Dynamique et mécanique membranaires de la signalisation intracellulaire

Publications de l’équipe

Année de publication : 2016

Blouin CM, Hamon Y, Gonnord P, Boularan C, Kagan J, Viaris de Lesegno C, Ruez R, Mailfert S, Bertaux N, Loew D, Wunder C, Johannes L,Vogt G, Contreras FX, Marguet D, Casanova JL, Galès C, He HT, Lamaze C. (2016 Dec 11)

Glycosylation-Dependent IFN-γR Partitioning in Lipid and Actin Nanodomains Is Critical for JAK Activation

Cell : Volume 166, Issue 4 : 920–934 : DOI : doi.org/10.1016/j.cell.2016.07.003 En savoir plus
Résumé

Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-γ receptor (IFN-γR) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-γR2 subunit, this mutation blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-γ could not occur. Removing IFN-γR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo. VIDEO ABSTRACT.

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Daniela Chmiest, Nanaocha Sharma, Natacha Zanin, Christine Viaris de Lesegno, Massiullah Shafaq-Zadah, Vonick Sibut, Florent Dingli, Philippe Hupé, Stephan Wilmes, Jacob Piehler, Damarys Loew, Ludger Johannes, Gideon Schreiber, Christophe Lamaze (2016 Dec 6)

Spatiotemporal control of interferon-induced JAK/STAT signalling and gene transcription by the retromer complex.

Nature communications : 13476 : DOI : 10.1038/ncomms13476 En savoir plus
Résumé

Type-I interferons (IFNs) play a key role in the immune defences against viral and bacterial infections, and in cancer immunosurveillance. We have established that clathrin-dependent endocytosis of the type-I interferon (IFN-α/β) receptor (IFNAR) is required for JAK/STAT signalling. Here we show that the internalized IFNAR1 and IFNAR2 subunits of the IFNAR complex are differentially sorted by the retromer at the early endosome. Binding of the retromer VPS35 subunit to IFNAR2 results in IFNAR2 recycling to the plasma membrane, whereas IFNAR1 is sorted to the lysosome for degradation. Depletion of VPS35 leads to abnormally prolonged residency and association of the IFNAR subunits at the early endosome, resulting in increased activation of STAT1- and IFN-dependent gene transcription. These experimental data establish the retromer complex as a key spatiotemporal regulator of IFNAR endosomal sorting and a new factor in type-I IFN-induced JAK/STAT signalling and gene transcription.

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Christophe Lamaze, Cédric M Blouin (2016 Oct 14)

Receptor Lipid nanodomain Partitioning and Signaling.

Cell cycle (Georgetown, Tex.) : 0 En savoir plus
Résumé

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Camille Kieffer, Ye Wang, Fatma Bagca, Christophe Lamaze (2016 Sep 13)

[Butterfly effect and cancer: how a mechanical pressure induced in vivo leads to tumorigenesis in neighboring healthy cells].

Médecine sciences : M/S : 713-5 : DOI : 10.1051/medsci/20163208017 En savoir plus
Résumé

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Cédric M Blouin, Yannick Hamon, Pauline Gonnord, Cédric Boularan, Jérémy Kagan, Christine Viaris de Lesegno, Richard Ruez, Sébastien Mailfert, Nicolas Bertaux, Damarys Loew, Christian Wunder, Ludger Johannes, Guillaume Vogt, Francesc-Xabier Contreras, Didier Marguet, Jean-Laurent Casanova, Céline Galès, Hai-Tao He, Christophe Lamaze (2016 Aug 9)

Glycosylation-Dependent IFN-γR Partitioning in Lipid and Actin Nanodomains Is Critical for JAK Activation.

Cell : 920-34 : DOI : 10.1016/j.cell.2016.07.003 En savoir plus
Résumé

Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-γ receptor (IFN-γR) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-γR2 subunit, this mutation blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-γ could not occur. Removing IFN-γR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo. VIDEO ABSTRACT.

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Vincent Mercier, Marine H Laporte, Olivier Destaing, Béatrice Blot, Cédric M Blouin, Karin Pernet-Gallay, Christine Chatellard, Yasmina Saoudi, Corinne Albiges-Rizo, Christophe Lamaze, Sandrine Fraboulet, Anne Petiot, Rémy Sadoul (2016 Jun 1)

ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling.

