Papers by Alexandre Benmerah
Journal of Biological Chemistry, 2002
Eps15 and Eps15R are constitutive components of clathrin-coated pits that are required for clathr... more Eps15 and Eps15R are constitutive components of clathrin-coated pits that are required for clathrin-dependent endocytosis. The most striking difference between these two related proteins is that Eps15R is also found in the nucleus, whereas Eps15 is excluded from this compartment at steady state. To better understand the individual functions of these two proteins, the mechanisms responsible for their different localization were investigated. Interestingly, some mutants of Eps15 were found in the nucleus. This nuclear localization was correlated with the loss of the last Ϸ100 amino acids of Eps15, suggesting the presence of a nuclear export signal (NES) within this region. As expected, the last 25 amino acids contain a leucine-rich sequence matching with classical NESs, show a leptomycin B-sensitive nuclear export activity, and bind to the exportin CRM1 in a leucine residue-dependent manner. In contrast, no NES could be found in Eps15R, a result in keeping with its constitutive nuclear localization that appears to be regulated by alternative splicing. Altogether, these results are the first characterization of nucleocytoplasmic shuttling signals for endocytic proteins. They also provide an explanation for the different nuclear localization of Eps15 and Eps15R and further evidence for a possible nuclear function for Eps15 protein family members.
Dynamic Interaction of HIV-1 Nef with the Clathrin-Mediated Endocytic Pathway at the Plasma Membrane
Traffic, 2007
The HIV-1 Nef protein perturbs the trafficking of membrane proteins such as CD4 by interacting wi... more The HIV-1 Nef protein perturbs the trafficking of membrane proteins such as CD4 by interacting with clathrin-adaptor complexes. We previously reported that Nef alters early/recycling endosomes, but its role at the plasma membrane is poorly documented. Here, we used total internal reflection fluorescence microscopy, which restricts the analysis to a approximately 100 nm region of the adherent surface of the cells, to focus on the dynamic of Nef at the plasma membrane relative to that of clathrin. Nef colocalized both with clathrin spots (CS) that remained static at the cell surface, corresponding to clathrin-coated pits (CCPs), and with approximately 50% of CS that disappeared from the cell surface, corresponding to forming clathrin-coated vesicles (CCVs). The colocalization of Nef with clathrin required the di-leucine motif essential for Nef binding to AP complexes and was independent of CD4 expression. Furthermore, analysis of Nef mutants showed that the capacity of Nef to induce internalization and downregulation of CD4 in T lymphocytes correlated with its localization into CCPs. In conclusion, this analysis shows that Nef is recruited into CCPs and into forming CCVs at the plasma membrane, in agreement with a model in which Nef uses the clathrin-mediated endocytic pathway to induce internalization of some membrane proteins from the surface of HIV-1-infected T cells.
Journal of Cell Science, 2010
Clathrin adaptor (AP) complexes facilitate membrane trafficking between subcellular compartments.... more Clathrin adaptor (AP) complexes facilitate membrane trafficking between subcellular compartments. One such compartment is the cilium, whose dysfunction underlies disorders classified as ciliopathies. Although AP-1mu subunit (UNC-101) is linked to cilium formation and targeting of transmembrane proteins (ODR-10) to nematode sensory cilia at distal dendrite tips, these functions remain poorly understood. Here, using Caenorhabditis elegans sensory neurons and mammalian cell culture models, we find conservation of AP-1 function in facilitating cilium morphology, positioning and orientation, and microtubule stability and acetylation. These defects appear to be independent of IFT, because AP-1-depleted cells possess normal IFT protein localisation and motility. By contrast, disruption of chc-1 (clathrin) or rab-8 phenocopies unc-101 worms, preventing ODR-10 vesicle formation and causing misrouting of ODR-10 to all plasma membrane destinations. Finally, ODR-10 colocalises with RAB-8 in cell soma and they cotranslocate along dendrites, whereas ODR-10 and UNC-101 signals do not overlap. Together, these data implicate conserved roles for metazoan AP-1 in facilitating cilium structure and function, and suggest cooperation with RAB-8 to coordinate distinct early steps in neuronal ciliary membrane sorting and trafficking.
