Dynamique et mécanique membranaires de la signalisation intracellulaire

Publications de l’équipe

Année de publication : 2021

Ivashenka# A, Wunder# C, Chambon# V, Sandhoff R, Jennemann R, Dransart# D, Podsypanina K, Lombard B, Loew D, Lamaze C, Poirier F, Gröne HJ, Johannes* L, Shafaq-Zadah*# M (2021 Feb 9)

Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes

COMMUNICATIONS BIOLOGYNature : 4 : 173 (2021) : DOI : 10.1038 En savoir plus
Résumé

Glycoproteins and glycolipids at the plasma membrane contribute to a range of functions from growth factor signaling to cell adhesion and migration. Glycoconjugates undergo endocytic trafficking. According to the glycolipid-lectin (GL-Lect) hypothesis, the construction of tubular endocytic pits is driven in a glycosphingolipid-dependent manner by sugar-binding proteins of the galectin family. Here, we provide evidence for a function of the GL-Lect mechanism in transcytosis across enterocytes in the mouse intestine. We show that galectin-3 (Gal3) and its newly identified binding partner lactotransferrin are transported in a glycosphingolipid-dependent manner from the apical to the basolateral membrane. Transcytosis of lactotransferrin is perturbed in Gal3 knockout mice and can be rescued by exogenous Gal3. Inside enterocytes, Gal3 is localized to hallmark structures of the GL-Lect mechanism, termed clathrin-independent carriers. These data pioneer the existence of GL-Lect endocytosis in vivo and strongly suggest that polarized trafficking across the intestinal barrier relies on this mechanism.

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Année de publication : 2020

Domingues, L., I. Hurbain, F. Gilles-Marsens, J. Sirés-Campos, N. André, M. Dewulf, M. Romao, C. Viaris de Lesegno, A.S. Macé, C. Blouin, C. Guéré, K. Vié, G. Raposo, C. Lamaze, and C. Delevoye (2020 Jun 12)

Coupling of melanocyte signaling and mechanics by caveolae is required for human skin pigmentation

Nature CommunicationNature : 11 : 2988 (2020) : DOI : 10.1038/s41467-020-16738-z En savoir plus
Résumé

Tissue homeostasis requires regulation of cell–cell communication, which relies on signaling molecules and cell contacts. In skin epidermis, keratinocytes secrete factors transduced by melanocytes into signaling cues promoting their pigmentation and dendrite outgrowth, while melanocytes transfer melanin pigments to keratinocytes to convey skin photoprotection. How epidermal cells integrate these functions remains poorly characterized. Here, we show that caveolae are asymmetrically distributed in melanocytes and particularly abundant at the melanocyte–keratinocyte interface in epidermis. Caveolae in melanocytes are modulated by ultraviolet radiations and keratinocytes-released factors, like miRNAs. Preventing caveolae formation in melanocytes increases melanin pigment synthesis through upregulation of cAMP signaling and decreases cell protrusions, cell–cell contacts, pigment transfer and epidermis pigmentation. Altogether, we identify that caveolae serve as molecular hubs that couple signaling outputs from keratinocytes to mechanical plasticity of pigment cells. The coordination of intercellular communication and contacts by caveolae is thus crucial to skin pigmentation and tissue homeostasis.

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Année de publication : 2019

Dewulf, M., D.V. Koster, B. Sinha, C. Viaris de Lesegno, V. Chambon, A. Bigot, M. Bensalah, E. Negroni, N. Tardif, J. Podkalicka, L. Johannes, P. Nassoy, G. Butler-Browne, C. Lamaze* and C.M. Blouin* (2019 Apr 29)

Dystrophy-associated caveolin-3 mutations reveal that caveolae couple IL6/STAT3 signaling with mechanosensing in human muscle cells

Nature CommunicationNature : 10:1974 : DOI : 10.1038/s41467-019-09405-5. En savoir plus
Résumé

Caveolin-3 is the major structural protein of caveolae in muscle. Mutations in the CAV3 gene cause different types of myopathies with altered membrane integrity and repair, expression of muscle proteins, and regulation of signaling pathways. We show here that myotubes from patients bearing the CAV3 P28L and R26Q mutations present a dramatic decrease of caveolae at the plasma membrane, resulting in abnormal response to mechanical stress. Mutant myotubes are unable to buffer the increase in membrane tension induced by mechanical stress. This results in impaired regulation of the IL6/STAT3 signaling pathway leading to its constitutive hyperactivation and increased expression of muscle genes. These defects are fully reversed by reassembling functional caveolae through expression of caveolin-3. Our study reveals that under mechanical stress the regulation of mechanoprotection by caveolae is directly coupled with the regulation of IL6/STAT3 signaling in muscle cells and that this regulation is absent in Cav3-associated dystrophic patients.

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Année de publication : 2018

Mani, C. Viaris de Lesegno, P. Bost, A. Grassart, D. Koster, C.A. Valades-Cruz, V. Chambon, L. Johannes, P. Pierobon, V. Soumelis, C. Coirault, S. Vassilopoulos, and C. Lamaze. (2018 Oct 22)

EHD2 is a mechanotransducer connecting caveolae dynamics with gene transcription

Journal of Cell BiologyJournal of Cell Biology : DOI : 10.1083/jcb.201801122 En savoir plus
Résumé

Caveolae are small invaginated pits that function as dynamic mechanosensors to buffer tension variations at the plasma membrane. Here we show that under mechanical stress, the EHD2 ATPase is rapidly released from caveolae, SUMOylated, and translocated to the nucleus, where it regulates the transcription of several genes including those coding for caveolae constituents. We also found that EHD2 is required to maintain the caveolae reservoir at the plasma membrane during the variations of membrane tension induced by mechanical stress. Metal-replica electron microscopy of breast cancer cells lacking EHD2 revealed a complete absence of caveolae and a lack of gene regulation under mechanical stress. Expressing EHD2 was sufficient to restore both functions in these cells. Our findings therefore define EHD2 as a central player in mechanotransduction connecting the disassembly of the caveolae reservoir with the regulation of gene transcription under mechanical stress.

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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.

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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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