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1. Compressive stress triggers fibroblasts spreading over cancer cells to generate carcinoma in situ organization

Author

Fabien Bertillot, Laetitia Andrique, Carlos Ureña Martin, Olivier Zajac, Ludmilla de Plater, Michael M. Norton, Aurélien Richard, Kevin Alessandri, Basile G. Gurchenkov, Florian Fage, Atef Asnacios, Christophe Lamaze, Moumita Das, Jean- Léon Maître, Pierre Nassoy, and Danijela Matic Vignjevic

Subjects
Biology (General), QH301-705.5
Abstract

Abstract At the early stage of tumor progression, fibroblasts are located at the outer edges of the tumor, forming an encasing layer around it. In this work, we have developed a 3D in vitro model where fibroblasts’ layout resembles the structure seen in carcinoma in situ. We use a microfluidic encapsulation technology to co-culture fibroblasts and cancer cells within hollow, permeable, and elastic alginate shells. We find that in the absence of spatial constraint, fibroblasts and cancer cells do not mix but segregate into distinct aggregates composed of individual cell types. However, upon confinement, fibroblasts enwrap cancer cell spheroid. Using a combination of biophysical methods and live imaging, we find that buildup of compressive stress is required to induce fibroblasts spreading over the aggregates of tumor cells. We propose that compressive stress generated by the tumor growth might be a mechanism that prompts fibroblasts to form a capsule around the tumor.

Published
2024
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2. Caveolin-1 protects endothelial cells from extensive expansion of transcellular tunnel by stiffening the plasma membrane

Author

Camille Morel, Eline Lemerle, Feng-Ching Tsai, Thomas Obadia, Nishit Srivastava, Maud Marechal, Audrey Salles, Marvin Albert, Caroline Stefani, Yvonne Benito, François Vandenesch, Christophe Lamaze, Stéphane Vassilopoulos, Matthieu Piel, Patricia Bassereau, David Gonzalez-Rodriguez, Cecile Leduc, and Emmanuel Lemichez

Subjects
caveolin-1, plasma membrane mechanics, transendothelial cell macroaperture, bacterial toxin, Medicine, Science, Biology (General), QH301-705.5
Abstract

Large transcellular pores elicited by bacterial mono-ADP-ribosyltransferase (mART) exotoxins inhibiting the small RhoA GTPase compromise the endothelial barrier. Recent advances in biophysical modeling point toward membrane tension and bending rigidity as the minimal set of mechanical parameters determining the nucleation and maximal size of transendothelial cell macroaperture (TEM) tunnels induced by bacterial RhoA-targeting mART exotoxins. We report that cellular depletion of caveolin-1, the membrane-embedded building block of caveolae, and depletion of cavin-1, the master regulator of caveolae invaginations, increase the number of TEMs per cell. The enhanced occurrence of TEM nucleation events correlates with a reduction in cell height due to the increase in cell spreading and decrease in cell volume, which, together with the disruption of RhoA-driven F-actin meshwork, favor membrane apposition for TEM nucleation. Strikingly, caveolin-1 specifically controls the opening speed of TEMs, leading to their dramatic 5.4-fold larger widening. Consistent with the increase in TEM density and width in siCAV1 cells, we record a higher lethality in CAV1 KO mice subjected to a catalytically active mART exotoxin targeting RhoA during staphylococcal bloodstream infection. Combined theoretical modeling with independent biophysical measurements of plasma membrane bending rigidity points toward a specific contribution of caveolin-1 to membrane stiffening in addition to the role of cavin-1/caveolin-1-dependent caveolae in the control of membrane tension homeostasis.

Published
2024
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3. A caveolin-1 dependent glucose-6-phosphatase trafficking contributes to hepatic glucose production

Author

Amandine Gautier-Stein, Julien Chilloux, Maud Soty, Bernard Thorens, Christophe Place, Carine Zitoun, Adeline Duchampt, Lorine Da Costa, Fabienne Rajas, Christophe Lamaze, and Gilles Mithieux

Subjects
Liver, Glucose-6 phosphatase, Gluconeogenesis, Intracellular glucose transport, Caveolin 1, Internal medicine, RC31-1245
Abstract

Objective: Deregulation of hepatic glucose production is a key driver in the pathogenesis of diabetes, but its short-term regulation is incompletely deciphered. According to textbooks, glucose is produced in the endoplasmic reticulum by glucose-6-phosphatase (G6Pase) and then exported in the blood by the glucose transporter GLUT2. However, in the absence of GLUT2, glucose can be produced by a cholesterol-dependent vesicular pathway, which remains to be deciphered. Interestingly, a similar mechanism relying on vesicle trafficking controls short-term G6Pase activity. We thus investigated whether Caveolin-1 (Cav1), a master regulator of cholesterol trafficking, might be the mechanistic link between glucose production by G6Pase in the ER and glucose export through a vesicular pathway. Methods: Glucose production from fasted mice lacking Cav1, GLUT2 or both proteins was measured in vitro in primary culture of hepatocytes and in vivo by pyruvate tolerance tests. The cellular localization of Cav1 and the catalytic unit of glucose-6-phosphatase (G6PC1) were studied by western blotting from purified membranes, immunofluorescence on primary hepatocytes and fixed liver sections and by in vivo imaging of chimeric constructs overexpressed in cell lines. G6PC1 trafficking to the plasma membrane was inhibited by a broad inhibitor of vesicular pathways or by an anchoring system retaining G6PC1 specifically to the ER membrane. Results: Hepatocyte glucose production is reduced at the step catalyzed by G6Pase in the absence of Cav1. In the absence of both GLUT2 and Cav1, gluconeogenesis is nearly abolished, indicating that these pathways can be considered as the two major pathways of de novo glucose production. Mechanistically, Cav1 colocalizes but does not interact with G6PC1 and controls its localization in the Golgi complex and at the plasma membrane. The localization of G6PC1 at the plasma membrane is correlated to glucose production. Accordingly, retaining G6PC1 in the ER reduces glucose production by hepatic cells. Conclusions: Our data evidence a pathway of glucose production that relies on Cav1-dependent trafficking of G6PC1 to the plasma membrane. This reveals a new cellular regulation of G6Pase activity that contributes to hepatic glucose production and glucose homeostasis.

Published
2023
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4. Neuronal activity-dependent ATP enhances the pro-growth effect of repair Schwann cell extracellular vesicles by increasing their miRNA-21 loading

Author

Cristian Saquel, Romina J. Catalan, Rodrigo Lopez-Leal, Ramon A. Ramirez, David Necuñir, Ursula Wyneken, Christophe Lamaze, and Felipe A. Court

Subjects
Schwann cell, extracellular vesicles, ATP, purinergic receptors, axonal growth, axonal regeneration, Neurosciences. Biological psychiatry. Neuropsychiatry, RC321-571
Abstract

Functional recovery after peripheral nerve injuries is critically dependent on axonal regeneration. Several autonomous and non-cell autonomous processes regulate axonal regeneration, including the activation of a growth-associated transcriptional program in neurons and the reprogramming of differentiated Schwann cells (dSCs) into repair SCs (rSCs), triggering the secretion of neurotrophic factors and the activation of an inflammatory response. Repair Schwann cells also release pro-regenerative extracellular vesicles (EVs), but is still unknown whether EV secretion is regulated non-cell autonomously by the regenerating neuron. Interestingly, it has been described that nerve activity enhances axonal regeneration by increasing the secretion of neurotrophic factors by rSC, but whether this activity modulates pro-regenerative EV secretion by rSC has not yet been explored. Here, we demonstrate that neuronal activity enhances the release of rSC-derived EVs and their transfer to neurons. This effect is mediated by activation of P2Y receptors in SCs after activity-dependent ATP release from sensory neurons. Importantly, activation of P2Y in rSCs also increases the amount of miRNA-21 present in rSC-EVs. Taken together, our results demonstrate that neuron to glia communication by ATP-P2Y signaling regulates the content of SC-derived EVs and their transfer to axons, modulating axonal elongation in a non-cell autonomous manner.

Published
2022
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5. Identification of a New Cholesterol‐Binding Site within the IFN‐γ Receptor that is Required for Signal Transduction

Author

Ornella Morana, Jon Ander Nieto‐Garai, Patrik Björkholm, Jorge Bernardino de la Serna, Oihana Terrones, Aroa Arboleya, Dalila Ciceri, Iratxe Rojo‐Bartolomé, Cédric M. Blouin, Christophe Lamaze, Maier Lorizate, and Francesc‐Xabier Contreras

Subjects
cholesterol, interferon gamma receptors, lipid nanodomains, protein–lipid interactions, signal transduction, Science
Abstract

Abstract The cytokine interferon‐gamma (IFN‐γ) is a master regulator of innate and adaptive immunity involved in a broad array of human diseases that range from atherosclerosis to cancer. IFN‐γ exerts it signaling action by binding to a specific cell surface receptor, the IFN‐γ receptor (IFN‐γR), whose activation critically depends on its partition into lipid nanodomains. However, little is known about the impact of specific lipids on IFN‐γR signal transduction activity. Here, a new conserved cholesterol (chol) binding motif localized within its single transmembrane domain is identified. Through direct binding, chol drives the partition of IFN‐γR2 chains into plasma membrane lipid nanodomains, orchestrating IFN‐γR oligomerization and transmembrane signaling. Bioinformatics studies show that the signature sequence stands for a conserved chol‐binding motif presented in many mammalian membrane proteins. The discovery of chol as the molecular switch governing IFN‐γR transmembrane signaling represents a significant advance for understanding the mechanism of lipid selectivity by membrane proteins, but also for figuring out the role of lipids in modulating cell surface receptor function. Finally, this study suggests that inhibition of the chol‐IFNγR2 interaction may represent a potential therapeutic strategy for various IFN‐γ‐dependent diseases.

Published
2022
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6. Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes

Author

Alena Ivashenka, Christian Wunder, Valerie Chambon, Roger Sandhoff, Richard Jennemann, Estelle Dransart, Katrina Podsypanina, Bérangère Lombard, Damarys Loew, Christophe Lamaze, Francoise Poirier, Hermann-Josef Gröne, Ludger Johannes, and Massiullah Shafaq-Zadah

Subjects
Biology (General), QH301-705.5
Abstract

Ivashenka et al. report that galectin-3 (Gal3) binding to lactotransferrin drives its transcytosis in enterocytes. Such trafficking is Gal3- and glycosphingolipid-dependent, and Gal3 is found in clathrin-independent carriers. These findings suggest that polarized trafficking across the intestinal barrier relies on this glycolipid and lectin (GL-Lect)-mediated endocytosis.

Published
2021
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7. Coupling of melanocyte signaling and mechanics by caveolae is required for human skin pigmentation

Author

Lia Domingues, Ilse Hurbain, Floriane Gilles-Marsens, Julia Sirés-Campos, Nathalie André, Melissa Dewulf, Maryse Romao, Christine Viaris de Lesegno, Anne-Sophie Macé, Cédric Blouin, Christelle Guéré, Katell Vié, Graça Raposo, Christophe Lamaze, and Cédric Delevoye

Subjects
Science
Abstract

Caveolae are plasma membrane invaginations playing crucial functions, like signal transduction and mechanoprotection. Here, the authors show that caveolae contribute to skin pigmentation by integrating the biochemical and mechanical response of epidermal melanocytes to extracellular cues.