Endocytose: chaque voie compte ! : Trafic intracellulaire
Ms Medecine Sciences, 2002
Protéines Nef du VIH et K3/K5 du virus associé au sarcoma de Kaposi : des « parasites »de la voie d’endocytose
médecine/sciences, 2003
The modulation of plasma membrane proteins involved in the communication with the immune system i... more The modulation of plasma membrane proteins involved in the communication with the immune system is a general mechanism developed by viruses to escape the immune response. Most of the studied examples have focused on viral proteins that missort cellular proteins during their biosynthesis. However, an increasing number of examples show that the down-modulation can also be achieved after membrane delivery by targeting into the endocytic pathway. For both human immunodeficiency virus (HIV) and Kaposi sarcoma-associated herpesvirus (KSHV), the proteins required for this process are identified, Nef and K3/K5 respectively. The extensive studies in this field have shown that the mechanisms by which these proteins "parasite" the endocytic pathway are completely different. Nef directly interacts with components of the cellular machinery involved in the vesicular transport between the endocytic compartments, mainly the clathrin adaptor complexes (AP), inducing the misrouting of numerous cellular proteins, including CD4, MHC-I, LIGHT, DC-SIGN, CD28 and MHC-II to the endosomal degradation compartment or the trans Golgi-network. The K3 and K5 proteins from KSHV act by inducing the ubiquitylation of the target proteins, such as CMH-I and B7.2, triggering their internalization and subsequent degradation by the highly conserved Tsg101/vps23 ubiquitin-dependent endosomal pathway. While these findings show that the strategies used by viruses to target cellular proteins to the endocytic pathway are extremely diverse, additional investigations are needed for the complete understanding of the specific roles of Nef and K3/K5 in the physiopathology of HIV and KSHV infections, respectively. In addition, these viral factors represent valuable tools to study the pathway they are perturbing.
Endocytose : chaque voie compte!
médecine/sciences, 2002
Adhesion Molecules on Mucosal T Lymphocytes
Essentials of Mucosal Immunology, 1996
Ligand stimulation induces clathrin- and Rab5-dependent downregulation of the kinase-dead EphB6 receptor preceded by the disruption of EphB6-Hsp90 interaction
Cellular Signalling, 2014
Ligand-induced internalisation and subsequent downregulation of receptor tyrosine kinases (RTKs) ... more Ligand-induced internalisation and subsequent downregulation of receptor tyrosine kinases (RTKs) serve to determine biological outputs of their signalling. Intrinsically kinase-deficient RTKs control a variety of biological responses, however, the mechanism of their downregulation is not well understood and its analysis is focused exclusively on the ErbB3 receptor. The Eph group of RTKs is represented by the EphA and EphB subclasses. Each bears one kinase-inactive member, EphA10 and EphB6, respectively, suggesting an important role for these molecules in the Eph signalling network. While EphB6 effects on cell behaviour have been assessed, the mechanism of its downregulation remains elusive. Our work reveals that EphB6 and its kinase-active relative, and signalling partner, EphB4, are downregulated in a similar manner in response to their common ligand, ephrin-B2. Following stimulation, both receptors are internalised through clathrin-coated pits and are degraded in lysosomes. Their targeting for lysosomal degradation relies on the activity of an early endosome regulator, the Rab5 GTPase, as this process is inhibited in the presence of a Rab5 dominant-negative mutant. EphB6 also interacts with the Hsp90 chaperone and EphB6 downregulation is preceded by their rapid dissociation. Moreover, the inhibition of Hsp90 results in EphB6 degradation, mimicking its ligand-induced downregulation. These processes appear to rely on overlapping mechanisms, since Hsp90 inhibition does not significantly enhance ligand-induced EphB6 elimination. Taken together, our observations define a novel mechanism for intrinsically kinase-deficient RTK downregulation and support an intriguing model, where Hsp90 dissociation acts as a trigger for ligand-induced receptor removal.
La poche ciliaire : fruit des liaisons du centrosome avec le trafic vésiculaire
médecine/sciences, 2014
The assembly of cilia, ciliogenesis, involves complex and conserved mechanisms during which the b... more The assembly of cilia, ciliogenesis, involves complex and conserved mechanisms during which the basal body has to dock onto cell membranes. Studies in the 1960s suggested that in many cell types such docking occurs in the cytoplasm and that cilia are formed within a vesicle before being delivered to the plasma membrane. This intracellular pathway, recently characterized at the molecular level, leads to the formation of a membrane domain at the basis of cilia, the ciliary pocket, which was involved in vesicular trafficking and signaling.