Scientific reports : 26986 : DOI : 10.1038/srep26986 En savoir plus
Résumé

The molecular mechanisms and the biological functions of clathrin independent endocytosis (CIE) remain largely elusive. Alix (ALG-2 interacting protein X), has been assigned roles in membrane deformation and fission both in endosomes and at the plasma membrane. Using Alix ko cells, we show for the first time that Alix regulates fluid phase endocytosis and internalization of cargoes entering cells via CIE, but has no apparent effect on clathrin mediated endocytosis or downstream endosomal trafficking. We show that Alix acts with endophilin-A to promote CIE of cholera toxin and to regulate cell migration. We also found that Alix is required for fast endocytosis and downstream signaling of the interleukin-2 receptor giving a first indication that CIE is necessary for activation of at least some surface receptors. In addition to characterizing a new function for Alix, our results highlight Alix ko cells as a unique tool to unravel the biological consequences of CIE.

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Année de publication : 2015

Massiullah Shafaq-Zadah, Carina S Gomes-Santos, Sabine Bardin, Paolo Maiuri, Mathieu Maurin, Julian Iranzo, Alexis Gautreau, Christophe Lamaze, Patrick Caswell, Bruno Goud, Ludger Johannes (2015 Dec 8)

Persistent cell migration and adhesion rely on retrograde transport of β(1) integrin.

Nature cell biology : 54-64 : DOI : 10.1038/ncb3287 En savoir plus
Résumé

Integrins have key functions in cell adhesion and migration. How integrins are dynamically relocalized to the leading edge in highly polarized migratory cells has remained unexplored. Here, we demonstrate that β1 integrin (known as PAT-3 in Caenorhabditis elegans), but not β3, is transported from the plasma membrane to the trans-Golgi network, to be resecreted in a polarized manner. This retrograde trafficking is restricted to the non-ligand-bound conformation of β1 integrin. Retrograde trafficking inhibition abrogates several β1-integrin-specific functions such as cell adhesion in early embryonic development of mice, and persistent cell migration in the developing posterior gonad arm of C. elegans. Our results establish a paradigm according to which retrograde trafficking, and not endosomal recycling, is the key driver for β1 integrin function in highly polarized cells. These data more generally suggest that the retrograde route is used to relocalize plasma membrane machinery from previous sites of function to the leading edge of migratory cells.

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Philippe Roudot, Charles Kervrann, Cedric M Blouin, Francois Waharte (2015 Oct 20)

Lifetime estimation of moving subcellular objects in frequency-domain fluorescence lifetime imaging microscopy.

Journal of the Optical Society of America. A, Optics, image science, and vision : 1821-35 : DOI : 10.1364/JOSAA.32.001821 En savoir plus
Résumé

Fluorescence lifetime is usually defined as the average nanosecond-scale delay between excitation and emission of fluorescence. It has been established that lifetime measurements yield numerous indications on cellular processes such as interprotein and intraprotein mechanisms through fluorescent tagging and Förster resonance energy transfer. In this area, frequency-domain fluorescence lifetime imaging microscopy is particularly appropriate to probe a sample noninvasively and quantify these interactions in living cells. The aim is then to measure the fluorescence lifetime in the sample at each location in space from fluorescence variations observed in a temporal sequence of images obtained by phase modulation of the detection signal. This leads to a sensitivity of lifetime determination to other sources of fluorescence variations such as intracellular motion. In this paper, we propose a robust statistical method for lifetime estimation for both background and small moving structures with a focus on intracellular vesicle trafficking.

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Christophe Lamaze, Stéphanie Torrino (2015 Sep 9)

Caveolae and cancer: A new mechanical perspective.

Biomedical journal : 367-79 : DOI : 10.4103/2319-4170.164229 En savoir plus
Résumé

Caveolae are small invaginations of the plasma membrane in cells. In addition to their classically described functions in cell signaling and membrane trafficking, it was recently shown that caveolae act also as plasma membrane sensors that respond immediately to acute mechanical stresses. Caveolin 1 (Cav1), the main component of caveolae, is a multifunctional scaffolding protein that can remodel the extracellular environment. Caveolae dysfunction, due to mutations in  caveolins, has been linked to several human diseases called « caveolinopathies, » including muscular dystrophies, cardiac disease, infection, osteoporosis, and cancer. The role of caveolae and/or Cav1 remains controversial particularly in tumor progression. Cav1 function has been associated with several steps of cancerogenesis such as tumor growth, cell migration, metastasis, and angiogenesis, yet it was observed that Cav1 could affect these steps in a positive or negative manner. Here, we discuss the possible function of caveolae and Cav1 in tumor progression in the context of their recently discovered role in cell mechanics.

