CD9 Tetraspanin Research Articles

The Tetraspanin CD53 Regulates Early B Cell Development by Promoting IL-7R Signaling.

The tetraspanin CD53 has been implicated in B cell development and function. CD53 is a transcriptional target of EBF1, a critical transcription factor for early B cell development. Further, human deficiency of CD53 results in recurrent infections and reduced serum Igs. Although prior studies have indicated a role for CD53 in regulating mature B cells, its role in early B cell development is not well understood. In this study, we show that CD53 expression, which is minimal on hematopoietic stem and progenitor cells, increases throughout bone marrow B cell maturation, and mice lacking CD53 have significantly decreased bone marrow, splenic, lymphatic, and peripheral B cells. Mixed bone marrow chimeras show that CD53 functions cell autonomously to promote B lymphopoiesis. Cd53-/- mice have reduced surface expression of IL-7Rα and diminished phosphatidylinositol 3 kinase and JAK/STAT signaling in prepro- and pro-B cells. Signaling through these pathways via IL-7R is essential for early B cell survival and transition from the pro-B to pre-B cell developmental stage. Indeed, we find increased apoptosis in developing B cells and an associated reduction in pre-B and immature B cell populations in the absence of CD53. Coimmunoprecipitation and proximity ligation studies demonstrate physical interaction between CD53 and IL-7R. Together, these data, to our knowledge, suggest a novel role for CD53 during IL-7 signaling to promote early B cell differentiation.

The Tetraspanin CD53 Regulates Early B Cell Development By Promoting IL-7R Signaling

The tetraspanin CD53 has been implicated in B cell development and function. Tetraspanins are a family of transmembrane proteins important for organization of the plasma membrane and regulation of cellular migration, adhesion, and activation. CD53 has been shown to be a transcriptional target of EBF1, a critical transcription factor for early B cell development. Additional signaling for early B cell development occurs through the IL-7 receptor (IL-7R), where ligation promotes continued B cell differentiation and pro-survival/anti-apoptotic gene expression. Human deficiency of CD53 results in recurrent infections and reduced serum immunoglobulins. While prior studies have implicated a role for CD53 in regulating mature B cells, its role in early B cell development is not well understood. Herein, we show that CD53 expression rapidly increases throughout B cell development, beginning at the pre-pro-B cell stage. With a CRISPR-generated knockout mouse, we show that Cd53-/- mice have significantly reduced bone marrow (25% fewer, p<0.005), splenic (35% fewer, p<0.05), lymphatic (65% fewer, p<0.0001), and peripheral (30% fewer, p<0.005) B cells compared to wild-type (WT) littermate controls. Mirroring the human phenotype, Cd53-/- mice have significantly reduced serum IgG and IgM (40% reduced, p<0.01). In addition, hematopoietic stem cells isolated from Cd53-/- mice give rise to 30% fewer B cells compared to controls in vitro (p=0.005). Analysis of bone marrow B cell development demonstrates that this loss of B cells originates with early B cell progenitors, which express nearly 50% less IL-7Ra than WT and reduced IL-7 signaling. Using mass cytometry, we identified differential signaling pathways downstream of IL-7R in B cell progenitors. Specifically, we observe impaired PI3K and STAT5 activation in pre-pro- and pro-B cells in the absence of CD53, with a consequent increase in apoptosis in these populations (p<0.01). Decreased STAT5 phosphorylation was confirmed by western blot. Finally, co-immunoprecipitation studies demonstrate a physical interaction between CD53 and IL-7Ra, suggesting that these proteins associate at the cell surface. Together, these data suggest a novel role for CD53 during IL-7 signaling to promote early B cell development. Ongoing studies are focused on determining the CD53 residues required for interaction with IL-7R. Disclosures No relevant conflicts of interest to declare.

JUNO, the receptor of sperm IZUMO1, is expressed by the human oocyte and is essential for human fertilisation.

