PubMed Health⌕ Search

Biomedical subjects

Alexandre Benmerah

Publications and source records attributed to Alexandre Benmerah.

At least 19 recordsLinked to original sources
PubMed2006

Dynamic interaction of HIV-1 Nef with the clathrin-mediated endocytic pathway at the plasma membrane.

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.

Amino Acid Motifs↗
PubMed2006

Dynamics of clathrin and adaptor proteins during endocytosis.

The endocytic adaptor complex AP-2 colocalizes with the majority of clathrin-positive spots at the cell surface. However, we previously observed that AP-2 is excluded from internalizing clathrin-coated vesicles (CCVs). The present studies quantitatively demonstrate that AP-2 disengages from sites of endocytosis seconds before internalization of the nascent CCV. In contrast, epsin, an alternate adaptor for clathrin at the plasma membrane, disappeared, along with clathrin. This suggests that epsin remains an integral part of the CCV throughout endocytosis. Clathrin spots at the cell surface represent a heterogeneous population: a majority (70%) of the spots disappeared with a time course of 4 min, whereas a minority (22%) remained static for > or =30 min. The static clathrin spots undergo constant subunit exchange, suggesting that although they are static structures, these spots comprise functional clathrin molecules, rather than dead-end aggregates. These results support a model where AP-2 serves a cargo-sorting function before endocytosis, whereas alternate adaptors, such as epsin, actually link cargo to the clathrin coat surrounding nascent endocytic vesicles. These data also support a role for static clathrin, providing a nucleation site for endocytosis.

Adaptor Protein Complex 2↗
PubMed2006

Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly.

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

Adaptor Protein Complex 2↗
PubMed2005

Homo- and hetero-oligomerization of beta-arrestins in living cells.

Arrestins are important proteins, which regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous beta-arrestins are believed to function exclusively as monomers, although self-association is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Here the oligomerization status of beta-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged beta-arrestins and resonance energy transfer (BRET and FRET) in living cells. At steady state and at physiological concentrations, beta-arrestins constitutively form both homo- and hetero-oligomers. Co-expression of beta-arrestin2 and beta-arrestin1 prevented beta-arrestin1 accumulation into the nucleus, suggesting that hetero-oligomerization may have functional consequences. Our data clearly indicate that beta-arrestins can exist as homo- and hetero-oligomers in living cells and raise the hypothesis that the oligomeric state may regulate their subcellular distribution and functions.

Animals↗
PubMed2005

Ubiquilin recruits Eps15 into ubiquitin-rich cytoplasmic aggregates via a UIM-UBL interaction.

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-S-transferase 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-fused-ubiquilin 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 UIM-containing 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.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Intracellular trafficking of CD23: differential regulation in humans and mice by both extracellular and intracellular exons.

In mouse models of food allergy, we recently characterized a new CD23b-derived splice form lacking extracellular exon 5, bDelta5, which undergoes constitutive internalization and mediates the transepithelial transport of free IgE, whereas classical CD23b is more efficient in transporting IgE/allergen complexes. These data suggested that regulation of endocytosis plays a central role in CD23 functions and drove us to systematically compare the intracellular trafficking properties of human and murine CD23 splice forms. We found that CD23 species show similar endocytic behaviors in both species; CD23a undergoes constitutive clathrin-dependent internalization, whereas CD23b is stable at the plasma membrane. However, the mechanisms controlling these similar behaviors appeared to be different. In mice, a positive internalization signal was localized in the cytoplasmic region shared by all CD23 splice forms. This positive signal was negatively regulated by the intracellular CD23b-specific exon. In addition, the fact that alternative splice forms lacking exons of the extracellular region (5, 6, 7, and/or 8) were all constitutively internalized suggested that endocytosis of murine CD23 is regulated by a process similar to the outside-in signaling of integrins. In humans, the internalization signal was mapped in the CD23a-specific intracellular exon. Interestingly, this signal also behaved as a basolateral targeting signal in polarized Madin-Darby canine kidney cells. The latter result and the fact that human intestinal cell lines were found to coexpress both CD23a and CD23b provide a molecular explanation for the initial observations that CD23 was found at the basolateral membrane of intestinal epithelial cells from allergic patients.