Replier
Barthélémy, F., C. Blouin, N. Wein, V. Mouly, S. Courrier, E. Dionnet, V. Kergourlay, Y. Mathieu, L. Garcia, G. Butler-Browne, C. Lamaze, N. Lévy, M. Krahn, M. Bartoli. (2015 Sep 1)

Exon 32 Skipping of Dysferlin Rescues Membrane Repair in Patients’ Cells

Journal of Neuromuscular Diseases : 2 : 281-290 : DOI : DOI: 10.3233/JND-150109 En savoir plus
Résumé

Dysferlinopathies are a family of disabling muscular dystrophies with LGMD2B and Miyoshi myopathy as the main phenotypes. They are associated with molecular defects in DYSF, which encodes dysferlin, a key player in sarcolemmal homeostasis. Previous investigations have suggested that exon skipping may be a promising therapy for a subset of patients with dysferlinopathies. Such an approach aims to rescue functional proteins when targeting modular proteins and specific tissues.

We sought to evaluate the dysferlin functional recovery following exon 32 skipping in the cells of affected patients. Exon skipping efficacy was characterized at several levels by use ofin vitro myotube formation assays and quantitative membrane repair and recovery tests. Data obtained from these assessments confirmed that dysferlin function is rescued by quasi-dysferlin expression in treated patient cells, supporting the case for a therapeutic antisense-based trial in a subset of dysferlin-deficient patients.

 

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Henri-François Renard, Maria Daniela Garcia-Castillo, Valérie Chambon, Christophe Lamaze, Ludger Johannes (2015 Jun 14)

Shiga toxin stimulates clathrin-independent endocytosis of the VAMP2, VAMP3 and VAMP8 SNARE proteins.

Journal of cell science : 2891-902 : DOI : 10.1242/jcs.171116 En savoir plus
Résumé

Endocytosis is an essential cellular process that is often hijacked by pathogens and pathogenic products. Endocytic processes can be classified into two broad categories, those that are dependent on clathrin and those that are not. The SNARE proteins VAMP2, VAMP3 and VAMP8 are internalized in a clathrin-dependent manner. However, the full scope of their endocytic behavior has not yet been elucidated. Here, we found that VAMP2, VAMP3 and VAMP8 are localized on plasma membrane invaginations and very early uptake structures that are induced by the bacterial Shiga toxin, which enters cells by clathrin-independent endocytosis. We show that toxin trafficking into cells and cell intoxication rely on these SNARE proteins. Of note, the cellular uptake of VAMP3 is increased in the presence of Shiga toxin, even when clathrin-dependent endocytosis is blocked. We therefore conclude that VAMP2, VAMP3 and VAMP8 are removed from the plasma membrane by non-clathrin-mediated pathways, in addition to by clathrin-dependent uptake. Moreover, our study identifies these SNARE proteins as the first transmembrane trafficking factors that functionally associate at the plasma membrane with the toxin-driven clathrin-independent invaginations during the uptake process.

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Maria Daniela Garcia-Castillo, Thi Tran, Alexandre Bobard, Henri-François Renard, Stefan J Rathjen, Estelle Dransart, Bahne Stechmann, Christophe Lamaze, Mike Lord, Jean-Christophe Cintrat, Jost Enninga, Eric Tartour, Ludger Johannes (2015 May 16)

Retrograde transport is not required for cytosolic translocation of the B-subunit of Shiga toxin.

Journal of cell science : 2373-87 : DOI : 10.1242/jcs.169383 En savoir plus
Résumé

Antigen-presenting cells have the remarkable capacity to transfer exogenous antigens to the cytosol for processing by proteasomes and subsequent presentation on major histocompatibility complex class-I (MHC-I) molecules, a process termed cross-presentation. This is the target of biomedical approaches that aim to trigger a therapeutic immune response. The receptor-binding B-subunit of Shiga toxin (STxB) has been developed as an antigen delivery tool for such immunotherapy applications. In this study, we have analyzed pathways and trafficking factors that are involved in this process. A covalent conjugate between STxB and saporin was generated to quantitatively sample the membrane translocation step to the cytosol in differentiated monocyte-derived THP-1 cells. We have found that retrograde trafficking to the Golgi complex was not required for STxB-saporin translocation to the cytosol or for STxB-dependent antigen cross-presentation. Depletion of endosomal Rab7 inhibited, and lowering membrane cholesterol levels favored STxB-saporin translocation. Interestingly, experiments with reducible and non-reducible linker-arm-STxB conjugates led to the conclusion that after translocation, STxB remains associated with the cytosolic membrane leaflet. In summary, we report new facets of the endosomal escape process bearing relevance to antigen cross-presentation.