Is JUNO protein present at the surface membrane of human oocytes and involved in the fertilisation process? JUNO protein is expressed on the plasma membrane of human oocytes and its inhibition by a monoclonal antibody completely blocks gamete fusion. Fusion of gamete membranes is the culminating event of the fertilisation process, but its molecular mechanisms are poorly understood. Until now, three molecules have been shown to be essential: CD9 tetraspanin in the oocyte, Izumo1 protein on the sperm and Juno, its corresponding receptor on the oocyte. Oocyte CD9 and sperm IZUMO1 have been identified in human gametes and their interaction is also well-conserved among several mammalian species. The presence of JUNO on human oocytes, however, has not yet been reported, nor has its role in fertilisation been investigated. We selected an anti-human JUNO antibody in order to investigate the presence of JUNO on the oocyte membrane surface and studied its potential involvement in gamete membrane interaction during fertilisation. Monoclonal antibodies against human JUNO (anti-hJUNO mAb) were produced by immunisation of mice with HEK cells transfected with the putative human JUNO sequence (HEK-hJUNO). These antibodies were used for immunostaining experiments and in vitro fertilisation assays with human gametes (GERMETHEQUE Biobank). Three hybridoma supernatants, verified by immunostaining, revealed specifically HEK-hJUNO cells. The three purified monoclonal antibodies, FJ2E4 (IgG1), FJ8E8 (IgG1) and FJ4F5 (IgG2a), recognised the soluble recombinant human JUNO protein and, in a western blot of HEK-hJUNO extracts, a protein with an expected MW of 25 kDa. In addition, soluble recombinant human IZUMO protein inhibited the binding of anti-hJUNO mAbs to cells expressing hJUNO. Using these anti-hJUNO mAbs in immunostaining, we identified the presence of JUNO protein at the plasma membrane of human oocytes. Furthermore, we revealed a progressive expression of JUNO according to oocyte maturity. Finally, we showed that human zona-free oocytes, inseminated in the presence of anti-hJUNO mAb, were not fertilised by human sperm. These results suggest that, as seen in the mouse, JUNO is indeed involved in human gamete membrane fusion during fertilisation. N/A. In accordance with French bioethics laws, functional tests were performed using zona-free oocytes, which of course does not fully encompass all normal in vivo physiological conditions. However, these in vitro tests do provide direct information regarding sperm-oocyte membrane interactions. Mechanisms of gamete fusion appear to be homologous between mice and humans. However, some differences do exist and analysing the human mechanisms is essential. In fact, this is the first report describing the presence of JUNO on human oocytes and its involvement in human fertilisation. This discovery allows further examination of the understanding of molecular mechanisms that drive gamete fusion: a crucial challenge at a time when infertility affects 16% of reproductively active couples. This work was supported by the Agence Nationale pour la Recherche, Grant no. ANR-13-BVS5-0004, and by Association Institut du Cancer et d'Immunogénétique (ICIG). There are no competing interests.

Regulation of Homeostatic and Malignant B Cell Development By the Tetraspanin CD53

