag. Here we report on the generation of anti-Tspan5 monoclonal antibodies and use them to investigate several aspects of Tspan5, including its expression profile, subcellular localization, and the interaction of the endogenous protein with ADAM10 and with the tetraspanin web. We also show that two of these mAbs inhibit ligand-induced Notch signaling. RESULTS Generation of antibodies recognizing Tspan5. To generate anti-Tspan5 mAbs, we immunized mice twice with U2OS cells stably expressing Tspan5-GFP, and twice with a Tspan5-GFP immunoprecipitate. Because the human, mouse and rat Tspan5 molecules are completely identical, Tspan5 knock-out mice were used. Hybridomas were screened by indirect labelling of live U2OS cells stably expressing Tspan5-GFP and flow-cytometry analysis. Out of more than 3000 clones tested, we isolated nine hybridomas stably secreting antibodies that stained U2OS-Tspan5 cells proportionally to the level of Tspan5-GFP expressed by the cells. Three examples are shown in Fig. 1A. As a control, the labeling by the CD81 antibody did not change according to the GFP signal, and the labelling by the antiADAM10 mAb 11G2 reached a plateau, as previously described (8,19). The characteristics of these antibodies are shown in Table 1. To validate that these antibodies indeed recognize Tspan5, GFP-Tspan5 was immunoprecipitated from U2OS/Tspan5 cells using GFP trap beads after lysis in RIPA buffer (Fig. 1B). This lysis buffer is known to dissociate from tetraspanins most if not all associated proteins. Indeed, under this condition, ADAM10 was no longer coimmunoprecipitated with Tspan5 whereas it is strongly co-immunoprecipitated after lysis with Brij 97 (Fig. 1B). All antibodies tested recognized by western-blot the immunoprecipitated Tspan5 GFP (see 3 examples in Fig. 1B), as shown by the strong ignal perfectly overlapping with the signal obtain with an anti GFP antibody. The mAb TS5-2 was selected for further studies because it gave in the initial characterization one of the strongest signal in western-blot and flow-cytometry. To validate that it recognized the endogenous Tspan5, we turned to colon cancer HCT116 cells which showed the strongest surface staining among various cell lines tested (data not shown). Silencing Tspan5 in these cells by two previously validated siRNA (19) reduced by 7080% the staining by TS5-2 in flow cytometry at ST A T E U N IV O F N E W Y O R K on A ril 2, 2017 hp://w w w .jb.org/ D ow nladed from Characterization of the tetraspanin Tspan5 3 experiments (Fig. 1C). In addition, the mAb TS5-2 recognized by western-blot endogenous levels of Tspan5 in HCT116 and CT26 (respectively a human and a mouse colon cancer cell lines) cells, but not in Tspan5-silenced cells (Fig. 1D,E). Moreover, this mAb immunoprecipitated a major ~27-34 kDa band and a fainter ~22 kDa thin band that were recognized by western-blot by the TS5-2 mAb (Fig. 1D). None of these bands were visualized after silencing Tspan5 further indicating that they both correspond to Tspan5 (possibly to different conformations or glycosylated forms), and that TS5-2 only recognizes Tspan5 in these cells. Specificity of Tspan5 antibodies and demonstration that they bind to the LEL. TspanC8 tetraspanins are characterized not only by the presence of 8 cysteines in the larger extracellular domain, but also by the presence of specific residues not present in other tetraspanins (8). It was therefore important to test whether these anti-Tspan5 antibodies recognized other TspanC8. In a first set of experiments we tested by western-blot whether these antibodies recognized Tspan14, Tspan15 or Tspan33 in the lysates of U2OS cells stably expressing GFPtagged versions of Tspan15, Tspan14 or Tspan33 (19), or after GFP immunoprecipitation. As shown in Fig.2A and summarized in Table I, none of the mAbs recognized these 3 tetraspanins. Similarly, none of these mAbs recognized Tspan17 or Tspan10 by western-blot after transfection in HeLa cells (Fig.2B). However, two antibodies, TS5/17 (Fig. 2) and 20E2 (Table I) strongly stained Tspan17transfected cells, either by flow cytometry analysis (data not shown) or immunostaining of saponin-permeabi