Alternative Splicing↗
PubMed2005

Differential role for CD23 splice forms in apical to basolateral transcytosis of IgE/allergen complexes.

The low affinity receptor for IgE (CD23) was recently implicated in the trans-epithelial transport of IgE-allergen complexes from the luminal side of enterocytes in animal models for intestinal allergy. Here, the respective functions of CD23 splice forms, b and bDelta5, in this apical to basolateral transport event have been investigated. First, the new bDelta5 splice form was further characterized, providing evidence that it binds IgE with high affinity, that its expression is induced by sensitization, and that bDelta5, unlike the classical b, undergoes constitutive internalization through clathrin-coated pits. These results suggested that the two CD23 splice forms were likely involved in different transcytotic events. MDCK cell lines expressing either b or bDelta5 were generated to directly test this hypothesis. In both cell lines, CD23 splice forms were localized at the apical membrane as in enterocytes from sensitized mice. Using mouse monoclonal IgE, we obtained evidence showing that bDelta5 mediates the apical to basolateral transport of free IgE, whereas classical b is much more efficient in mediating the transcytosis of IgE/allergen complexes. The present results shed new light on the role of CD23 species in IgE/allergen transepithelial transport and provide a new powerful physiological tool to study apical to basolateral transcytosis, a process which remains poorly characterized.

Allergens↗
PubMed2005

Analysis of the AP-2 adaptor complex and cargo during clathrin-mediated endocytosis.

Previously, we reported that the hetero-tetrameric adaptor complex AP-2 co-localizes with the static population of clathrin spots, whereas it is excluded from clathrin spots that disappear from the plasma membrane (forming clathrin-coated vesicles). More recently however, another group provided evidence that AP-2 markers could be observed coincident with disappearing clathrin spots. Thus, we tested several possible explanations for the apparent discrepancies in these two studies. We evaluated the potential contribution of nonred emission of clathrin-dsRed (used in both studies) in the simultaneous measurement of AP-2 and clathrin at various times. Additionally, we directly compared two different green fluorescent protein-tagged AP-2 constructs (similar to those used in the previous reports). These studies demonstrated that the duration of expression time greatly influences the subcellular localization of the AP-2 markers. Furthermore, we quantitatively evaluated the AP-2 fluorescence at the sites of numerous static and disappearing clathrin spots (at least 80 per group) and confirmed our initial observation that while AP-2 is present in nearly all static clathrin spots, it is excluded from the disappearing population of clathrin spots. Finally, in order to verify that clathrin spot disappearance represents clathrin-coated vesicle internalization, we simultaneously imaged clathrin and the cargo molecule transferrin at the cell surface.

Biomarkers↗
PubMed2004

Nef-induced alteration of the early/recycling endosomal compartment correlates with enhancement of HIV-1 infectivity.

human immunodeficiency virus type 1 (HIV-1) Nef interacts with the clathrin-associated AP-1 and AP-3 adaptor complexes, stabilizing their association with endosomal membranes. These findings led us to hypothesize a general impact of this viral protein on the endosomal system. Here, we have shown that Nef specifically disturbs the morphology of the early/recycling compartment, inducing a redistribution of early endosomal markers and a shortening of the tubular recycling endosomal structures. Furthermore, Nef modulates the trafficking of the transferrin receptor (TfR), the prototypical recycling surface protein, indicating that it also disturbs the function of this compartment. Nef reduces the rate of recycling of TfR to the plasma membrane, causing TfR to accumulate in early endosomes and reducing its expression at the cell surface. These effects depend on the leucine-based motif of Nef, which is required for the membrane stabilization of AP-1 and AP-3 complexes. Since we show that this motif is also required for the full infectivity of HIV-1 virions, these results indicate that the positive influence of Nef on viral infectivity may be related to its general effects on early/recycling endosomal compartments.

Amino Acid Motifs↗
PubMed2004

Food allergens are protected from degradation during CD23-mediated transepithelial transport.