Published
2020
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8. An Abl-FBP17 mechanosensing system couples local plasma membrane curvature and stress fiber remodeling during mechanoadaptation

Author

Asier Echarri, Dácil M. Pavón, Sara Sánchez, María García-García, Enrique Calvo, Carla Huerta-López, Diana Velázquez-Carreras, Christine Viaris de Lesegno, Nicholas Ariotti, Ana Lázaro-Carrillo, Raffaele Strippoli, David De Sancho, Jorge Alegre-Cebollada, Christophe Lamaze, Robert G. Parton, and Miguel A. Del Pozo

Subjects
Science
Abstract

Mechanical forces are sensed by cells and can alter plasma membrane properties, but biochemical changes underlying this are not clear. Here the authors show tension is sensed by c-Abl and FBP17, which couples changes in mechanical tension to remodelling of the plasma membrane and actin cytoskeleton.

Published
2019
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9. Dystrophy-associated caveolin-3 mutations reveal that caveolae couple IL6/STAT3 signaling with mechanosensing in human muscle cells

Author

Melissa Dewulf, Darius Vasco Köster, Bidisha Sinha, Christine Viaris de Lesegno, Valérie Chambon, Anne Bigot, Mona Bensalah, Elisa Negroni, Nicolas Tardif, Joanna Podkalicka, Ludger Johannes, Pierre Nassoy, Gillian Butler-Browne, Christophe Lamaze, and Cedric M. Blouin

Subjects
Science
Abstract

Caveolae are mechanosensors and mutations of their coat proteins are implicated in muscle disorders, but molecular mechanisms are unclear. Here, the authors show that caveolae can regulate IL6/STAT3 signaling in muscle cells under stress, and that dystrophy related Cav3 mutant myotubes have reduced caveolae and upregulated IL6 signaling.

Published
2019
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10. Interferon Receptor Trafficking and Signaling: Journey to the Cross Roads

Author

Natacha Zanin, Christine Viaris de Lesegno, Christophe Lamaze, and Cedric M. Blouin

Subjects
transmembrane receptor, interferon, endocytosis, intracellular signaling, traffic, JAK - STAT signaling pathway, Immunologic diseases. Allergy, RC581-607
Abstract

Like most plasma membrane proteins, type I interferon (IFN) receptor (IFNAR) traffics from the outer surface to the inner compartments of the cell. Long considered as a passive means to simply control subunits availability at the plasma membrane, an array of new evidence establishes IFNAR endocytosis as an active contributor to the regulation of signal transduction triggered by IFN binding to IFNAR. During its complex journey initiated at the plasma membrane, the internalized IFNAR complex, i.e. IFNAR1 and IFNAR2 subunits, will experience post-translational modifications and recruit specific effectors. These finely tuned interactions will determine not only IFNAR subunits destiny (lysosomal degradation vs. plasma membrane recycling) but also the control of IFN-induced signal transduction. Finally, the IFNAR system perfectly illustrates the paradigm of the crosstalk between membrane trafficking and intracellular signaling. Investigating the complexity of IFN receptor intracellular routes is therefore necessary to reveal new insight into the role of IFNAR membrane dynamics in type I IFNs signaling selectivity and biological activity.

Published
2021
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11. Spatiotemporal control of interferon-induced JAK/STAT signalling and gene transcription by the retromer complex

Author

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, and Christophe Lamaze

Subjects
Science
Abstract

Endocytosis of the type I interferon receptor (IFNAR) is required for JAK/STAT signalling. Here the authors show that the internalized IFNAR1 and IFNAR2 subunits are differentially sorted by the retromer complex at the early endosome and this controls JAK/STAT signalling and gene transcription.

Published
2016
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12. Oxidative stress induces caveolin 1 degradation and impairs caveolae functions in skeletal muscle cells.

Author

Alexis Mougeolle, Sylvie Poussard, Marion Decossas, Christophe Lamaze, Olivier Lambert, and Elise Dargelos

Subjects
Medicine, Science
Abstract

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.

Published
2015
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13. The dynamin chemical inhibitor dynasore impairs cholesterol trafficking and sterol-sensitive genes transcription in human HeLa cells and macrophages.

Author

Emmanuelle Girard, Jean Louis Paul, Natalie Fournier, Philippe Beaune, Ludger Johannes, Christophe Lamaze, and Benoît Védie

Subjects
Medicine, Science
Abstract

Intracellular transport of cholesterol contributes to the regulation of cellular cholesterol homeostasis by mechanisms that are yet poorly defined. In this study, we characterized the impact of dynasore, a recently described drug that specifically inhibits the enzymatic activity of dynamin, a GTPase regulating receptor endocytosis and cholesterol trafficking. Dynasore strongly inhibited the uptake of low-density lipoprotein (LDL) in HeLa cells, and to a lower extent in human macrophages. In both cell types, dynasore treatment led to the abnormal accumulation of LDL and free cholesterol (FC) within the endolysosomal network. The measure of cholesterol esters (CE) further showed that the delivery of regulatory cholesterol to the endoplasmic reticulum (ER) was deficient. This resulted in the inhibition of the transcriptional control of the three major sterol-sensitive genes, sterol-regulatory element binding protein 2 (SREBP-2), 3-hydroxy-3-methyl-coenzymeA reductase (HMGCoAR), and low-density lipoprotein receptor (LDLR). The sequestration of cholesterol in the endolysosomal compartment impaired both the active and passive cholesterol efflux in HMDM. Our data further illustrate the importance of membrane trafficking in cholesterol homeostasis and validate dynasore as a new pharmacological tool to study the intracellular transport of cholesterol.

Published
2011
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14. Caveolae contribute to the apoptosis resistance induced by the alpha(1A)-adrenoceptor in androgen-independent prostate cancer cells.

Author

Maria Katsogiannou, Charbel El Boustany, Florian Gackiere, Philippe Delcourt, Anne Athias, Pascal Mariot, Etienne Dewailly, Nathalie Jouy, Christophe Lamaze, Gabriel Bidaux, Brigitte Mauroy, Natalia Prevarskaya, and Christian Slomianny

Subjects
Medicine, Science
Abstract

BACKGROUND:During androgen ablation prostate cancer cells' growth and survival become independent of normal regulatory mechanisms. These androgen-independent cells acquire the remarkable ability to adapt to the surrounding microenvironment whose factors, such as neurotransmitters, influence their survival. Although findings are becoming evident about the expression of alpha(1A)-adrenoceptors in prostate cancer epithelial cells, their exact functional role in androgen-independent cells has yet to be established. Previous work has demonstrated that membrane lipid rafts associated with key signalling proteins mediate growth and survival signalling pathways in prostate cancer cells. METHODOLOGY/PRINCIPAL FINDINGS:In order to analyze the membrane topology of the alpha(1A)-adrenoceptor we explored its presence by a biochemical approach in purified detergent resistant membrane fractions of the androgen-independent prostate cancer cell line DU145. Electron microscopy observations demonstrated the colocalization of the alpha(1A)-adrenoceptor with caveolin-1, the major protein component of caveolae. In addition, we showed that agonist stimulation of the alpha(1A)-adrenoceptor induced resistance to thapsigargin-induced apoptosis and that caveolin-1 was necessary for this process. Further, immunohistofluorescence revealed the relation between high levels of alpha(1A)-adrenoceptor and caveolin-1 expression with advanced stage prostate cancer. We also show by immunoblotting that the TG-induced apoptosis resistance described in DU145 cells is mediated by extracellular signal-regulated kinases (ERK). CONCLUSIONS/SIGNIFICANCE:In conclusion, we propose that alpha(1A)-adrenoceptor stimulation in androgen-independent prostate cancer cells via caveolae constitutes one of the mechanisms contributing to their protection from TG-induced apoptosis.

Published
2009
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16. PSL Chemical Biology Symposia Third Edition: A Branch of Science in its Explosive Phase

Author

Leeroy Baron, Justine Hadjerci, Leishemba Thoidingjam, Marina Plays, Romain Bucci, Nolwenn Morris, Sebastian Müller, Fabien Sindikubwabo, Stéphanie Solier, Tatiana Cañeque, Ludovic Colombeau, Cedric M. Blouin, Christophe Lamaze, Alain Puisieux, Yannick Bono, Christine Gaillet, Luca Laraia, Boris Vauzeilles, Frédéric Taran, Sébastien Papot, Philippe Karoyan, Romain Duval, Florence Mahuteau‐Betzer, Paola Arimondo, Kevin Cariou, Gilles Guichard, Laurent Micouin, Mélanie Ethève‐Quelquejeu, Daniela Verga, Antoine Versini, Gilles Gasser, Cong Tang, Philippe Belmont, Andreas Linkermann, Claudia Bonfio, Dennis Gillingham, Thomas Poulsen, Marco Di Antonio, Marie Lopez, Dominique Guianvarc'h, Christophe Thomas, Géraldine Masson, Arnaud Gautier, Ludger Johannes, and Raphaël Rodriguez

Subjects
Organic Chemistry, Molecular Medicine, Molecular Biology, Biochemistry
Published
2023

17. Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system

Author

Fidel-Nicolás Lolo, Nikhil Walani, Eric Seemann, Dobryna Zalvidea, Dácil María Pavón, Gheorghe Cojoc, Moreno Zamai, Christine Viaris de Lesegno, Fernando Martínez de Benito, Miguel Sánchez-Álvarez, Juan José Uriarte, Asier Echarri, Daniel Jiménez-Carretero, Joan-Carles Escolano, Susana A. Sánchez, Valeria R. Caiolfa, Daniel Navajas, Xavier Trepat, Jochen Guck, Christophe Lamaze, Pere Roca-Cusachs, Michael M. Kessels, Britta Qualmann, Marino Arroyo, Miguel A. del Pozo, Universitat Politècnica de Catalunya. Departament d'Enginyeria Civil i Ambiental, Universitat Politècnica de Catalunya. LACÀN - Mètodes Numèrics en Ciències Aplicades i Enginyeria, Unión Europea. Comisión Europea. H2020, Marie Curie, Ministerio de Ciencia e Innovación (España), European Union Next Generation, Asociación Española Contra el Cáncer, Comunidad de Madrid (España), Fundación La Marató TV3, Fundación La Caixa, Ministerio de Ciencia e Innovación. Centro de Excelencia Severo Ochoa (España), and Fundación ProCNIC

Subjects
Biomathematics, Biologia, Biomatemàtica, 92 Biology and other natural sciences::92B Mathematical biology in general [Classificació AMS], Matemàtiques i estadística::Matemàtica aplicada a les ciències [Àrees temàtiques de la UPC], Cell Biology, Biology, 92 Biology and other natural sciences::92C Physiological, cellular and medical topics [Classificació AMS]
Abstract