Syncytins are envelope genes from endogenous retroviruses, "captured" for a role in placentation. They mediate cell-cell fusion, resulting in the formation of a syncytium (the syncytiotrophoblast) at the fetomaternal interface. These genes have been found in all placental mammals in which they have been searched for. Cell-cell fusion is also pivotal for muscle fiber formation and repair, where the myotubes are formed from the fusion of mononucleated myoblasts into large multinucleated structures. Here we show, taking advantage of mice knocked out for syncytins, that these captured genes contribute to myoblast fusion, with a >20% reduction in muscle mass, mean muscle fiber area and number of nuclei per fiber in knocked out mice for one of the two murine syncytin genes. Remarkably, this reduction is only observed in males, which subsequently show muscle quantitative traits more similar to those of females. In addition, we show that syncytins also contribute to muscle repair after cardiotoxin-induced injury, with again a male-specific effect on the rate and extent of regeneration. Finally, ex vivo experiments carried out on murine myoblasts demonstrate the direct involvement of syncytins in fusion, with a >40% reduction in fusion index upon addition of siRNA against both syncytins. Importantly, similar effects are observed with primary myoblasts from sheep, dog and human, with a 20-40% reduction upon addition of siRNA against the corresponding syncytins. Altogether, these results show a direct contribution of the fusogenic syncytins to myogenesis, with a demonstrated male-dependence of the effect in mice, suggesting that these captured genes could be responsible for the muscle sexual dimorphism observed in placental mammals.

Antiserum from rabbits immunised with pure human fibrinogen was affinity purified on immobilised fibrin fragment E (FFE). This FFE antibody (Ab) induced significant growth inhibition of a human cancer xenograft in mice and suppression of tumour angiogenesis, leaving no formed vessels and only CD31-staining endothelial fragments in place. Tubule formation of HUVEC on Matrigel (TM) was also significantly inhibited by FFE Ab. Since Matrigel (TM) is fibrin-free, this effect implicated a different FFE Ab binding site than FFE. Flow cytometry of HUVEC showed that FFE Ab bound to HUVEC, but with a broad range of 55-98%. Immunofluorescent staining of HUVEC explained this range, since FFE Ab was seen not to bind to human umbilical vein endothelial cells (HUVEC) directly but instead to a matrix protein variably adherent to HUVEC. This protein was identified as fibronectin (FN) by appearance, staining with FN Ab, and by a FN knockdown study. Neither HUVEC nor matrix reacted with fibrin D-dimer (DD) Ab. Immunofluorescent stains of HUVEC matrix with FFE and FN Ab's showed that these Ab's bound to the same epitopes on FN, as also seen on Western blots of purified FN. These findings indicate the presence of an antigenic determinant in fibrinogen/FFE that is homologous with an epitope(s) in FN recognised by FFEAb, and critical for angiogenesis in this xenograft. The FN epitope(s) remains to be identified, but the present findings can be used for the selection of the appropriate clones from mice immunised with fibrinogen which can facilitate this identification, and which may also be of clinical use.

The metalloprotease ADAM10 mediates the shedding of the ectodomain of various cell membrane proteins, including APP, the precursor of the amyloid peptide Aβ, and Notch receptors following ligand binding. ADAM10 associates with the members of an evolutionary conserved subgroup of tetraspanins, referred to as TspanC8, which regulate its exit from the endoplasmic reticulum. Here we show that 4 of these TspanC8 (Tspan5, Tspan14, Tspan15 and Tspan33) which positively regulate ADAM10 surface expression levels differentially impact ADAM10-dependent Notch activation and the cleavage of several ADAM10 substrates, including APP, N-cadherin and CD44. Sucrose gradient fractionation, single molecule tracking and quantitative mass-spectrometry analysis of the repertoire of molecules co-immunoprecipitated with Tspan5, Tspan15 and ADAM10 show that these two tetraspanins differentially regulate ADAM10 membrane compartmentalization. These data represent a unique example where several tetraspanins differentially regulate the function of a common partner protein through a distinct membrane compartmentalization.

Tetraspanins are a family of proteins with four transmembrane domains that play a role in many aspects of cell biology and physiology; they are also used by several pathogens for infection and regulate cancer progression. Many tetraspanins associate specifically and directly with a limited number of proteins, and also with other tetraspanins, thereby generating a hierarchical network of interactions. Through these interactions, tetraspanins are believed to have a role in cell and membrane compartmentalization. In this Cell Science at a Glance article and the accompanying poster, we describe the basic principles underlying tetraspanin-based assemblies and highlight examples of how tetraspanins regulate the trafficking and function of their partner proteins that are required for the normal development and function of several organs, including, in humans, the eye, the kidney and the immune system.