Acute lymphoblastic leukemia (ALL) is the most frequent pediatric malignancy, most commonly originating from the transformation of progenitor cells of the B cell lineage (B cell precursor-ALL; BCP-ALL). Treatment of patients with high-risk or relapsed disease is difficult and prognosis remains poor in pediatric patients, with an even worse survival rate for adult BCP-ALL. Previous studies have shown an association of enhanced CD53 expression with many B cell malignancies, suggesting upregulation of CD53 may be implicated in carcinogenesis or maintenance of malignant cells. CD53 is a member of the tetraspanin family of transmembrane proteins, classically involved in cell adhesion, proliferation, and survival, and expressed exclusively on hematopoietic cells. While several studies have implicated a role for CD53 in regulating mature B cell proliferation, its role in early B cell development is not yet known. To elucidate the contribution of CD53 to normal and malignant B cell development, we have generated a CD53 knockout mouse.In our CD53-/- mouse, we observe no differences in total white blood cell counts, yet the fraction of peripheral blood B cells is significantly reduced by 31% compared to wild-type (WT) controls (28.3% vs. 19.5%; p<0.005). During homeostatic B lymphopoiesis, CD53 increases through development, beginning at the pre-pro-B cell stage and reaching highest expression on mature B cells. Further investigation into the loss of B cells revealed that immature pre-B cells in the bone marrow and mature B cells in the spleen and lymph nodes are significantly diminished upon loss of CD53, resulting from increased apoptosis in CD53-/- mice. B cell differentiation of CD53-/- hematopoietic stem cells (HSCs) in vitro corroborates the dependence on CD53 for normal differentiation, as CD53-/- cultures have 26% fewer B cells than controls (p=0.033). Investigation into the signaling differences between WT and CD53-/- B cell progenitors by mass cytometry (CyTOF) suggests that decreased PI3K/Akt and MAPK signaling could be driving this loss.With the observed loss of both B cell progenitors and mature B cells in CD53-deficient mice, CD53-/- mice were recently crossed to Eμ-Myc transgenic mice, a model of B-lineage leukemia/lymphoma, to generate WT, CD53-/-, Eμ-Myc+;CD53+/+, and Eμ-Myc+;CD53-/- groups to assess whether loss of CD53 alters the pathology or survival of these mice. As observed in human patients, moribund Eμ-Myc+ mice significantly upregulate CD53 on malignant cells, suggesting a potential role for CD53 during pathogenesis. Ongoing experiments are aimed at elucidating the mechanism by which CD53 promotes homeostatic B cell development and determining the potential of CD53 as a therapeutic target for B lineage malignancies. DisclosuresNo relevant conflicts of interest to declare.

CD9 Tetraspanin: A New Pathway for the Regulation of Inflammation?

CD9 belongs to the tetraspanin superfamily. Depending on the cell type and associated molecules, CD9 has a wide variety of biological activities such as cell adhesion, motility, metastasis, growth, signal transduction, differentiation, and sperm–egg fusion. This review focuses on CD9 expression by hematopoietic cells and its role in modulating cellular processes involved in the regulation of inflammation. CD9 is functionally very important in many diseases and is involved either in the regulation or in the mediation of the disease. The role of CD9 in various diseases, such as viral and bacterial infections, cancer and chronic lung allograft dysfunction, is discussed. This review focuses also on its interest as a biomarker in diseases. Indeed CD9 is primarily known as a specific exosome marker however, its expression is now recognized as an anti-inflammatory marker of monocytes and macrophages. It was also described as a marker of murine IL-10-competent Breg cells and IL-10-secreting CD9+ B cells were associated with better allograft outcome in lung transplant patients, and identified as a new predictive biomarker of long-term survival. In the field of cancer, CD9 was both identified as a favorable prognostic marker or as a predictor of metastatic potential depending on cancer types. Finally, this review discusses strategies to target CD9 as a therapeutic tool. Because CD9 can have opposite effects depending on the situation, the environment and the pathology, modulating CD9 expression or blocking its effects seem to be a new promising therapeutic strategy.

3160 - The Tetraspanin CD53 Regulates the Function of Mobilized HSCs

3160 - The Tetraspanin CD53 Regulates the Function of Mobilized HSCs

The Impact of the CD9 Tetraspanin on Lentivirus Infectivity and Exosome Secretion.