Cell Reports, 2013
Transforming growth factor b (TGF-b) signaling is regulated by clathrin-dependent endocytosis (CD... more Transforming growth factor b (TGF-b) signaling is regulated by clathrin-dependent endocytosis (CDE) for the control of cellular processes during development and in tissue homeostasis. The primary cilium coordinates several signaling pathways, and the pocket surrounding the base and proximal part of the cilium is a site for CDE. We report here that TGF-b receptors localize to the ciliary tip and endocytic vesicles at the ciliary base in fibroblasts and that TGF-b stimulation increases receptor localization and activation of SMAD2/3 and ERK1/2 at the ciliary base. Inhibition of CDE reduced TGF-b-mediated signaling at the cilium, and TGF-b signaling and CDE activity are reduced at stunted primary cilia in Tg737 orpk fibroblasts. Similarly, TGF-b signaling during cardiomyogenesis correlated with accumulation of TGF-b receptors and activation of SMAD2/3 at the ciliary base. Our results indicate that the primary cilium regulates TGF-b signaling and that the ciliary pocket is a compartment for CDE-dependent regulation of signal transduction.
Journal of Cell Science, 2005
Eps15 and its related protein Eps15R are key components of the clathrin-mediated endocytic pathwa... more Eps15 and its related protein Eps15R are key components of the clathrin-mediated endocytic pathway. We searched for new binding partners of Eps15 using a yeast two-hybrid screen. We report here that ubiquilin (hPLIC1), a type-2 ubiquitin-like protein containing a ubiquitin-like domain (UBL) and a ubiquitin-associated domain (UBA), interacts with both Eps15 and Eps15R. Using glutathione-Stransferase pull-down experiments, we show that the first ubiquitin-interacting motif of Eps15 (UIM1) interacts directly with the UBL domain of ubiquilin, whereas it does not bind to ubiquitinated proteins. The second UIM of Eps15 (UIM2) binds poorly to the UBL domain but does bind to ubiquitinated proteins. Two other UIM-containing endocytic proteins, Hrs and Hbp, also interact with ubiquilin in a UIM-dependent manner, whereas epsin does not. Immunofluorescence analysis showed that endogenous Eps15 and Hrs, but not epsin, colocalize with green-fluorescent-protein-fused ubiquilin in cytoplasmic aggregates that are not endocytic compartments. We have characterized these green-fluorescent-protein-fusedubiquilin aggregates as ubiquitin-rich intracytoplasmic inclusions that are recruited to aggresomes upon proteasome inhibition. Moreover, we show that endogenous Eps15 and endogenous ubiquilin colocalize to cytoplasmic aggregates and aggresomes. Finally, we show that the recruitment of Eps15 into ubiquilin-positive aggregates is UIM dependent. Altogether, our data identify ubiquilin as the first common UIM-binding partner of a subset of UIMcontaining endocytic proteins. We propose that this UIM/UBL-based interaction is responsible for the sequestration of certain UIM-containing endocytic proteins into cytoplasmic ubiquitin-rich protein aggregates.
Quiescent melanocytes form primary cilia
Experimental Dermatology, 2014
We show, for the first time, that melanocytes can form a primary cilium in vitro, corresponding t... more We show, for the first time, that melanocytes can form a primary cilium in vitro, corresponding to an immotile or sensory cilium. Such cilia are observed when melanocytes reach confluence or when medium nutrient levels are insufficient. This observation should greatly improve our understanding of the signal transduction processes potentially occurring in these cells during embryonic development, homeostasis in adulthood and melanomagenesis.
The ciliary pocket
Current Opinion in Cell Biology, 2013
Cilia are fascinating highly conserved organelles shared by very different organisms from unicell... more Cilia are fascinating highly conserved organelles shared by very different organisms from unicellular eukaryotes to vertebrates where they are involved in motility and sensory functions. In vertebrates, the function of the primary cilium, a unique nonmotile cilium found at the surface of most cell types during development, remained mysterious during 40 years until its crucial function in the control of key signaling cascades during development and its involvement in complex genetic disorders now called ciliopathies were uncovered. Recent studies have focused on a specific membrane domain found at the base of primary cilia in most cell types which was already mentioned in the first descriptions of these cilia but did not raise much interest during 50 years. This membrane domain, the 'ciliary pocket', also found at the base of some motile cilia, may act as a platform for cilia-associated vesicular trafficking and as an interface with the actin cytoskeleton but also likely in additional important functions which remain to be discovered.
Novel NEK8 Mutations Cause Severe Syndromic Renal Cystic Dysplasia through YAP Dysregulation
PLoS genetics, 2016
Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primar... more Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, >500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of >1200 ciliary genes. NEK8/NPHP9 mutations were identified in five cases with severe overlapping phenotypes including renal cystic dysplasia/hypodysplasia, situs inversus, cardiopathy with hypertrophic septum and bile duct paucity. These cases highlight a genotype-phenotype correlation, with missense and nonsense mutations associated with hypodysplasia and enlarged cystic organs, respectively. Functional analyses of NEK8 mutations in patient fibroblasts and mIMCD3 cells showed that these mutations differentially affect ciliogenesis, prolif...