Skeletal muscle regeneration after injury follows a remarkable sequence of synchronized events. However, the mechanisms regulating the typical organization of the regenerating muscle at different stages remain largely unknown. Here we show that muscle regeneration in mice lacking either CD9 or CD81 is abnormal and characterized by the formation of discrete giant dystrophic myofibres, which form more quickly in the absence of both tetraspanins. We also show that, in myoblasts, these two tetraspanins associate with the immunoglobulin domain molecule CD9P-1 (EWI-F/FPRP), and that grafting of CD9P-1-depleted myoblasts in regenerating muscles also leads to abnormal regeneration. In vitro myotubes lacking CD9P-1 or both CD9 and CD81 fuse with a higher frequency than normal myotubes. Our study unveils a mechanism preventing inappropriate fusion of myotubes that has an important role in the restitution of normal muscle architecture during muscle regeneration.

Cargo sorting to intraluminal vesicles (ILVs) of multivesicular endosomes is required for lysosome-related organelle (LRO) biogenesis. PMEL—a component of melanocyte LROs (melanosomes)—is sorted to ILVs in an ESCRT-independent manner, where it is proteolytically processed and assembled into functional amyloid fibrils during melanosome maturation. Here we show that the tetraspanin CD63 directly participates in ESCRT-independent sorting of the PMEL luminal domain, but not of traditional ESCRT-dependent cargoes, to ILVs. Inactivating CD63 in cell culture or in mice impairs amyloidogenesis and downstream melanosome morphogenesis. Whereas CD63 is required for normal PMEL luminal domain sorting, the disposal of the remaining PMEL transmembrane fragment requires functional ESCRTs but not CD63. In the absence of CD63, the PMEL luminal domain follows this fragment and is targeted for ESCRT-dependent degradation. Our data thus reveal a tight interplay regulated by CD63 between two distinct endosomal ILV sorting processes for a single cargo during LRO biogenesis.

T-cell receptor (TCR) signalling is triggered and tuned at immunological synapses by the generation of signalling complexes that associate into dynamic microclusters. Microcluster movement is necessary to tune TCR signalling, but the molecular mechanism involved remains poorly known. We show here that the membrane-microfilament linker ezrin has an important function in microcluster dynamics and in TCR signalling through its ability to set the microtubule network organization at the immunological synapse. Importantly, ezrin and microtubules are important to down-regulate signalling events leading to Erk1/2 activation. In addition, ezrin is required for appropriate NF-AT activation through p38 MAP kinase. Our data strongly support the notion that ezrin regulates immune synapse architecture and T-cell activation through its interaction with the scaffold protein Dlg1. These results uncover a crucial function for ezrin, Dlg1 and microtubules in the organization of the immune synapse and TCR signal down-regulation. Moreover, they underscore the importance of ezrin and Dlg1 in the regulation of NF-AT activation through p38.

Despite high expression levels at the plasma membrane or in intracellular vesicles, tetraspanins remain among the most mysterious transmembrane molecules 20 years after their discovery. Several genetic studies in mammals and invertebrates have demonstrated key physiological roles for some of these tetraspanins, in particular in the immune response, sperm-egg fusion, photoreceptor function and the normal function of certain epithelia. Other studies have highlighted their ability to modulate cell migration and metastasis formation. Their role in the propagation of infectious agents has drawn recent attention, with evidence for HIV budding in tetraspanin-enriched plasma membrane domains. Infection of hepatocytic cells by two major pathogens, the hepatitis C virus and the malaria parasite, also requires the tetraspanin CD81. The function of tetraspanins is thought to be linked to their ability to associate with one another and a wealth of other integral proteins, thereby building up an interacting network or 'tetraspanin web'. On the basis of the biochemical dissection of the tetraspanin web and recent analysis of the dynamics of some of its constituents, we propose that tetraspanins tightly regulate transient interactions between a variety of molecules and as such favour the efficient assembly of specialized structures upon proper stimulation.

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. They associate with each other in multimolecular complexes containing numerous membrane proteins. As a first step towards the study of the supramolecular organization of tetraspanin complexes, we have implemented a proteomic approach based on in situ protein cross-linking on living cells followed by affinity purification of tetraspanin complexes. This allowed observing the presence of high molecular weight protein complexes that were characterized as containing CD9P-1/CD315 using LC-MS/MS. Western blot analyses and the use of different tags demonstrated the presence of CD9P-1 oligomer in cis-association at cell surface. A significant amount of CD9P-1 oligomer was observed on various cell types. We have shown that CD9P-1 self-associates independently from its association with tetraspanins. However, the expression level of CD9 or CD81 that associate directly and specifically with CD9P-1, positively modulates the cross-linking efficiency of CD9P-1. Thus, tetraspanins can play a role on CD9P-1 oligomerization status.