Efficient transduction tools are a hallmark for both research and therapy development. Here, we introduce new insights into the generation of lentiviral vectors with improved performance by utilizing producer cells with increased production rates of extracellular vesicles through CD9 overexpression. Most human cells secrete small vesicles from their surface (microvesicles) or intraluminal endosome-derived membranes (exosomes). In particular, enhanced levels of the tetraspanin CD9 result in significantly increased numbers of extracellular vesicles with exosome-like features that were secreted from four different human cell lines. Intriguingly, exosomes and their biogenesis route display similarities to lentivirus and we examined the impact of CD9 expression on release and infectivity of recombinant lentiviral vectors. Although the titers of released viral particles were not increased upon production in high CD9 cells, we observed improved performance in terms of both speed and efficiency of lentiviral gene delivery into numerous human cell lines, including HEK293, HeLa, SH-SY5Y, as well as B and T lymphocytes. Here, we demonstrate that enhanced CD9 enables lentiviral transduction in the absence of any pseudotyping viral glycoprotein or fusogenic molecule. Our findings indicate an important role of CD9 for lentiviral vector and exosome biogenesis and point out a remarkable function of this tetraspanin in membrane fusion, viral infectivity, and exosome-mediated horizontal information transfer.

Transcriptomics Identify CD9 as a Marker of Murine IL-10-Competent Regulatory B Cells.

Regulatory B cells (Breg) have immune suppressive functions in various autoimmune/inflammation models and diseases and are found to be enriched in diverse B cell subsets. The lack of a unique marker or set of markers efficiently identifying Breg cells impedes detailed investigation into their origin, development, and immunological roles. Here, we perform transcriptome analysis of IL-10-expressing B cells to identify key regulators for Breg biogenesis and function and identify CD9, a tetraspanin-family transmembrane protein, as a key surface marker for most mouse IL-10(+) B cells and their progenitors. CD9 plays a role in the suppressive function of IL-10(+) B cells in ex vivo T cell proliferation assays through a mechanism that is dependent upon B/T cell interactions. CD9(+) B cells also demonstrate inhibition of Th1-mediated contact hypersensitivity in an in vivo model system. Taken together, our findings implicate CD9 in the immunosuppressive activity of regulatory B cells.

The tetraspanin web revisited by super-resolution microscopy.

The spatial organization of membrane proteins in the plasma membrane is critical for signal transduction, cell communication and membrane trafficking. Tetraspanins organize functional higher-order protein complexes called ‘tetraspanin-enriched microdomains (TEMs)’ via interactions with partner molecules and other tetraspanins. Still, the nanoscale organization of TEMs in native plasma membranes has not been resolved. Here, we elucidated the size, density and distribution of TEMs in the plasma membrane of human B cells and dendritic cells using dual color stimulated emission depletion (STED) microscopy. We demonstrate that tetraspanins form individual nanoclusters smaller than 120 nm and quantified that a single tetraspanin CD53 cluster contains less than ten CD53 molecules. CD53 and CD37 domains were adjacent to and displayed only minor overlap with clusters containing tetraspanins CD81 or CD82. Moreover, CD53 and CD81 were found in closer proximity to their partners MHC class II and CD19 than to other tetraspanins. Although these results indicate that tetraspanin domains are adjacently positioned in the plasma membrane, they challenge the current view of the tetraspanin web of multiple tetraspanin species organized into a single domain. This study increases the molecular understanding of TEMs at the nanoscale level which is essential for comprehending tetraspanin function in cell biology.

Epididymosomes: a heterogeneous population of microvesicles with multiple functions in sperm maturation and storage.