Eps15 and Epsin1 Are Crucial for Enteropathogenic Escherichia coli Pedestal Formation Despite the Absence of Adaptor Protein 2
Journal of Infectious Diseases, 2011
Enteropathogenic Escherichia coli (EPEC) are primarily extracellular pathogens that generate acti... more Enteropathogenic Escherichia coli (EPEC) are primarily extracellular pathogens that generate actin-rich structures known as pedestals during their pathogenesis. Surprising evidence has demonstrated that despite maintaining an extracellular location, EPEC require the endocytic protein, clathrin, for pedestal formation. To evaluate the strategies EPEC use to usurp endocytic machinery, we investigated the roles of a number of clathrin-coated pits components, adaptor protein 2 (AP-2), Eps15 and epsin1, during EPEC infections. We demonstrated that in conjunction with clathrin, pedestal formation also required the recruitment of Eps15 and epsin1 but not AP-2. Because AP-2 orchestrates the recruitment of clathrin, Eps15, and epsin1, as well as other adaptors, during assembly of clathrin-coated pits at the plasma membrane, our findings reveal a novel internalization subversion strategy employed by EPEC. These results further emphasize the recent paradigm that endocytic proteins are important for EPEC-mediated disease.
Interaction of the EPS15 protein with the AP2 complex is required for receptor mediated endocytosis
International Journal of Cell Biology, 1998
Plos One, 2008
Background: The primary cilium is a sensory organelle generated from the centrosome in quiescent ... more Background: The primary cilium is a sensory organelle generated from the centrosome in quiescent cells and found at the surface of most cell types, from where it controls important physiological processes. Specific sets of membrane proteins involved in sensing the extracellular milieu are concentrated within cilia, including G protein coupled receptors (GPCRs). Most GPCRs are regulated by b-arrestins, barr1 and barr2, which control both their signalling and endocytosis, suggesting that barrs may also function at primary cilium.
M S Medecine Sciences, 2004
Montréal. Il a pour mission la promotion et la valorisation de la recherche. Érudit offre des ser... more Montréal. Il a pour mission la promotion et la valorisation de la recherche. Érudit offre des services d'édition numérique de documents scientifiques depuis 1998. Pour communiquer avec les responsables d'Érudit : info@erudit.org Article « Endocytose des récepteurs couplés aux protéines G » Mark G.H. Scott, Alexandre Benmerah et Stefano Marullo M/S : médecine sciences, vol. 20, n° 1, 2004, p. 78-83. Pour citer cet article, utiliser l'information suivante : URI: http://id.erudit.org/iderudit/007525ar Note : les règles d'écriture des références bibliographiques peuvent varier selon les différents domaines du savoir.
Homotypic aggregation of CD103 (aE�7)+ lymphocytes by an anti-CD103 antibody, HML-4
Eur J Immunol, 1994
One monoclonal antibody, HML-4, directed against the αEβ7 integrin (CD103), an integrin preferent... more One monoclonal antibody, HML-4, directed against the αEβ7 integrin (CD103), an integrin preferentially expressed on human intestinal intraepithelial lymphocytes (IEL), induced the homotypic aggregation of IEL and of a CD103+ MOLT16 cell line. Aggregation was an active adhesion event dependent on an intact cytoskeleton, on tyrosine phosphorylation but not on activation of protein kinase C. It was blocked by four other anti-CD103 antibodies but by none of the antibodies blocking known adhesion lymphocyte pathways. It was associated with a redistribution of the CD103 integrin in the areas of cell-cell contacts. These results indicated that HML-4zx-induced homotypic adhesion was mediated via CD103 and resulted from the binding of the integrin to an as yet undefined ligand expressed by CD103+ cells. This ligand was distinct from the epithelial ligand of CD103: in contrast with homotypic adhesion, heterotypic adhesion of CD103+ MOLT16 cells on two epithelial intestinal cell lines (DLD1 and HT29) was dependent on the presence of divalent cations, was not enhanced by HML-4, was inhibited by HML-1 but not by the three other antibodies with an inhibitory effect on homotypic adhesion. Finally, the study of conjugates between CD103+ and CD103- sublines derived from the MOLT16 cell line suggested that HML-4-induced homotypic aggregation resulted from homophilic CD103-CD103 interactions.
Uploads
Papers by Alexandre Benmerah