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations-dolines-capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli. We thank R. Parton (Institute for Molecular Biosciences, Queensland), P. Pilch (Boston University School of Medicine) and L. Liu (Boston University School of Medicine) for kindly providing PTRFKO cells and reagents, S. Casas Tintó for kindly providing SH-Sy5y cells, P. Bassereau (Curie Institute, Paris) for kindly providing OT setup, V. Labrador Cantarero from CNIC microscopy Unit for helping with ImageJ analysis, O. Otto and M. Herbig for providing help with RTDC experiments, S. Berr and K. Gluth for technical assistance in cell culture, F. Steiniger for support in electron tomography, and A. Norczyk Simón for providing pCMV-FLAG-PTRF construct. This project received funding from the European Union Horizon 2020 Research and Innovation Programme through Marie Sklodowska-Curie grant 641639; grants from the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033): SAF2014-51876-R, SAF2017-83130-R co-funded by ‘ERDF A way of making Europe’, PID2020-118658RB-I00, PDC2021-121572-100 co-funded by ‘European Union NextGenerationEU/PRTR’, CSD2009- 0016 and BFU2016-81912-REDC; and the Asociación Española Contra el Cáncer foundation (PROYE20089DELP) all to M.A.d.P. M.A.d.P. is member of the Tec4Bio consortium (ref. S2018/NMT¬4443; Comunidad Autónoma de Madrid/FEDER, Spain), co-recipient with P.R.-C. of grants from Fundació La Marató de TV3 (674/C/2013 and 201936- 30-31), and coordinator of a Health Research consortium grant from Fundación Obra Social La Caixa (AtheroConvergence, HR20-00075). M.S.-A. is recipient of a Ramón y Cajal research contract from MCIN (RYC2020-029690-I). The CNIC Unit of Microscopy and Dynamic Imaging is supported by FEDER ‘Una manera de hacer Europa’ (ReDIB ICTS infrastructure TRIMA@CNIC, MCIN). We acknowledge the support from Deutsche Forschungsgemeinschaft through grants to M.M.K. (KE685/7-1) and B.Q. (QU116/6-2 and QU116/9-1). Work in D.N. laboratory was supported by grants from the European Union Horizon 2020 Research and Innovation Programme through Marie Sklodowska-Curie grant 812772 and MCIN (DPI2017-83721-P). Work in C.L. laboratory was supported by grants from Curie, INSERM, CNRS, Agence Nationale de la Recherche (ANR-17-CE13-0020-01) and Fondation ARC pour la Recherche (PGA1-RF20170205456). Work in P.R.-C. lab is funded by the MCIN (PID2019-110298GB-I00), the EC (H20 20-FETPROACT-01-2016-731957). Work in X.T. lab is funded by the MICIN (PID2021-128635NB-I00), ERC (Adv-883739) and La Caixa Foundation (LCF/PR/HR20/52400004; co-recipient with P.R.-C.). IBEC is recipient of a Severo Ochoa Award of Excellence from the MINECO. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MCIN and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). Sí

Published
2022

18. Caveolae govern plasma membrane mechanics to protect cells against EDIN B-induced transcellular tunnel formation and lethality from S. aureus septicaemia

Author

Camille Morel, Eline Lemerle, Feng-Ching Tsai, Thomas Obadia, Nishit Srivastava, Maud Marechal, Audrey Salles, Marvin Albert, Caroline Stefani, Christophe Lamaze, Stéphane Vassilopoulos, Matthieu Piel, Patricia Bassereau, David Gonzalez-Rodriguez, Cécile Leduc, and Emmanuel Lemichez

Abstract

The spontaneous opening of large transendothelial cell macroaperture (TEM) tunnels can accompany leukocyte diapedesis and is triggered by bacterial exoenzymes that inhibit RhoA-driven cytoskeleton contractility. Modelling the dynamics of TEM via a theoretical framework used for soft matter physics allowed us to depict the essential driving forces at play on the membrane to enlarge TEMs. In this study, we conducted multidisciplinary experiments to characterize the role respectively played by cavin-1-structured caveolae and non-caveolar caveolin-1 in plasma membrane mechanics and identify their functional effects on TEM size. The results pointed towards a contributing role for non-caveolar caveolin-1 in the membrane bending rigidity, a mechanical parameter we quantified in a model system of tubes pulled from plasma membrane spheres. Depletion of cavin-1-structured caveolae showed no effect on membrane rigidity, whereas caveolae controlled cell height favouring TEM nucleation. Hence, caveolae confer protection against exoenzyme EDIN-B in mice with staphylococcal septicaemia.

Published
2022

21. Small Molecule Inhibitors of Interferon-Induced JAK-STAT Signalling

Author

Leishemba K. Thoidingjam, Cédric M. Blouin, Christine Gaillet, Aurélien Brion, Stéphanie Solier, Supaporn Niyomchon, Ahmed El Marjou, Sara Mouasni, Fernando E. Sepulveda, Geneviève de Saint Basile, Christophe Lamaze, and Raphaël Rodriguez

Subjects
Interferon-gamma, General Medicine, General Chemistry, Interferons, Phosphorylation, Catalysis, Janus Kinases, Signal Transduction
Abstract

Interferons (IFN) are cytokines which, upon binding to cell surface receptors, trigger a series of downstream biochemical events including Janus Kinase (JAK) activation, phosphorylation of Signal Transducer and Activator of Transcription protein (STAT), translocation of pSTAT to the nucleus and transcriptional activation. Dysregulated IFN signalling has been linked to cancer progression and auto-immune diseases. Here, we report the serendipitous discovery of a small molecule that blocks IFNγ activation of JAK-STAT signalling. Further lead optimisation gave rise to a potent and more selective analogue that exerts its activity by a mechanism consistent with direct IFNγ targeting in vitro, which reduces bleeding in model of haemorrhagic colitis in vivo. This first-in-class small molecule also inhibits type I and III IFN-induced STAT phosphorylation in vitro. Our work provides the basis for the development of pan-IFN inhibitory drugs.

Published
2022

22. Caveolae promote successful abscission by controlling intercellular bridge tension during cytokinesis

Author

Virginia Andrade, Jian Bai, Neetu Gupta-Rossi, Ana Joaquina Jimenez, Cédric Delevoye, Christophe Lamaze, Arnaud Echard, Trafic membranaire et Division cellulaire - Membrane Traffic and Cell Division, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Sorbonne Université (SU), Dynamique de l’organisation intra-cellulaire, Biologie Cellulaire et Cancer, Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), BioImaging Cell and Tissue Core Facility (PICT-IBiSA), Institut Curie [Paris], Structure et compartimentation membranaire, Compartimentation et dynamique cellulaires (CDC), Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), This work was supported by Institut Pasteur (A.E.), CNRS (A.E.), ANR Cytosign, ANR SeptScort (A.E.), ANR MOTICAV (C.L.), ANR CAV-SM (C.L.), Fondation pour le Recherche Médicale: Recherche soutenue par la FRM EQU202103012627 (A.E.), French National Research Agency through the 'Investments for the Future' program, France-BioImaging, ANR-10-INSB-04 (C.D.), Cell and Tissue Imaging core facility (PICT-IBiSA), member of the France-BioImaging national research infrastructure, supported by the Labex Cell(n)Scale (ANR-10-LBX-0038) part of the IDEX PSL (ANR-10-IDEX-0001-02 PSL) (C.D. and C.L.), Doctoral School Complexité du Vivant ED515, contrat n° 2828/2017 (V.A.), La Ligue Contre le Cancer (4ème année de thèse) (V.A.), Pasteur-Paris University (PPU) international PhD program (J.B.), Fondation ARC pour la recherche sur le cancer (DOC20180507410) (J.B.), Programme Labellisé PGA1-RF20170205456 (C.L.), and CEFIPRA, project 6301-1., ANR-16-CE13-0004,CYTOSIGN,Polarité épithéliale et signalisation au cours de la cytodiérèse des cellules animales(2016), ANR-20-CE11-0014,SeptScort,Interaction spatiale et fonctionnelle entre septines et Escrt pendant la cytocinèse: une approche multi-échelle(2020), ANR-17-CE13-0020,MOTICAV,Cavéoles et Tension Membranaire dans la Migration Cellulaire(2017), ANR-20-CE13-0002,CAV-SM,Un rôle nouveau de la cavéoline dans le transport de la sphingomyéline à la membrane plasmique(2020), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-10-IDEX-0001,PSL,Paris Sciences et Lettres(2010), Collège Doctoral, Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), and Agence Nationale de la Recherche: CytosignAgence Nationale de la Recherche: SeptScortAgence Nationale de la Recherche: ANR-10-INSB-04Agence Nationale de la Recherche: ANR-10-LBX-0038Agence Nationale de la Recherche: ANR-10-IDEX-0001-02 PSLInstitut PasteurFondation ARC pour la Recherche sur le Cancer: DOC20180507410La Ligue Contre Le CancerDoctoral School Complexité du Vivant ED515: 2828/2017CNRSPasteur-Paris University (PPU) international PhD program

Subjects
Multidisciplinary, [SDV]Life Sciences [q-bio], macromolecular substances
Abstract

During cytokinesis, the intercellular bridge (ICB) connecting the daughter cells experiences pulling forces, which delay abscission by preventing the assembly of the ESCRT scission machinery. Abscission is thus triggered by tension release, but how ICB tension is controlled is unknown. Here, we report that caveolae, which are known to control membrane tension upon mechanical stress in interphase cells, are located at the midbody, at the abscission site and at the ICB/cell interface in dividing cells. Functionally, the loss of caveolae delays ESCRT-III recruitment during cytokinesis and impairs abscission. This is the consequence of a 2-fold increase of ICB tension measured by laser ablation, associated with a local increase in myosin II activity at the ICB/cell interface. We thus propose that caveolae buffer membrane tension and limit contractibility at the ICB to promote ESCRT-III assembly and cytokinetic abscission. Altogether, this work reveals an unexpected connection between caveolae and the ESCRT machinery and the first role of caveolae in cell division.TEASERCaveolae limit the tension in the intercellular bridge during cytokinesis to enable ESCRT-III assembly and successful abscission.

Published
2022

23. Membrane tension buffering by caveolae: a role in cancer?

Author

Christophe Lamaze and Vibha Singh

Subjects
0301 basic medicine, Cancer Research, Angiogenesis, Caveolin 1, Biology, Caveolae, Mechanotransduction, Cellular, Metastasis, 03 medical and health sciences, Mechanobiology, 0302 clinical medicine, Neoplasms, medicine, Animals, Humans, Cell growth, Cell Membrane, Cell migration, medicine.disease, Cell biology, 030104 developmental biology, Oncology, 030220 oncology & carcinogenesis, Cancer cell, Function (biology)
Abstract

Caveolae are bulb-like invaginations made up of two essential structural proteins, caveolin-1 and cavins, which are abundantly present at the plasma membrane of vertebrate cells. Since their discovery more than 60 years ago, the function of caveolae has been mired in controversy. The last decade has seen the characterization of new caveolae components and regulators together with the discovery of additional cellular functions that have shed new light on these enigmatic structures. Early on, caveolae and/or caveolin-1 have been involved in the regulation of several parameters associated with cancer progression such as cell migration, metastasis, angiogenesis, or cell growth. These studies have revealed that caveolin-1 and more recently cavin-1 have a dual role with either a negative or a positive effect on most of these parameters. The recent discovery that caveolae can act as mechanosensors has sparked an array of new studies that have addressed the mechanobiology of caveolae in various cellular functions. This review summarizes the current knowledge on caveolae and their role in cancer development through their activity in membrane tension buffering. We propose that the role of caveolae in cancer has to be revisited through their response to the mechanical forces encountered by cancer cells during tumor mass development.