BACKGROUND: CD23 (FcepsilonRII) is expressed by intestinal epithelial cells (IEC) following allergic stimulation and increases the uptake of IgE/allergen complexes. The aim of this study was to further analyze the role of CD23 in the intraepithelial processing of food allergens during transepithelial transport. METHODS: Balb-C mice were sensitized intraperitoneally with horseradish peroxidase (HRP) or beta-lactoglobulin (beta-LG) in the presence of pertussis toxin. In control and sensitized mice, 3H-HRP, intact HRP, or 14C-beta-LG fluxes were measured across jejunal segments mounted in Ussing chambers, in the presence or absence of mucosal anti-CD23 antibodies. HPLC analysis of serosal buffer was performed to detect HRP- or beta-LG-derived radiolabelled metabolites generated during transepithelial transport. RESULTS: In HRP-sensitized mice, 3H-HRP fluxes and intact HRP fluxes (3,836 +/- 476 and 290 +/- 86 ng/h x cm2, respectively) were significantly increased compared to control mice (1,677 +/- 297 ng/h x cm2, p < 0.01, and 106 +/- 23 ng/h x cm2, p < 0.02, respectively). HPLC analysis indicated the presence of intact HRP in the serosal compartment already 10 min after addition of HRP to the mucosal compartment, a result not observed in the control mice. In the presence of anti-CD23 antibodies, intact HRP fluxes were significantly decreased (131 +/- 27 ng/h x cm2) compared to control values in sensitized mice (290 +/- 86 ng/h x cm2, p < 0.02), suggesting that CD23 is involved is this 'protected' transport pathway. A similar protection during intestinal transport was observed for beta-LG in beta-LG sensitized mice. CONCLUSIONS: These results confirm that CD23 is involved in the rapid transepithelial transport of intact allergens in sensitized animals, and indicate that CD23 opens a 'protected' transport pathway in IECs.

Allergens↗
PubMed2004

Studies on endocytic mechanisms of the Menkes copper-translocating P-type ATPase (ATP7A; MNK). Endocytosis of the Menkes protein.

The human X-linked recessive copper deficiency disorder, Menkes disease, is caused by mutations in the ATP7A (MNK) gene, which encodes a transmembrane copper-transporting P-type ATPase (MNK). The MNK protein is localised to the Golgi apparatus and relocalises to the plasma membrane when copper levels are elevated. Previous studies have identified a C-terminal di-leucine endocytic motif (L1487L1488) in MNK, thought to direct it into the clathrin-mediated endocytic pathway. To determine whether MNK is internalised via clathrin-dependent endocytosis, this pathway was blocked in MNK-overexpressing HeLa cells by the transient expression of dominant negative dynamin and Eps15 mutants. MNK internalisation was not inhibited in such cells. MNK internalisation was inhibited in cells treated with hypertonic sucrose that not only blocked clathrin-mediated endocytosis but also fluid-phase endocytosis. These studies, together with earlier studies on the requirement for L1487L1488, suggest that MNK can utilise both clathrin-dependent and clathrin-independent endocytosis in HeLa cells.

Adenosine Triphosphatases↗
PubMed2004

Plasma membrane targeting of podocin through the classical exocytic pathway: effect of NPHS2 mutations.

Podocytes are specialized epithelial cells of the glomerulus in the kidney, which interconnect at the top of the glomerular basement membrane through the slit diaphragm, an adherens-like junction that plays a crucial role in the glomerular filtration process. Podocin, a plasma membrane anchored stomatin-like protein, is expressed in lipid rafts at the insertion of the slit diaphragm in podocytes. Mutations in NPHS2, the gene encoding podocin, are associated with inherited and sporadic cases of steroid-resistant nephrotic syndrome. Here, we show that brefeldin A induces accumulation of newly synthesized podocin in the endoplasmic reticulum, suggesting that podocin biosynthesis follows the classical secretory pathway, and we study the effect of 12 NPHS2 mutations associated with steroid-resistant nephrotic syndrome on the trafficking of the protein. We found that 9 podocin mutants were not targeted to the plasma membrane, 8 being retained in the endoplasmic reticulum and one being localized in late endosomes. Furthermore, by screening our database of patients with NPHS2 mutations, we found that podocin mutants retained in the endoplasmic reticulum are associated with earlier onset of the disease than those correctly targeted to the cell membrane. Our data suggest that most of NPHS2 mutations lead to retention of podocin in the endoplasmic reticulum and therefore provide a rationale for devising therapeutic approaches aimed at correcting the protein processing defect.