Extracellular microvesicles present in the epididymal fluid have been named epididymosomes. Many epididymosome-associated proteins are transferred to spermatozoa during their maturation in the excurrent duct. Epididymosomes are heterogeneous, with their size varying between 50 and 250 nm. Two distinct population of epididymosomes characterized by different protein compositions and diameters have been isolated from the bovine epididymal fluid using different centrifugation protocols. One subpopulation of epididymosomes was characterized by CD9 and other tetraspanin partners. Transfer of proteins from these epididymosomes to maturing spermatozoa in co-incubation experiments was inhibited by antibodies against tetraspanin proteins. This suggests that this subpopulation of epididymosomes is involved in the acquisition of proteins involved in maturation by spermatozoa in the epididymis. The other population of epididymosomes was characterized by ELSPBP1 (epididymal sperm binding protein 1), known for its affinity for the phospholipid choline group. Flow cytometric analyses showed that ELSPBP1-positive epididymosomes only interacted with dying or dead epididymal spermatozoa in a Zn2 +-dependent manner. BLVRA (biliverdin reductase) was identified as a partner of ELSPBP1. This enzyme reduces biliverdin to bilirubin: two molecules with powerful anti-oxidant properties. We hypothesize that BLVRA is involved in an ROS-scavenging mechanism protecting live epididymal spermatozoa against detrimental molecules (ROS) released by dying cells. Therefore, it appears that there are at least two epididymosome population with distinct functions: targeting specific proteins to transiting spermatozoa by tetraspanin-mediated membrane fusion, and protection of epididymal spermatozoa against ROS released from dying cells. Further work is needed to understand functions of epididymosomes in epididymal physiology and sperm maturation and storage.

The tetraspanin CD53 modulates responses from activating NK cell receptors, promoting LFA-1 activation and dampening NK cell effector functions.

NK cells express several tetraspanin proteins, which differentially modulate NK cell activities. The tetraspanin CD53 is expressed by all resting NK cells and was previously shown to decrease NK cell cytotoxicity upon ligation. Here, we show that CD53 ligation reduced degranulation of rat NK cells in response to tumour target cells, evoked redirected inhibition of killing of Fc-bearing targets, and reduced the IFN-γ response induced by plate-bound antibodies towards several activating NK cell receptors (Ly49s3, NKR-P1A, and NKp46). CD53 induced activation of the β2 integrin LFA-1, which was further enhanced upon co-stimulation with activating NK cell receptors. Concordant with a role for CD53 in increasing NK cell adhesiveness, CD53 ligation induced a strong homotypic adhesion between NK cells. Further, the proliferative capacity of NK cells to a suboptimal dose of IL-2 was enhanced by CD53 ligation. Taken together, these data suggest that CD53 may shift NK cell responses from effector functions towards a proliferation phase.

Identification of alternative transcripts of rat CD9 expressed by tumorigenic neural cell lines and in normal tissues

CD9 is the best-studied member of the tetraspanin family of transmembrane proteins. It is involved in various fundamental cellular processes and its altered expression is a characteristic of malignant cells of different origins. Despite numerous investigations confirming its fundamental role, the heterogeneity of CD9 or other tetraspanin proteins was considered only to be caused by posttranslational modification, rather than alternative splicing. Here we describe the first identification of CD9 transcript variants expressed by cell lines derived from fetal rat brain cells. Variant mRNA-B lacks a potential translation initiation codon in the alternative exon 1 and seems to be characteristic of the tumorigenic BT cell lines. In contrast, variant mRNA-C can be translated from a functional initiation codon located in its extended exon 2, and substantial amounts of this form detected in various tissues suggest a contribution to CD9 functions. From the alternative sequence of variant C, a different membrane topology (5 transmembrane domains) and a deviating spectrum of functions can be expected.

Immunophenotypic Identification and Characterization of Tumor Cells and Infiltrating Cell Populations in Meningiomas

Meningiomas are primary tumors of the central nervous system composed of both neoplastic and other infiltrating cells. We determined the cellular composition of 51 meningioma samples by multiparameter flow cytometric (MFC) immunophenotyping and investigated the potential relationship between mRNA and protein expression levels of neoplastic cells. For immunophenotypic, morphologic, and cytogenetic characterization of individual cell populations, a large panel of markers was used together with phagocytic/endocytic functional assays and MFC sorting. Overall, our results revealed coexistence of CD45(-) neoplastic cells and CD45(+) immune infiltrating cells in all meningiomas. Infiltrating cells included tissue macrophages, with an HLA-DR(+)CD14(+)CD45(+)CD68(+)CD16(-/+)CD33(-/+) phenotype and high phagocytic/endocytic activity, and a small proportion of cytotoxic lymphocytes (mostly T CD8(+) and natural killer cells). Tumor cells expressed multiple cell adhesion proteins, tetraspanins, HLA-I/HLA-DR molecules, complement regulatory proteins, cell surface ectoenzymes, and growth factor receptors. Noteworthy, the relationship between mRNA and protein levels was variable, depending on the proteins evaluated and the level of infiltration by immune cells. In summary, our results indicate that MFC immunophenotyping provides a reliable tool for the characterization of the patterns of protein expression of different cell populations coexisting in meningioma samples, with a more accurate measure of gene expression profiles of tumor cells at the functional/protein level than conventional mRNA microarray, independently of the degree of infiltration of the tumor by immune cells.