Published
2020

24. An Abl-FBP17 mechanosensing system couples local plasma membrane curvature and stress fiber remodeling during mechanoadaptation

Author

Christine Viaris de Lesegno, Maria Garcia-Garcia, David De Sancho, Raffaele Strippoli, Enrique Calvo, Christophe Lamaze, Ana Lazaro-Carrillo, Sara Sanchez, Carla Huerta-Lopez, Nicholas Ariotti, Asier Echarri, Miguel A. del Pozo, Jorge Alegre-Cebollada, Dacil Maria Pavon, Diana Velázquez-Carreras, Robert G. Parton, Ministerio de Economía, Industria y Competitividad (España), European Regional Development Fund, Fundación La Marató TV3, Worldwide Cancer Research, Unión Europea. Comisión Europea, Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid (España), Agence Nationale de la Recherche (Francia), Fondation ARC pour la recherche sur le cancer, National Health and Medical Research Council (Australia), Instituto de Salud Carlos III, and Fundación ProCNIC

Subjects
0301 basic medicine, Stress fiber, Osmotic shock, Molecular biology, Science, General Physics and Astronomy, macromolecular substances, Caveolae, Fatty Acid-Binding Proteins, Mechanotransduction, Cellular, Article, General Biochemistry, Genetics and Molecular Biology, Membrane bending, Gene Knockout Techniques, 03 medical and health sciences, 0302 clinical medicine, Stress Fibers, Humans, Phosphorylation, RNA, Small Interfering, Mechanotransduction, Proto-Oncogene Proteins c-abl, Cytoskeleton, skin and connective tissue diseases, lcsh:Science, FBP17 mechanoadaptation membrane curvature, Multidisciplinary, Chemistry, food and beverages, General Chemistry, Fibroblasts, Microscopy, Electron, HEK293 Cells, 030104 developmental biology, Membrane curvature, Biophysics, Mechanosensitive channels, lcsh:Q, Stress, Mechanical, sense organs, 030217 neurology & neurosurgery, Cell signalling, HeLa Cells
Abstract

Cells remodel their structure in response to mechanical strain. However, how mechanical forces are translated into biochemical signals that coordinate the structural changes observed at the plasma membrane (PM) and the underlying cytoskeleton during mechanoadaptation is unclear. Here, we show that PM mechanoadaptation is controlled by a tension-sensing pathway composed of c-Abl tyrosine kinase and membrane curvature regulator FBP17. FBP17 is recruited to caveolae to induce the formation of caveolar rosettes. FBP17 deficient cells have reduced rosette density, lack PM tension buffering capacity under osmotic shock, and cannot adapt to mechanical strain. Mechanistically, tension is transduced to the FBP17 F-BAR domain by direct phosphorylation mediated by c-Abl, a mechanosensitive molecule. This modification inhibits FBP17 membrane bending activity and releases FBP17-controlled inhibition of mDia1-dependent stress fibers, favoring membrane adaptation to increased tension. This mechanoprotective mechanism adapts the cell to changes in mechanical tension by coupling PM and actin cytoskeleton remodeling., Mechanical forces are sensed by cells and can alter plasma membrane properties, but biochemical changes underlying this are not clear. Here the authors show tension is sensed by c-Abl and FBP17, which couples changes in mechanical tension to remodelling of the plasma membrane and actin cytoskeleton.

Published
2019

25. Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes

Author

Hermann-Josef Gröne, Bérangère Lombard, Roger Sandhoff, Françoise Poirier, Katrina Podsypanina, Ludger Johannes, Christian Wunder, Valérie Chambon, Alena Ivashenka, Massiullah Shafaq-Zadah, Richard Jennemann, Damarys Loew, Christophe Lamaze, and Estelle Dransart

Subjects
QH301-705.5, Physiology, Galectin 3, Galectins, Endocytic cycle, Medicine (miscellaneous), Endocytosis, Article, Glycosphingolipids, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, 0302 clinical medicine, Animals, Biology (General), Cell adhesion, 030304 developmental biology, Epithelial polarity, Galectin, Mice, Knockout, 0303 health sciences, biology, Chemistry, Lectin, Blood Proteins, Cell biology, carbohydrates (lipids), Mice, Inbred C57BL, Lactotransferrin, Lactoferrin, Enterocytes, Jejunum, Transcytosis, biology.protein, lipids (amino acids, peptides, and proteins), General Agricultural and Biological Sciences, 030217 neurology & neurosurgery
Abstract

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., Ivashenka et al. report that galectin-3 (Gal3) binding to lactotransferrin drives its transcytosis in enterocytes. Such trafficking is Gal3- and glycosphingolipid-dependent, and Gal3 is found in clathrin-independent carriers. These findings suggest that polarized trafficking across the intestinal barrier relies on this glycolipid and lectin (GL-Lect)-mediated endocytosis.

Published
2021

26. Interferon Receptor Trafficking and Signaling: Journey to the Cross Roads

Author

Christine Viaris de Lesegno, Natacha Zanin, Cédric M. Blouin, and Christophe Lamaze

Subjects
lcsh:Immunologic diseases. Allergy, 0301 basic medicine, Glycosylation, Immunology, Review, Endosomes, Receptor, Interferon alpha-beta, Endocytosis, 03 medical and health sciences, Cytosol, JAK - STAT signaling pathway, 0302 clinical medicine, Protein Domains, Interferon, medicine, Animals, Humans, endocytosis, Immunology and Allergy, transmembrane receptor, Phosphorylation, Receptor, traffic, Endosomal Sorting Complexes Required for Transport, Chemistry, Cell Membrane, intracellular signaling, Membrane Proteins, JAK-STAT signaling pathway, interferon, Protein-Tyrosine Kinases, Phosphoproteins, Rats, Cell biology, Protein Transport, STAT Transcription Factors, Crosstalk (biology), 030104 developmental biology, Membrane protein, Interferons, Signal transduction, Lysosomes, lcsh:RC581-607, Protein Processing, Post-Translational, 030217 neurology & neurosurgery, Intracellular, Signal Transduction, medicine.drug
Abstract

Like most plasma membrane proteins, type I interferon (IFN) receptor (IFNAR) traffics from the outer surface to the inner compartments of the cell. Long considered as a passive means to simply control subunits availability at the plasma membrane, an array of new evidence establishes IFNAR endocytosis as an active contributor to the regulation of signal transduction triggered by IFN binding to IFNAR. During its complex journey initiated at the plasma membrane, the internalized IFNAR complex, i.e. IFNAR1 and IFNAR2 subunits, will experience post-translational modifications and recruit specific effectors. These finely tuned interactions will determine not only IFNAR subunits destiny (lysosomal degradation vs. plasma membrane recycling) but also the control of IFN-induced signal transduction. Finally, the IFNAR system perfectly illustrates the paradigm of the crosstalk between membrane trafficking and intracellular signaling. Investigating the complexity of IFN receptor intracellular routes is therefore necessary to reveal new insight into the role of IFNAR membrane dynamics in type I IFNs signaling selectivity and biological activity.

Published
2021

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

Author

Anne-Sophie Macé, Katell Vié, Cédric M. Blouin, Christelle Guéré, Lia Domingues, Graça Raposo, Julia Sirés-Campos, Christophe Lamaze, Floriane Gilles-Marsens, Melissa Dewulf, Ilse Hurbain, Maryse Romao, Christine Viaris de Lesegno, Cédric Delevoye, Nathalie André, Structure and Membrane Compartments [Paris], Biologie Cellulaire et Cancer, Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), BioImaging Cell and Tissue Core Facility (PICT-IBiSA), Institut Curie [Paris], Génétique et Neurobiologie de C. Elegans [Lyon], Institut NeuroMyoGène (INMG), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire Clarins [Pontoise], Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC), We acknowledge the Nikon Imaging Center at the Institut Curie/Centre National de la Recherche Scientifique and the PICT-IBiSA, a member of the France-BioImaging national research infrastructure (ANR-10-INBS-04). This work has received support under the program 'Investissement d’Avenir' launched by the French Government and implemented by the Agence Nationale de la Recherche (ANR) with the references ANR-10-LBX-0038 and ANR-10-IDEX-0001-02 PSL, Fondation pour la Recherche Médicale (Equipe FRM DEQ20140329491 Team label to G.R.), Agence Nationale de la Recherche ('MOTICAV' ANR-17-CE13-0020-01 to C.L., and 'MYOACTIONS' ANR-17-CE11-0029-03 to C.D.), Fondation ARC pour la Recherche sur le Cancer (PJA20161204965 to C.D., and Programme Labellisé PGA1-RF20170205456 to C.L.). This work received support from the grants ANR-11-LABX-0038, ANR-10-IDEX-0001-02 (Labex CelTisPhyBio to L.D.), Groupe Clarins, Institut Curie, CNRS and INSERM., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-10-IDEX-0001,PSL,Paris Sciences et Lettres(2010), ANR-17-CE13-0020,MOTICAV,Cavéoles et Tension Membranaire dans la Migration Cellulaire(2017), ANR-17-CE11-0029,MyoActions,Rôle des partenaires pour définir la fonction cellulaire de la myosine VI(2017), Bodescot, Myriam, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, Initiative d'excellence - Paris Sciences et Lettres - - PSL2010 - ANR-10-IDEX-0001 - IDEX - VALID, Cavéoles et Tension Membranaire dans la Migration Cellulaire - - MOTICAV2017 - ANR-17-CE13-0020 - AAPG2017 - VALID, Rôle des partenaires pour définir la fonction cellulaire de la myosine VI - - MyoActions2017 - ANR-17-CE11-0029 - AAPG2017 - VALID, Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Keratinocytes, Cell signaling, Ultraviolet Rays, Science, Caveolin 1, General Physics and Astronomy, Human skin, Skin Pigmentation, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Cell Communication, Melanocyte, Caveolae, General Biochemistry, Genetics and Molecular Biology, Article, Melanin, 03 medical and health sciences, 0302 clinical medicine, Downregulation and upregulation, Microscopy, Electron, Transmission, medicine, Humans, lcsh:Science, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Tissue homeostasis, Cells, Cultured, Skin, Multidisciplinary, Epidermis (botany), integumentary system, Chemistry, General Chemistry, Coculture Techniques, Cell biology, 030104 developmental biology, medicine.anatomical_structure, Epidermal Cells, Microscopy, Fluorescence, 030220 oncology & carcinogenesis, Cell polarity, Melanocytes, lcsh:Q, sense organs, Epidermis, Cell signalling, HeLa Cells, Signal Transduction
Abstract

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., Caveolae are plasma membrane invaginations playing crucial functions, like signal transduction and mechanoprotection. Here, the authors show that caveolae contribute to skin pigmentation by integrating the biochemical and mechanical response of epidermal melanocytes to extracellular cues.