Animals↗
PubMed2004

[G protein coupled receptor endocytosis].

Most G protein-coupled receptors (GPCR) are rapidly internalized upon agonist stimulation. From the large number of studies available to date on receptor endocytosis, substantial differences seem to exist among GPCRs in terms of both the molecular pathways of receptor internalization and the biological significance of this process. The aim of this review is to outline common themes in GPCR endocytosis and to delineate true phenotypic variations, which reflect specific necessities for receptor's function.

Endocytosis↗
PubMed2004

Understanding living clathrin-coated pits.

Most knowledge of clathrin-mediated endocytosis has been gained by biochemical fractionation and in vitro assays. Recently, the study of endocytosis has extended into the living cell. The tracking of individual clathrin-coated pits and vesicles (CCPs and CCVs) has provided new insight into understanding the dynamic nature of CCPs. The use of total internal reflection fluorescence microscopy (TIR-FM), also termed evanescent field microscopy, has enabled the direct observation of events occurring within a restricted area of the cell adjacent to and including the adherent plasma membrane. TIR-FM is now actively being pursued in the study of endocytic processes. The direct observation of CCP-associated proteins including clathrin itself, dynamin and, most recently, AP-2 has considerably challenged old models, confirming some points but raising very interesting new questions.

Adaptor Protein Complex 2↗
PubMed2003

The AP-2 complex is excluded from the dynamic population of plasma membrane-associated clathrin.

Numerous biologically relevant substrates are selectively internalized via clathrin-mediated endocytosis. At the plasma membrane the AP-2 complex plays a major role in clathrin coat formation, interacting with both cargo and clathrin. Utilizing simultaneous dual-channel total internal reflection fluorescence microscopy we have analyzed components of the AP-2 complex (alpha- and beta 2-adaptin) during clathrin-mediated endocytosis. Although in static images enhanced green fluorescent protein-tagged AP-2 markers significantly co-localized with clathrin and other components of clathrin-coated pits, AP-2 did not seem to be present in clathrin spots that appeared to undergo internalization or motility parallel to the plane of the plasma membrane. Two populations of clathrin at the plasma membrane seem to exist, the dynamic and the static, and AP-2 appears to be only found within the latter. These results suggest that colocalized clathrin/AP-2 puncta may represent loci for coated pit production and that previous models that assumed AP-2 was retained within clathrin coats during endocytosis may need to be re-evaluated.

Adaptor Protein Complex 2↗
PubMed2003

Intestinal epithelial CD23 mediates enhanced antigen transport in allergy: evidence for novel splice forms.

We previously demonstrated enhanced transepithelial antigen transport in the intestine of allergic rodents associated with elevated expression of the low-affinity IgE receptor CD23 on enterocytes. Here, we examined the role of CD23 in the transport phenomenon using CD23-/- mice and characterized the isoform of intestinal epithelial CD23. Jejunal segments of sensitized mice were challenged with antigen. Enhanced transepithelial antigen transport and transmucosal antigen flux were found in the intestine of sensitized CD23+/+ but not CD23-/- mice. RT-PCR showed that enterocytes expressed only the isoform b of CD23. Sequencing revealed classic and alternative CD23b transcripts lacking exon 5 (bDelta5) or 6, all of which were translated into functional IgE receptors. The protein encoded by bDelta5 but not the classic b transcript was able to mediate the uptake of anti-CD23 or IgE, whereas both CD23 proteins were internalized after binding to IgE/antigen complexes. Our results suggest that the classic and alternative forms of CD23b display distinct endocytic properties, suggesting that they are likely to play different roles in transepithelial transport of IgE and allergens.

Animals↗
PubMed2003

Nuclear functions for plasma membrane-associated proteins?

There are a growing number of observations that proteins, which were initially thought to perform a specific function in a given subcellular compartment, may also play additional roles in different locations within the cell. Proteins found in adhesion and endocytic structures of the plasma membrane and which also traffic to the nucleus perhaps represent the more spectacular examples of this phenomenon. The mechanisms involved in the transport of these molecules through the nuclear pores and their potential nuclear functions are discussed.

Adherens Junctions↗