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Alexis Mougeolle, Sylvie Poussard, Marion Decossas, Christophe Lamaze, Olivier Lambert, Elise Dargelos (2015 Mar 24)

Oxidative stress induces caveolin 1 degradation and impairs caveolae functions in skeletal muscle cells.

PloS one : e0122654 : DOI : 10.1371/journal.pone.0122654 En savoir plus
Résumé

Increased level of oxidative stress, a major actor of cellular aging, impairs the regenerative capacity of skeletal muscle and leads to the reduction in the number and size of muscle fibers causing sarcopenia. Caveolin 1 is the major component of caveolae, small membrane invaginations involved in signaling and endocytic trafficking. Their role has recently expanded to mechanosensing and to the regulation of oxidative stress-induced pathways. Here, we increased the amount of reactive oxidative species in myoblasts by addition of hydrogen peroxide (H2O2) at non-toxic concentrations. The expression level of caveolin 1 was significantly decreased as early as 10 min after 500 μM H2O2 treatment. This reduction was not observed in the presence of a proteasome inhibitor, suggesting that caveolin 1 was rapidly degraded by the proteasome. In spite of caveolin 1 decrease, caveolae were still able to assemble at the plasma membrane. Their functions however were significantly perturbed by oxidative stress. Endocytosis of a ceramide analog monitored by flow cytometry was significantly diminished after H2O2 treatment, indicating that oxidative stress impaired its selective internalization via caveolae. The contribution of caveolae to the plasma membrane reservoir has been monitored after osmotic cell swelling. H2O2 treatment increased membrane fragility revealing that treated cells were more sensitive to an acute mechanical stress. Altogether, our results indicate that H2O2 decreased caveolin 1 expression and impaired caveolae functions. These data give new insights on age-related deficiencies in skeletal muscle.

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Renard, H.F., M. Simunovic, J. Lemiere, E. Boucrot, M.D. Garcia-Castillo, S. Arumugam, V. Chambon, C. Lamaze, C. Wunder, A.K. Kenworthy, A.A. Schmidt, H.T. McMahon, C. Sykes, P. Bassereau, L. Johannes (2015 Jan 22)

Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis

Nature : 517 : 493-496 : DOI : doi:10.1038/nature14064 En savoir plus
Résumé

During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers1. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect234, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin56. Clathrin-independent endocytic events are often less reliant on dynamin7, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we show, in human and other mammalian cell lines, that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which are both clathrin-independent endocytic cargoes8. In controlled in vitro systems, endoA2 reshapes membranes before scission. Furthermore, we demonstrate that endoA2, dynamin and actin contribute in parallel to the scission of Shiga-toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling-force-driven dynamic scission.

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Année de publication : 2014

Lakshminarayan, R., C. Wunder, U. Becken, M.T. Howes, C. Benzing, S. Arumugam, S. Sales, N. Ariotti, V. Chambon, C. Lamaze, D. Loew, A. Shevchenko, K. Gaus, R.G. Parton, L. Johannes (2014 May 18)

Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers

Nat Cell Biol : 16 : 595-606 : DOI : DOI: 10.1038/ncb2970 En savoir plus
Résumé

Several cell surface molecules including signalling receptors are internalized by clathrin-independent endocytosis. How this process is initiated, how cargo proteins are sorted and membranes are bent remains unknown. Here, we found that a carbohydrate-binding protein, galectin-3 (Gal3), triggered the glycosphingolipid (GSL)-dependent biogenesis of a morphologically distinct class of endocytic structures, termed clathrin-independent carriers (CLICs). Super-resolution and reconstitution studies showed that Gal3 required GSLs for clustering and membrane bending. Gal3 interacted with a defined set of cargo proteins. Cellular uptake of the CLIC cargo CD44 was dependent on Gal3, GSLs and branched N-glycosylation. Endocytosis of β1-integrin was also reliant on Gal3. Analysis of different galectins revealed a distinct profile of cargoes and uptake structures, suggesting the existence of different CLIC populations. We conclude that Gal3 functionally integrates carbohydrate specificity on cargo proteins with the capacity of GSLs to drive clathrin-independent plasma membrane bending as a first step of CLIC biogenesis.

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