Published
2020

28. Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2

Author

Raphaël Sierocki, Julien Barbier, Adam D. Linstedt, Ludger Johannes, Alison Forrester, Stefan J. Rathjen, Mathilde Munier, Sylvain Pichard, Daniel Gillet, Henri-François Renard, Christophe Lamaze, Damarys Loew, Maria Daniela Garcia-Castillo, Jennifer Martinez, Livia Tepshi, Jean-Christophe Cintrat, Collin Bachert, Audrey Couhert, Florent Dingli, Cesar Augusto Valades-Cruz, Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), ANR-19-CE13-0001,GlycoTopoSwitch,La topologie des glycans comme interrupteur moléculaire pour l'activité des intérgines(2019), Carnegie Mellon University [Pittsburgh] (CMU), Service de Chimie Bio-Organique et de Marquage (SCBM), Centre de recherche de l'Institut Curie [Paris], Institut Curie [Paris], Human Frontier Science Program RGP0029-2014, Swedish Research Council European Commission K2015-99X-22877-01-6, Joint Ministerial Program of R&D against CBRNE Risks, Ile de France Région DIM Malinf initiative 140101, Région Ile-de-France, Fondation pour la Recherche Médicale, French Atomic Energy Commission, ANR-11-BSV2-0018,RETROscreen,Génétique chimique de la voie du transport rétrograde(2011), ANR-14-CE16-0004,AntiHUS,Preuve de concept in vivo de molécules contre le syndrome hémolytique et urémique(2014), European Project: 340485,EC:FP7:ERC,ERC-2013-ADG,GALECTCOMPART(2014), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC), Serva, Camille, BLANC - Génétique chimique de la voie du transport rétrograde - - RETROscreen2011 - ANR-11-BSV2-0018 - BLANC - VALID, Appel à projets générique - Preuve de concept in vivo de molécules contre le syndrome hémolytique et urémique - - AntiHUS2014 - ANR-14-CE16-0004 - Appel à projets générique - VALID, La topologie des glycans comme interrupteur moléculaire pour l'activité des intérgines - - GlycoTopoSwitch2019 - ANR-19-CE13-0001 - AAPG2019 - VALID, and Endocytic Membrane Compartmentalization by Galectins - GALECTCOMPART - - EC:FP7:ERC2014-04-01 - 2019-03-31 - 340485 - VALID

Subjects
Retrograde transport, [SDV]Life Sciences [q-bio], Vesicular Transport Proteins, Golgi Apparatus, Endoplasmic Reticulum, Shiga Toxins, chemistry.chemical_compound, anterograde trafficking, COPII, mass spectrometry, 0303 health sciences, biology, Qa-SNARE Proteins, Chemistry, trans-Golgi network, 030302 biochemistry & molecular biology, Shiga-like toxin, Shiga toxin, Transport protein, Cell biology, Protein Transport, chemical genetics, Ricin, SNARE, Benzamides, click chemistry, symbols, Retro-2, GPP130, biological phenomena, cell phenomena, and immunity, retention using selective hooks, syntaxin-5, endocrine system, Sec16A, Endosome, small molecule, chemical biology, Endosomes, Thiophenes, STxB, 03 medical and health sciences, symbols.namesake, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, proximity ligation assay, Molecular Biology, 030304 developmental biology, Endoplasmic reticulum, Biological Transport, Cell Biology, Golgi apparatus, nervous system, Chaperone (protein), Axoplasmic transport, biology.protein, HeLa Cells
Abstract

International audience; The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.

Published
2020

29. EHD2 is a Predictive Biomarker of Chemotherapy Efficacy in Triple Negative Breast Carcinoma

Author

Sophie Vacher, Wei-Wei Shen, Anne Vincent-Salomon, Ivan Bièche, Laetitia Fuhrmann, Christophe Lamaze, Stéphanie Torrino, Membrane Dynamics and Mechanics of Intracellular Signaling team [Paris], Institut Curie [Paris]-Université Paris sciences et lettres (PSL), Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC), Pharmacogenomics Unit [Paris], Department of Genetics [Paris], Institut Curie [Paris]-Institut Curie [Paris], Department of Pathology [Paris], Institut Curie [Paris], Unité de génétique et biologie des cancers (U830), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de pharmacologie moléculaire et cellulaire (IPMC), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), The Johannes and Lamaze teams, the PICT-IBiSA/Nikon Imaging Centre at Institut Curie-CNRS and the France-BioImaging infrastructure are members of Labex Cell(n)Scale (N° ANR-11-LBX-0038) and of IDEX PSL (N° ANR-10-IDEX-0001-02 PSL)., ANR-10-IDEX-0001,PSL,Paris Sciences et Lettres(2010), Bodescot, Myriam, Initiative d'excellence - Paris Sciences et Lettres - - PSL2010 - ANR-10-IDEX-0001 - IDEX - VALID, Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)

Subjects
Oncology, medicine.medical_treatment, lcsh:Medicine, Triple Negative Breast Neoplasms, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, 0302 clinical medicine, Breast cancer, Caveolae, Antineoplastic Combined Chemotherapy Protocols, Neoplasm Metastasis, lcsh:Science, Triple-negative breast cancer, Cancer, 0303 health sciences, Multidisciplinary, Predictive marker, Middle Aged, Prognosis, Immunohistochemistry, 3. Good health, Gene Expression Regulation, Neoplastic, Cytokine, Treatment Outcome, 030220 oncology & carcinogenesis, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Triple-Negative Breast Carcinoma, Female, Adult, medicine.medical_specialty, [SDV.CAN]Life Sciences [q-bio]/Cancer, Article, 03 medical and health sciences, [SDV.CAN] Life Sciences [q-bio]/Cancer, Internal medicine, medicine, Biomarkers, Tumor, Humans, 030304 developmental biology, Aged, Neoplasm Staging, Chemotherapy, business.industry, lcsh:R, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, medicine.disease, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, lcsh:Q, Neoplasm Grading, business, Carrier Proteins
Abstract

EHD2 is a mechanotransducing ATPase localized in caveolae invaginations at the plasma membrane. EHD2 has recently been associated with several human cancers, however the significance of EHD2 transcript levels in cancer prognosis remains debated. Breast cancer is the most commonly occurring cancer in women and prognosis is variable depending on the subtypes. Triple negative breast cancer (TNBC) often has a poor therapeutic response. The aim of this study was to assess the prognostic significance of EHD2 transcripts and protein expression levels in breast carcinomas. We found that low EHD2 levels were associated with enhanced proliferation, migration and invasion of TNBC cells. EHD2 expression was significantly reduced in TNBC tissues and the loss of EHD2 led to higher expression of the pro-tumoral cytokine IL-8. In apparent contradiction with in vitro data, multivariate analysis of two independent cohorts of breast cancer patients revealed that low EHD2 was in fact associated with good prognosis in the highly proliferative TNBC subtype. Accordingly, TNBC low EHD2 expressers were found to benefit the most from chemotherapy when compared to all subtypes of breast cancers. Our study validates EHD2 expression level as an independent prognostic factor of metastasis-free survival and as a new predictive marker of chemotherapy efficacy in TNBC patients.

Published
2019

30. A promotive effect for halofuginone on membrane repair and synaptotagmin-7 levels in muscle cells of dysferlin-null mice

Author

Mark Pines, Hila Barzilai-Tutsch, Gillian Butler Browne, Melissa Dewulf, Christophe Lamaze, and Orna Halevy

Subjects
0301 basic medicine, Dysferlinopathy, Muscle Fibers, Skeletal, Myoblasts, Dysferlin, Mice, Phosphatidylinositol 3-Kinases, Synaptotagmins, 03 medical and health sciences, Piperidines, Lysosome, Genetics, medicine, Animals, Humans, Myocyte, Muscle, Skeletal, Protein kinase A, Molecular Biology, Genetics (clinical), Quinazolinones, Mice, Knockout, Halofuginone, biology, Myogenesis, Skeletal muscle, General Medicine, medicine.disease, Cell biology, Disease Models, Animal, 030104 developmental biology, medicine.anatomical_structure, Muscular Dystrophies, Limb-Girdle, biology.protein, medicine.drug
Abstract

In the absence of dysferlin, skeletal muscle cells fail to reseal properly after injury, resulting in slow progress of the dysferlinopathy muscular dystrophy (MD). Halofuginone, a leading agent in preventing fibrosis in MDs, was tested for its effects on membrane resealing post-injury. A hypo-osmotic shock assay on myotubes derived from wild-type (Wt) and dysferlin-null (dysf-/-) mice revealed that pre-treatment with halofuginone reduces the percentage of membrane-ruptured myotubes only in dysf-/- myotubes. In laser-induced injury of isolated myofibers, halofuginone decreased the amount of FM1-43 at the injury site of dysf-/- myofibers while having no effect on Wt myofibers. These results implicate halofuginone in ameliorating muscle-cell membrane integrity in dysf-/- mice. Halofuginone increased lysosome scattering across the cytosol of dysf-/- primary myoblasts, in a protein kinase/extracellular signal-regulated protein kinase and phosphoinositide 3 kinase/Akt-dependent manner, in agreement with an elevation in lysosomal exocytotic activity in these cells. A spatial- and age-dependent synaptotagmin-7 (Syt-7) expression pattern was shown in dysf-/- versus Wt mice, suggesting that these pattern alterations are related to the disease progress and that sytnaptotagmin-7 may be compensating for the lack of dysferlin at least with regard to membrane resealing post-injury. While halofuginone did not affect patch-repair-complex key proteins, it further enhanced Syt-7 levels and its spread across the cytosol in dysf-/- myofibers and muscle tissue, and increased its co-localization with lysosomes. Together, the data imply a novel role for halofuginone in membrane-resealing events with Syt-7 possibly taking part in these events.

Published
2018

31. Meeting after meeting: 20 years of discoveries by the members of the Exocytosis-Endocytosis Club

Author

Florence Niedergang, Claire Desnos, Christophe Lamaze, Stéphane Gasman, and Nicolas Vitale

Subjects
0301 basic medicine, 03 medical and health sciences, 030104 developmental biology, education, Library science, Cell Biology, General Medicine, Club, Biology, Endocytosis, humanities, Exocytosis
Abstract

Twenty years ago, a group of French cell biologists merged two scientific clubs with the aim of bringing together researchers in the fields of Endocytosis and Exocytosis. Founded in 1997, the first annual meeting of the Exocytosis Club was held in 1998. The Endocytosis Club held quarterly meetings from its founding in 1999. The first joint annual meeting of the Exocytosis-Endocytosis Club took place in Paris in April, 2001. What started as a modest gathering of enthusiastic scientists working in the field of cell trafficking has gone from strength to strength, rapidly becoming an unmissable yearly meeting, vividly demonstrating the high quality of science performed in our community and beyond. On the occasion of the 20th meeting of our club, we want to provide historic insight into the fields of exocytosis and endocytosis, and by extension, to subcellular trafficking, highlighting how French scientists have contributed to major advances in these fields. Today, the Exocytosis-Endocytosis Club represents a vibrant and friendly community that will hold its 20th meeting at the Presqu'Ile de Giens, near Toulon in the South of France, on May 11-13, 2017.