Comparative expression profiling identifies differential roles for Myogenin and p38α MAPK signaling in myogenesis

Skeletal muscle differentiation is mediated by a complex gene expression program requiring both the muscle-specific transcription factor Myogenin (Myog) and p38α MAPK (p38α) signaling. However, the relative contribution of Myog and p38α to the formation of mature myotubes remains unknown. Here, we have uncoupled the activity of Myog from that of p38α to gain insight into the individual roles of these proteins in myogenesis. Comparative expression profiling confirmed that Myog activates the expression of genes involved in muscle function. Furthermore, we found that in the absence of p38α signaling, Myog expression leads to the down-regulation of genes involved in cell cycle progression. Consistent with this, the expression of Myog is sufficient to induce cell cycle exit. Interestingly, p38α-defective, Myog-expressing myoblasts fail to form multinucleated myotubes, suggesting an important role for p38α in cell fusion. Through the analysis of p38α up-regulated genes, the tetraspanin CD53 was identified as a candidate fusion protein, a role confirmed both ex vivo in primary myoblasts, and in vivo during myofiber regeneration in mice. Thus, our study has revealed an unexpected role for Myog in mediating cell cycle exit and has identified an essential role for p38α in cell fusion through the up-regulation of CD53.

Membrane transfer from oocyte to sperm occurs in two CD9-independent ways that do not supply the fertilising ability of Cd9-deleted oocytes

Spermatozoa undergo regulation of their functions along their lifespan through exchanges via vesicles or interactions with epithelial cells, in the epididymis, in the seminal fluid and in the female genital tract. Two different ways of oocyte membrane transfer to spermatozoa have been described: trogocytosis and exosomes. We here report an analysis of in vitro exchanges between the membranes of unfertilised oocytes and capacitated spermatozoa. We showed that optimum conditions are fulfilled when unfertilised oocytes interact with acrosome-reacted spermatozoa, a scenario mimicking the events occurring when the fertilising spermatozoon is inside the perivitelline space. Although CD9 tetraspanin is an essential molecule for fertilisation, exosome and trogocytosis transfer persists in Cd9-null oocytes in spite of their dramatic fusion failure. These exchanges are CD9 tetraspanin independent. We also confirm that mice sperm express CD9 tetraspanin and that when Cd9-null oocytes were inseminated with sperm covered with oocyte membrane materials, including CD9 tetraspanin, no rescue of the oocytes' fertilisability could be obtained. Thus, the existence of two ways of exchange between gametes during fertilisation suggests that these events could be of a physiological importance in this process.

CD9 Tetraspanin Interacts with CD36 on the Surface of Macrophages: A Possible Regulatory Influence on Uptake of Oxidized Low Density Lipoprotein