Published
2017

32. Dynamiques moléculaire et membranaire du récepteur de l’interféron gamma : pour un sucre de trop !

Author

Cédric M. Blouin, Yannick Hamon, Hai-Tao He, Christophe Lamaze, Centre d'Immunologie de Marseille - Luminy (CIML), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), and Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, 03 medical and health sciences, 030104 developmental biology, Chemistry, [SDV]Life Sciences [q-bio], General Medicine, General Biochemistry, Genetics and Molecular Biology
Abstract

International audience; No abstract available

Published
2017

33. Caveolae: The FAQs

Author

Anne K. Kenworthy, Christophe Lamaze, Anne Camus, Richard Lundmark, Soazig Le Lay, William C. Sessa, Stéphane Vassilopoulos, Ivan R. Nabi, Miguel A. del Pozo, Cédric M. Blouin, Robert G. Parton, Institute for Molecular Bioscience [Queensland, Australia], University of Queensland [Brisbane], Centre for Microscopy and Microanalysis [Queensland, Australia], Mechanoadaptation & Caveolae Biology Lab [Madrid, Spain] (Cell & Developmental Biology Area), Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Centre de recherche en myologie, Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Department of Cellular and Physiological Sciences [Vancouver, BC, Canada] (Life Sciences Institute), University of British Columbia (UBC)-Life Sciences Institute [Vancouver, BC, Canada], Stress Oxydant et Pathologies Métaboliques (SOPAM), Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Integrative Medical Biology [Umeå, Sweden], Umeå University, Center for Membrane and Cell Physiology [Charlottesville, VA, USA] (School of Medicine), University of Virginia [Charlottesville], Regenerative Medicine and Skeleton research lab (RMeS), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Vascular Biology and Therapeutics Program [New Haven, CT, USA] (Department of Pharmacology), Yale University School of Medicine, Centro Nacional de Investigaciones Cardiovasculares Carlos III [Madrid, Spain] (CNIC), Instituto de Salud Carlos III [Madrid] (ISC)-Instituto de Salud Carlos III [Madrid] (ISC), Centre de Recherche en Myologie, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC), Centre de recherche en Myologie – U974 SU-INSERM, Institut de Myologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Jehan, Frederic, University of Virginia, Regenerative Medicine and Skeleton (RMeS), École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Yale School of Medicine [New Haven, Connecticut] (YSM)

Subjects
muscular dystrophy, lipodystrophy, [SDV]Life Sciences [q-bio], [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], cavins, Biology, Caveolae, Biochemistry, Article, 03 medical and health sciences, 0302 clinical medicine, Structural Biology, Genetics, [SDV.MHEP.PHY]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Animals, endocytosis, Molecular Biology, membrane, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, caveolins, 0303 health sciences, [SDV.MHEP.RSOA] Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, [SDV.MHEP.GEG] Life Sciences [q-bio]/Human health and pathology/Geriatry and gerontology, [SDV.MHEP.GEG]Life Sciences [q-bio]/Human health and pathology/Geriatry and gerontology, EHD2, Cell Biology, Cell biology, Cell and molecular biology, [SDV.MHEP.RSOA]Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, cell membrane, 030217 neurology & neurosurgery
Abstract

International audience; At the first EMBO conference dedicated to caveolae held in Le Pouliguen, France, May 12-16 (http://meetings.embo.org.gate2.inist.fr/event/19-caveolae), round-table discussions were used to address some of the long-standing issues in the field and to decide upon a consensus view regarding key aspects of caveola biology. Here we summarise some of the frequently asked questions (FAQs) posed by cell biologists about caveolae and provide some brief consensual answers based on these discussions. This article is protected by copyright. All rights reserved.

Published
2019

34. Caveolae coupling of melanocytes signaling and mechanics is required for human skin pigmentation

Author

Floriane Gilles-Marsens, Cédric M. Blouin, Katell Vié, Graça Raposo, Christine Viaris de Lesegno, Christophe Lamaze, Cédric Delevoye, Ilse Hurbain, Lia Domingues, Maryse Romao, Christelle Guéré, Melissa Dewulf, and Nathalie André

Subjects
0303 health sciences, Cell signaling, Epidermis (botany), integumentary system, Chemistry, Cell, Human skin, Cell biology, Melanin, 03 medical and health sciences, 0302 clinical medicine, medicine.anatomical_structure, Caveolae, medicine, sense organs, 030217 neurology & neurosurgery, Tissue homeostasis, Intracellular, 030304 developmental biology
Abstract

SummaryTissue homeostasis requires regulation of cell-cell communication, which relies on signaling molecules and cell contacts. In skin epidermis, keratinocytes secrete specific factors transduced by melanocytes into signaling cues to promote 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 found that caveolae polarize in melanocytes and are particularly abundant at melanocyte-keratinocyte interface. Caveolae in melanocytes are sensitive to ultra-violet radiations and miRNAs released by keratinocytes. Preventing caveolae formation in melanocytes results in increased production of intracellular cAMP and melanin pigments, but decreases cell protrusions, cell-cell contacts, pigment transfer and epidermis pigmentation. Altogether, our data establish that, in melanocytes, caveolae serve as key molecular hubs that couple signaling outputs from keratinocytes to mechanical plasticity. This process is crucial to maintain cell-cell contacts and intercellular communication, skin pigmentation and tissue homeostasis.

Published
2019
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35. UBTD1 is a mechano‐regulator controlling cancer aggressiveness

Author

Damien Ambrosetti, Amel Mettouchi, Frédéric Bost, Christophe Lamaze, Maeva Gesson, Stéphanie Torrino, Jay P. Uhler, Emmanuel Lemichez, Lisa Kaminski, Stephan Clavel, Kathiane Laurent, Jean-François Michiels, Sabrina Pisano, François-René Roustan, Thomas Bertero, Maeva Dufies, Cedric Gaggioli, Centre méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), Centre National de la Recherche Scientifique (CNRS), Institut de Recherche sur le Cancer et le Vieillissement (IRCAN), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Hôpital Pasteur [Nice] (CHU), University of Gothenburg (GU), Toxines bactériennes - Bacterial Toxins, Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), This work has been supported by the French government, through the UCA‐JEDI Investments in the Future project managed by the National Research Agency (ANR) with the reference number ANR‐15‐IDEX‐01. S.T. is a recipient of a post‐doctoral fellowship from 'La Fondation de France'. L.K. is a recipient of a doctoral fellowship from the French ministry of research. F.B. is a CNRS researcher., We thank the GIS‐IBISA multi‐sites platform 'Microscopie Imagerie Côte d'Azur' (MICA), and particularly the imaging site of C3M and IRCAN (PICMI), which are supported by the 'Conseil General 06' and 'Conseil Départemental 06'. The PICMI AFM was supported by the Association pour la Recherche sur le Cancer (ARC) and by the 'Conseil General 06 de la Région Provence Alpes‐Côte'., ANR-15-IDEX-0001,UCA JEDI,Idex UCA JEDI(2015), Université Nice Sophia Antipolis (... - 2019) (UNS), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Dynamique et mécanique membranaires de la signalisation intracellulaire, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris sciences et lettres (PSL), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Côte d'Azur (UCA), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Paris Sciences et Lettres (PSL), and ANR: 15-IDEX-0001,UCA JEDI,Idex UCA JEDI(2015)

Subjects
RHOA, Cell, Regulator, Cell Cycle Proteins, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Mechanotransduction, Cellular, Biochemistry, 0302 clinical medicine, Ubiquitin, Neoplasms, matrix stiffness, ROCK2, Insulin-Like Growth Factor I, beta Catenin, 0303 health sciences, biology, Chemistry, Articles, Prognosis, 3. Good health, Cell biology, Ubiquitin ligase, Gene Expression Regulation, Neoplastic, medicine.anatomical_structure, Disease Progression, Disease Susceptibility, YAP, Protein Binding, Signal Transduction, [SDV.CAN]Life Sciences [q-bio]/Cancer, Protein Serine-Threonine Kinases, Models, Biological, UBTD1, 03 medical and health sciences, Cell Adhesion, Genetics, medicine, Humans, Hippo Signaling Pathway, Ubiquitins, Molecular Biology, 030304 developmental biology, Cancer, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, β‐TrCP, beta-Transducin Repeat-Containing Proteins, medicine.disease, Cancer cell, biology.protein, rhoA GTP-Binding Protein, Protein Processing, Post-Translational, 030217 neurology & neurosurgery, Transcription Factors
Abstract

International audience; Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with β-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase β-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.

Published
2019

36. STAM Interaction with Hrs Controls JAK/STAT Activation by Interferon-α at the Early Endosome

Author

Cédric M. Blouin, Ludger Johannes, Christophe Lamaze, Chmiest D, Zanin N, and de Lesegno Cv

Subjects
0303 health sciences, Chemistry, Endosome, JAK-STAT signaling pathway, Endocytosis, stat, Cell biology, 03 medical and health sciences, 0302 clinical medicine, Tyrosine kinase 2, STAT protein, Janus kinase, Receptor, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

Activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway by type I interferons (IFN) requires clathrin-dependent endocytosis of the IFN-α/β receptor (IFNAR). The molecular machinery that brings about the selective activation of IFN-α/β-induced JAK/STAT signaling on endosomes remains unknown. Here we show that the constitutive association of STAM with IFNAR1 and the TYK2 Janus kinase at the plasma membrane prevents the activation of TYK2 by type I IFNs. IFN-α stimulated endocytosis leads to the interaction of IFNAR1 with Hrs on early endosomes, which then relieves TYK2 inhibition by STAM and thereby allows for TYK2 and IFNAR signaling. In contrast, IFN-β stimulation results in sorting of IFNAR to a distinct endosomal subdomain where the receptor is activated independently from Hrs. Our results identify the molecular machinery that controls the spatiotemporal activation of TYK2 and establish the central role of endosomal sorting in the differential regulation of JAK/STAT signaling by IFN-α and IFN-β.SummaryThe spatiotemporal activation of JAK/STAT signaling by IFN-α is controlled by STAM association with Hrs at the early endosome.

Published
2019

37. EHD2 is a mechanotransducer connecting caveolae dynamics with gene transcription

Author

Darius Vasco Köster, Cédric M. Blouin, Paolo Pierobon, Vassili Soumelis, Satish Kailasam Mani, Christine Viaris de Lesegno, Ludger Johannes, Alexandre Grassart, Valérie Chambon, Wei-Wei Shen, Stéphanie Torrino, Catherine Coirault, Cesar Augusto Valades-Cruz, Stéphane Vassilopoulos, Pierre Bost, Christophe Lamaze, Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC), Immunité et cancer (U932), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Pathogénie microbienne moléculaire, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Cell and Developmental Biology [Coventry, UK], Division of Biomedical Sciences [Coventry, UK], Warwick Medical School, University of Warwick [Coventry]-University of Warwick [Coventry]-Warwick Medical School, University of Warwick [Coventry]-University of Warwick [Coventry], Trafic endocytique et ciblage intracellulaire (CBMCT - UMR 3666 / U1143), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Myologie, Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Sorbonne Université (SU), The AIC is jointly supported by the Gordon and Betty Moore Foundation and Howard Hughes Medical Institute. The electron microscope facility was supported by the ANR through 'Investments for the Future' program (France-BioImaging, ANR-10-INSB-04). This work was supported by institutional grants from the Institut Curie, Institut National de la Santé et de la Recherche Médicale, and Centre National de la Recherche Scientifique, and by specific grants from ANR (MECANOCAV, ANR-12-BSV2-0011, DECAV-RECAV, ANR-14-CE09-0008-03, and MOTICAV, ANR-17-CE-0013), Institut National du Cancer (INCa PLBIO12-203), Fondation de France, Marie Curie Actions—Networks for Initial Training (H2020-MSCA-ITN-2014), Association Française contre les Myopathies (15717 and 16754), and Program labellisé, Fondation ARC pour la Recherche sur le Cancer (PGA1 RF20170205456) to C. Lamaze, ANR (ANR-14-CE14-0002-02), Human Frontier Science Program (RGP0029-2014), and European Research Council (advanced grant 340485) to L. Johannes, ANR young researcher grant (EndoMechano ANR-14-CE12-0001-01) to S. Vassilopoulos. A. Grassart is supported by a Pasteur-Roux Fellowship. P. Pierobon is supported by grants from ANR (ANR-10-JCJC-1504-Immuphy) and PIC (Programme Incitatif et Collaboratif) Curie, 'Cell polarity, division, growth and cancer.' S. Torrino and W.-W. Shen were supported by a postdoctoral fellowship from Fondation ARC pour la Recherche sur le Cancer and Fondation de France, respectively. D. Köster and W.-W. Shen were supported by the Labex CelTisPhyBio Grants Program. The Johannes and Lamaze teams, the PICT-IBiSA/Nikon Imaging Centre at Institut Curie-Centre National de la Recherche Scientifique, and the France-BioImaging infrastructure are members of Labex CelTisPhyBio (ANR, ANR-10-LBX-0038) and of Initiatives d’Excellence (IDEX) PSL (ANR, ANR-10-IDEX-0001-02 PSL)., The facilities as well as scientific and technical assistance from the PICT-IBiSA/Nikon Imaging Centre staff at Institut Curie-Centre National de la Recherche Scientifique and the France-BioImaging infrastructure (Agence Nationale de la Recherche [ANR], ANR-10-INSB-04) are acknowledged. LLSM was performed at the Advanced Imaging Center (AIC) at Howard Hughes Medical Institute Janelia Research Campus., Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Curie [Paris], Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Compartimentation et dynamique cellulaires (CDC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie-Université Pierre et Marie Curie - Paris 6 (UPMC), Biologie du Développement et Reproduction (BDR), Institut National de la Recherche Agronomique (INRA), Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Cell and Developmental Biology [Coventry, UK] (Warwick Medical School Biomedical Sciences), University of Warwick [Coventry], Laboratoire Trafic, Signalisation et Ciblage Intracellulaires, Institut Curie, Physico-Chimie-Curie (PCC), Centre de recherche en myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), and Coirault, Catherine