CD36 is a type 2 scavenger receptor with multiple functions. CD36 binding to oxidized LDL triggers signaling cascades that are required for macrophage foam cell formation, but the mechanisms by which CD36 signals remain incompletely understood. Mass spectrometry analysis of anti-CD36 immuno-precipitates from macrophages identified the tetraspanin CD9 as a CD36 interacting protein. Western blot showed that CD9 was precipitated from mouse macrophages by anti-CD36 monoclonal antibody and CD36 was likewise precipitated by anti-CD9, confirming the mass spectrometry results. Macrophages from cd36 null mice were used to demonstrate specificity. Membrane associations of the two proteins on intact cells was analyzed by confocal immunofluorescence microscopy and by a novel cross linking assay that detects proteins in close proximity (<40 nm). Functional significance was determined by assessing lipid accumulation, foam cell formation and JNK activation in wt, cd9 null and cd36 null macrophages exposed to oxLDL. OxLDL uptake, lipid accumulation, foam cell formation, and JNK phosphorylation were partially impaired in cd9 null macrophages. The present study demonstrates that CD9 associates with CD36 on the macrophage surface and may participate in macrophage signaling in response to oxidized LDL.

CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization

CD9 tetraspanin is the only egg membrane protein known to be essential for fertilization. To investigate its role, we have measured, on a unique acrosome reacted sperm brought in contact with an egg, the adhesion probability and strength with a sensitivity of a single molecule attachment. Probing the binding events at different locations of wild-type egg we described different modes of interaction. Here, we show that more gamete adhesion events occur on Cd9 null eggs but that the strongest interaction mode disappears. We propose that sperm-egg fusion is a direct consequence of CD9 controlled sperm-egg adhesion properties. CD9 generates adhesion sites responsible for the strongest of the observed gamete interaction. These strong adhesion sites impose, during the whole interaction lifetime, a tight proximity of the gamete membranes, which is a requirement for fusion to take place. The CD9-induced adhesion sites would be the actual location where fusion occurs.

α2β1 integrin controls association of Rac with the membrane and triggers quiescence of endothelial cells

Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that alpha2beta1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, alpha2beta1 engagement alters assembly of mature focal adhesions by alpha5beta1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of alpha5beta1 to signal for GTP loading of Rac is preserved, the joint engagement of alpha2beta1 interferes with membrane anchorage of Rac. Adapting the 'split-ubiquitin' sensor to screen for membrane-proximal alpha2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by alpha2beta1 integrin. Altogether, our data establish that alpha2beta1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.

Sperm‐egg interaction: is there a link between tetraspanin(s) and GPI‐anchored protein(s)?

Both female mice deficient in CD9 tetraspanin- and oocyte-specific glycosyl-phosphatidylinositol-anchored family proteins showed severely reduced fertility due to the failure of sperm-egg fusion. This raises the question of a link between these two groups of proteins at the oocyte membrane. We propose two hypotheses to explain why the absence of one of these proteins from the oocyte membrane results in the same phenotype. The first hypothesis envisages different levels of control by these molecules of the common induced signaling cascade. The second relies on the known involvement of these molecules in the overall organization of the plasma membrane. Their disappearance could thus prevent sperm-egg fusion either by disruption of the signaling cascade and/or by an important disorganization of the oocyte membrane. In this review, describing their structural and functional characteristics, and using published results on the oocyte, we try to analyze how these two protein families could interact.

Transfer of oocyte membrane fragments to fertilizing spermatozoa

Several families of molecules are implicated in the membrane fusion process between sperm and oocyte. Among these, CD9 tetraspanin, a membrane-organizing molecule, plays a crucial role, since the fertilizing ability of CD9-/- oocytes is dramatically impaired. CD9 controls alpha6-beta1 integrin relocation involved in the membrane reorganization that occurs on oocyte fertilization but is not expressed on sperm. We report here that, together with several other proteins, the CD9 tetraspanin is transferred from the oocyte to the fertilizing spermatozoa present in the perivitelline space before fertilization. Transfer of CD9 from oocyte to sperm from CD9-/- male mice still occurs. CD9 acquisition by sperm results from a transfer of membrane fragments from the plasma membrane of the oocyte, in a process similar to trogocytosis, the recently described mechanism of intercellular exchange of membrane patches. Acquisition of CD9 by the sperm may be crucial for the membrane reorganization in sperm required for fusion with the oocyte, a process that is similar to the role CD9 plays in oocyte membrane reorganization.