Subjects
0301 basic medicine, Cell signaling, Transcription, Genetic, MESH: Mechanotransduction, Cellular, ATPase, [SDV]Life Sciences [q-bio], [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Caveolae, Mechanotransduction, Cellular, MESH: Stress, Mechanical, Cell membrane, 03 medical and health sciences, Transcription (biology), MESH: Carrier Proteins/genetics, MESH: Carrier Proteins/metabolism, Report, medicine, Humans, Mechanotransduction, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Research Articles, MESH: Transcription, Genetic, Cancer, Regulation of gene expression, Trafficking, MESH: Humans, biology, MESH: Caveolae/metabolism, Cell Biology, QP, 3. Good health, Cell biology, [SDV] Life Sciences [q-bio], Cell nucleus, 030104 developmental biology, medicine.anatomical_structure, MESH: HeLa Cells, biology.protein, Stress, Mechanical, Carrier Proteins, Nucleus, HeLa Cells
Abstract

Caveolae are dynamic mechanosensors. Torrino et al. show that EHD2 plays a crucial role in the adaptation to mechanical perturbations by maintaining the caveolae reservoir at the plasma membrane after changes in membrane tension and connecting caveolae mechanosensing at the plasma membrane with the regulation of gene transcription., 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.

Published
2018

38. Lack of functional caveolae in Cav3 mutated human dystrophic myotubes results in deficient mechanoprotection and IL6/STAT3 mechanosignaling

Author

Gillian Butler-Browne, Nicolas Tardif, Cédric M. Blouin, Christine Viaris de Lesegno, Ludger Johannes, Anne Bigot, Darius Koester, Pierre Nassoy, Valérie Chambon, Melissa Dewulf, Bidisha Sinha, and Christophe Lamaze

Subjects
Chemistry, Myogenesis, Caveolae, Mutant, Wild type, Myocyte, Muscle disorder, Signal transduction, Stat3 Signaling Pathway, Cell biology
Abstract

Caveolin-3 is the major structural protein of caveolae in muscle cells. Mutations in the CAV3 gene cause different type of muscle disorders mostly characterized by defects in membrane integrity and repair, deregulation in the expression of various muscle proteins and deregulation of several muscle associated signaling pathways. We show here that myotubes derived from patients bearing the CAV3 P28L and R26Q mutations present a lack of functional caveolae at the plasma membrane which results in an abnormal mechanoresponse. Mutant myotubes can no longer buffer the increase of membrane tension induced by mechanical stress and present an hyperactivation of the IL6/STAT3 signaling pathway at rest and under mechanical stress. The impaired mechanical regulation of the IL6/STAT3 signaling pathway by caveolae leads to chronic activation and a higher expression of muscle specific genes. These defects could be reversed by reassembling a pool of functional caveolae through expression of wild type Cav3. Our findings bring more mechanistic insight into human Cav3 associated muscle disorders and show a general defect in the mechanoresponse of CAV3 P28L and R26Q myotubes.

Published
2018

39. Caveolin-Assisted Sphingolipid Transport to the Plasma Membrane

Author

Aurélie Di Cicco, Daniel Lévy, Patricia Bassereau, Manuela Dezi, John Manzi, Christophe Lamaze, Joanna Podkalicka, Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC), Physico-Chimie-Curie (PCC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC), Paris-Jourdan Sciences Economiques (PSE), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Recherche Agronomique (INRA)-École des hautes études en sciences sociales (EHESS)-École des Ponts ParisTech (ENPC)-Centre National de la Recherche Scientifique (CNRS), Compartimentation et dynamique cellulaires (CDC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC), Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Curie-Université Pierre et Marie Curie - Paris 6 (UPMC), and École normale supérieure - Paris (ENS Paris)-Institut National de la Recherche Agronomique (INRA)-École des hautes études en sciences sociales (EHESS)-École des Ponts ParisTech (ENPC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Membrane, Chemistry, Caveolin, Biophysics, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Sphingolipid, ComputingMilieux_MISCELLANEOUS, Cell biology
Abstract

International audience

Published
2018

40. Membrane Tension Orchestrates Rear Retraction in Matrix Directed Cell Migration

Author

Vanesa Sokleva, Oana Dobre, Ewa K. Paluch, Henry De Belly, Patrick T. Caswell, Joe Swift, Aránzazu del Campo, Nils Gauthier, Christophe Lamaze, Joseph H. R. Hetmanski, Roshna V. Nair, Angus J.M. Cameron, and Jean-Marc Schwartz

Subjects
Durotaxis, RHOA, Interstitial matrix, biology, Chemistry, Caveolae, digestive, oral, and skin physiology, biology.protein, Cell migration, Matrix (biology), Cytoskeleton, Wound healing, Cell biology
Abstract

In development, wound healing, and cancer metastasis, vertebrate cells move through 3D interstitial matrix, responding to chemical and physical guidance cues. Protrusion at the cell front has been extensively studied, but the retraction phase of the migration cycle is not well understood. Here, we show that fast-moving cells guided by matrix cues establish positive feedback control of rear retraction by sensing membrane tension. We reveal a mechanism of rear retraction in 3D matrix and durotaxis controlled by caveolae, which form in response to low membrane tension at the cell rear. Caveolae activate RhoA-ROCK1/PKN2 signaling via the RhoA guanidine nucleotide exchange factor (GEF) Ect2 to control local F-actin organization and contractility in this subcellular region and promote translocation of the cell rear. A positive feedback loop between cytoskeletal signaling and membrane tension leads to rapid retraction to complete the migration cycle in fast-moving cells, providing directional memory to drive persistent cell migration in complex matrices.

Published
2018

41. Endocytosis and Signaling

Author

Christophe Lamaze, Ian Prior, Christophe Lamaze, and Ian Prior

Subjects
Cellular signal transduction, Endocytosis
Abstract

This book focuses on the context dependency of cell signaling by showing how the endosomal system helps to structure and regulate signaling pathways. The location and concentration of signaling nodes regulate their activation cycles and engagement with distinct effector pathways. Whilst many cell signaling pathways are initiated from the cell surface, endocytosis provides an opportunity for modulating signaling networks'output. In this book, first a series of reviews describe the endocytic and endosomal system and show how these subcellular platforms sort and regulate a wide range of signaling pathway components and phenotypic outputs. The book then reviews the latest scientific insights into how endocytic trafficking and subcellular location modulate a set of major pathways that are essential to normal cellular function and organisms'development.

Published
2018

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

Author

Anne K. Kenworthy, Mijo Simunovic, Anne A. Schmidt, Valérie Chambon, Patricia Bassereau, Henri-François Renard, Christian Wunder, Cécile Sykes, Joël Lemière, Emmanuel Boucrot, Christophe Lamaze, Harvey T. McMahon, Maria Daniela Garcia-Castillo, Senthil Arumugam, Ludger Johannes, Bondidier, Martine, Chimie biologique des membranes et ciblage thérapeutique ( CBMCT - UMR 3666 / U1143 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut Curie-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Physico-Chimie-Curie ( PCC ), Centre National de la Recherche Scientifique ( CNRS ) -INSTITUT CURIE-Université Pierre et Marie Curie - Paris 6 ( UPMC ), Department of Chemistry, University of Chicago, Université Paris Diderot - Paris 7 ( UPD7 ), Institute of Structural and Molecular Biology, Birkbeck College, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine. Nashville, Vanderbilt University School of Medicine. Nashville-Vanderbilt University School of Medicine. Nashville, Institut Jacques Monod ( IJM ), Université Paris Diderot - Paris 7 ( UPD7 ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratory of Molecular Biology, Division of Cell Biology, Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, UK, Agence Nationale pour la Recherche : (ANR-09-BLAN-283, ANR-10-LBX-0038, ANR-11 BSV2 014 03, ANR-12-BSV5-0014), Indo-French Centre for the Promotion of Advanced Science (project no. 3803), Marie Curie Actions — Networks for Initial Training (FP7-PEOPLE-2010-ITN), Marie Curie International Reintegration Grant (FP7-RG-277078), European Research Council advanced grant (project 340485), Royal Society (RG120481), Fondation ARC pour la Recherche sur le Cancer (DEQ20120323737), National Institutes of Health (RO1 GM106720), Ligue contre le Cancer, Comité de Paris (RS08/75-89), Fondation ARC pour la Recherche sur le Cancer, AXA Research Funds, Biological Sciences Research Council, Chateaubriand fellowship, France and Chicago Collaborating in the Sciences grant, Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Physico-Chimie-Curie (PCC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7), Vanderbilt University School of Medicine [Nashville], Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), and Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)

Subjects
MESH : Cell Line, MESH: Rats, MESH : Endocytosis, MESH : Cell Membrane, MESH: Shiga Toxin, Endocytic cycle, MESH : Actins, MESH : Dynamins, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, macromolecular substances, MESH: Acyltransferases, Biology, MESH: Actins, Endocytosis, environment and public health, Clathrin, Cell membrane, 03 medical and health sciences, 0302 clinical medicine, medicine, BAR domain, MESH: Animals, MESH: Clathrin, Endophilin-A2, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, MESH: Cholera Toxin, 030304 developmental biology, Dynamin, 0303 health sciences, MESH: Humans, MESH : Clathrin, Multidisciplinary, MESH : Rats, MESH : Cholera Toxin, [ SDV.BC ] Life Sciences [q-bio]/Cellular Biology, MESH : Humans, MESH: Cell Line, Cell biology, MESH: Dynamins, medicine.anatomical_structure, MESH : Shiga Toxin, MESH: Endocytosis, Amphiphysin, biology.protein, MESH : Animals, MESH : Acyltransferases, 030217 neurology & neurosurgery, MESH: Cell Membrane
Abstract

International audience; 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 carriers. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin. Clathrin-independent endocytic events are often less reliant on dynamin, 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 cargoes. 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.

Published
2014

43. Effet papillon et cancer : ou comment une pression mécanique induitein vivoactive la tumorigenèse des cellules voisines saines

Author

Christophe Lamaze, Ye Wang, Camille Kieffer, and Fatma Bagca

Subjects
0301 basic medicine, Programmed cell death, NADPH oxidase, biology, Chemistry, Cell, General Medicine, medicine.disease_cause, Molecular biology, General Biochemistry, Genetics and Molecular Biology, Liver regeneration, 03 medical and health sciences, 030104 developmental biology, medicine.anatomical_structure, Gene expression, biology.protein, medicine, Epidermal growth factor receptor, Receptor, Carcinogenesis
Abstract

713 m/s n° 8-9, vol. 32, aout-septembre 2016 DOI : 10.1051/medsci/20163208017 5. Factor VM, Seo D, Ishikawa T, et al. Loss of c-Met disrupts gene expression program required for G2/M progression during liver regeneration in mice. PLoS One 2010 ; 5 : e12739. 6. Lopez-Luque J, Caballero-Diaz D, MartinezPalacian A, et al. Dissecting the role of epidermal growth factor receptor catalytic activity during liver regeneration and hepatocarcinogenesis. Hepatology 2016 ; 63 : 604-19 7. Carmona-Cuenca I, Herrera B, Ventura JJ, et al. EGF blocks NADPH oxidase activation by TGF-beta in fetal rat hepatocytes, impairing oxidative stress, and cell death. J Cell Physiol 2006 ; 207 : 322-30. 8. Natarajan A, Wagner B, Sibilia M. The EGF receptor is required for efficient liver regeneration. Proc Natl Acad Sci USA 2007 ; 104 : 17081-6. LIENS D’INTERET Les auteurs declarent n’avoir aucun lien d’interet concernant les donnees publiees dans cet article.

Published
2016

44. The caveolae dress code: structure and signaling

Author

Cédric M. Blouin, Stéphane Vassilopoulos, Melissa Dewulf, Nicolas Tardif, Christophe Lamaze, Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Mécanique des Contacts et des Structures [Villeurbanne] (LaMCoS), Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut de Myologie, and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Structural organization, [SDV]Life Sciences [q-bio], Cell Membrane, Cellular functions, Context (language use), Cell Biology, Biology, Endocytosis, Caveolae, Caveolins, Cell biology, 03 medical and health sciences, 030104 developmental biology, Organelle, Animals, Humans, Signal transduction, Signal Transduction
Abstract

International audience; Over the past decade, interest in caveolae biology has peaked. These small bulb-shaped plasma membrane invaginations of 50-80nm diameter present in most cell types have been upgraded from simple membrane structures to a more complex bona fide organelle. However, although caveolae are involved in several essential cellular functions and pathologies, the underlying molecular mechanisms remain poorly defined. Following the identification of caveolins and cavins as the main caveolae constituents, recent studies have brought new insight into their structural organization as a coat. In this review, we discuss how these new data on caveolae can be integrated in the context of their role in signaling and pathophysiology.

Published
2017

45. Rab12 Localizes to Shiga Toxin-Induced Plasma Membrane Invaginations and Controls Toxin Transport

Author

Winfried Römer, Ludger Johannes, Henri-François Renard, Gustaf E. Rydell, Florent Dingli, Maria Daniela Garcia-Castillo, Damarys Loew, and Christophe Lamaze

Subjects
biology, Toxin, media_common.quotation_subject, Toxin transport, Shiga toxin, Cell Biology, Transfection, medicine.disease_cause, Biochemistry, Clathrin, Cell biology, Structural Biology, Stable isotope labeling by amino acids in cell culture, Genetics, biology.protein, medicine, Internalization, Receptor, Molecular Biology, media_common
Abstract

Several exogenous and endogenous cargo proteins are internalized independently of clathrin, including the bacterial Shiga toxin. The mechanisms underlying early steps of clathrin-independent uptake remain largely unknown. In this study, we have designed a protocol to obtain gradient fractions containing Shiga toxin internalization intermediates. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative mass spectrometry, Rab12 was found in association with these very early uptake carriers. The localization of the GTPase on Shiga toxin-induced plasma membrane invaginations was shown by fluorescence microscopy in cells transfected with GFP-Rab12. Furthermore, using a quantitative biochemical assay, it was found that the amount of receptor-binding B-subunit of Shiga toxin reaching the trans-Golgi/TGN membranes was decreased in Rab12-depleted cells, and that cells were partially protected against intoxication by Shiga-like toxin 1 under these conditions. These findings demonstrate the functional importance of Rab12 for retrograde toxin trafficking. Among several other intracellular transport pathways, only the steady-state localizations of TGN46 and cation-independent mannose-6-phosphate receptor were affected. These data thus strongly suggest that Rab12 functions in the retrograde transport route.

Published
2014

46. Rab7 Is Functionally Required for Selective Cargo Sorting at the Early Endosome

Author

Natalie Fournier, Daniela Chmiest, Benoît Vedie, Ludger Johannes, Jean-Louis Paul, Emmanuelle Girard, and Christophe Lamaze

Subjects
Endosome, Effector, Cell Biology, GTPase, Biology, Endocytosis, Biochemistry, Cell biology, Structural Biology, Epidermal growth factor, Genetics, Transcriptional regulation, Rab, Receptor, Molecular Biology
Abstract

The small GTPases of the Rab family act as a molecular switch regulating various aspects of membrane trafficking through the selective recruitment of effector proteins. Whereas Rab7 has been classically involved in the regulation of transport within the endolysosomal network, persistent controversy remains as to whether Rab7 also plays a role in earlier steps of endosomal trafficking. In this study, we show that Rab7 depletion or inactivation results in enlargement of both early and late endosomes. Rab7 depletion led to the retention of a significant fraction of internalized low-density lipoproteins (LDL) mainly in enlarged early endosomes (EE). As a result, LDL processing and the transcriptional regulation of sterol-sensitive genes were impaired. We found that Rab7 activity was also required for the sorting of the mannose-6-phosphate receptor, the interferon alpha-receptor and the Shiga toxin B-subunit. In contrast, epidermal growth factor (EGF) sorting at the EE or the recycling of transferrin and LDL-R were not affected by Rab7 depletion. Our findings demonstrate that in addition to regulating late endosomes (LE) to lysosomes transport, Rab7 plays a functional role in the selective sorting of distinct cargos at the EE and that the Rab5 to Rab7 exchange occurs early in the endosomal maturation process.

Published
2014

47. Receptor lipid nanodomain partitioning and signaling

Author

Cédric M. Blouin and Christophe Lamaze

Subjects
0301 basic medicine, Glycosylation, Receptors, Cell Surface, Biology, Editorials: Cell Cycle Features, Models, Biological, Cell membrane, 03 medical and health sciences, chemistry.chemical_compound, Interferon-gamma, 0302 clinical medicine, medicine, Interferon gamma, Receptor, Molecular Biology, Galectin, Cell Membrane, JAK-STAT signaling pathway, Cell Biology, Lipids, Cell biology, 030104 developmental biology, medicine.anatomical_structure, chemistry, 030220 oncology & carcinogenesis, Nanoparticles, Signal transduction, Cytokine receptor, Developmental Biology, medicine.drug, Signal Transduction
Published
2016

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

Author

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

Subjects
0301 basic medicine, Retromer, Transcription, Genetic, Endosome, Science, Vesicular Transport Proteins, General Physics and Astronomy, Endosomes, Receptor, Interferon alpha-beta, Biology, Models, Biological, General Biochemistry, Genetics and Molecular Biology, stat, Article, Cell Line, 03 medical and health sciences, VPS35, Lysosome, medicine, Humans, STAT1, Janus Kinases, Multidisciplinary, Cell Membrane, JAK-STAT signaling pathway, Interferon-alpha, General Chemistry, Interferon-beta, Endocytosis, Cell biology, Retromer complex, Protein Subunits, Protein Transport, STAT Transcription Factors, 030104 developmental biology, medicine.anatomical_structure, rab GTP-Binding Proteins, Multiprotein Complexes, biology.protein, Protein Binding, Signal Transduction
Abstract

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., Endocytosis of the type I interferon receptor (IFNAR) is required for JAK/STAT signalling. Here the authors show that the internalized IFNAR1 and IFNAR2 subunits are differentially sorted by the retromer complex at the early endosome and this controls JAK/STAT signalling and gene transcription.

Published
2016

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

Author

Cédric M. Blouin, Rémy Sadoul, Béatrice Blot, Yasmina Saoudi, Christine Chatellard, Olivier Destaing, Christophe Lamaze, Karin Pernet-Gallay, Corinne Albiges-Rizo, Vincent Mercier, Marine H. Laporte, Anne Petiot, Sandrine Fraboulet, Unité de Recherches Intégrées - Gotheron (GOTH RI UERI), Institut National de la Recherche Agronomique (INRA), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Laboratoire de Biologie Moléculaire de la Cellule (LBMC), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Grenoble Institute of Neuroscience, Université Joseph Fourier - Grenoble 1 (UJF), Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Grenoble Institut des Neurosciences (GIN), Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Grenoble-Université Joseph Fourier - Grenoble 1 (UJF), Organisation Fonctionnelle du Cytosquelette, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR27, Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Neurodegenerescence et Plasticite, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Unité Expérimentale Recherches Intégrées - Gotheron (UERI), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), [GIN] Grenoble Institut des Neurosciences (GIN), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
0301 basic medicine, Cholera Toxin, Endosome, media_common.quotation_subject, [SDV]Life Sciences [q-bio], Primary Cell Culture, Gene Expression, Cell Cycle Proteins, Plasma protein binding, Endosomes, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Endocytosis, Clathrin, Article, 03 medical and health sciences, Mice, 0302 clinical medicine, Cell Movement, Calcium-binding protein, Cell Line, Tumor, Animals, Humans, Internalization, media_common, Mice, Knockout, Neurons, B-Lymphocytes, Multidisciplinary, biology, Endosomal Sorting Complexes Required for Transport, Calcium-Binding Proteins, Receptors, Interleukin-2, Receptor-mediated endocytosis, Fibroblasts, Embryo, Mammalian, Cell biology, 030104 developmental biology, biology.protein, Signal transduction, 030217 neurology & neurosurgery, Acyltransferases, Protein Binding, Signal Transduction
Abstract

International audience; 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. The plasma membrane of all eukaryotic cells undergoes constant renewal through repeated cycles of endocyto-sis and exocytosis. During endocytosis, cell surface proteins and lipids are internalized forming vesicular carriers which then merge with early endosomes, a process central to the regulation of nutrient uptake, cell surface receptor signaling, plasma membrane remodeling, cellular mobility and synaptic vesicle recycling 1. Most of these processes rely on clathrin-mediated endocytosis (CME) based on the clathrin machinery for shaping endocytic vesicles. However alternative pathways, collectively referred to as clathrin-independent endocytosis (CIE), also occur at the plasma membrane, although the molecular mechanisms leading to membrane bending and fission, as well as the biological significance of these pathways have yet to be clarified 2. An important advance in defining the molecular players involved in CIE came from two recent reports demonstrating that endophilin-A controls CIE of activated receptors including those for epidermal growth factor (EGF) and interleukin-2 (IL2), and that this pathway is hijacked by shiga-or cholera bacterial toxins 3,4. The three endophilin-A isoforms (A1, A2, A3) contain a Src Homology 3 (SH3) domain binding to both dynamin and synaptojanin 5 and a N-BAR (Bin/amphiphysin/Rvs) domain capable of sensing and generating membrane curvature 6. At synapses, Endophilins-A are known to be involved in both CME 7-9 and CIE 10. We have shown previously that besides dynamin and synaptojanin, a major interacting partner of endophilin-A is Alix (ALG-2-interacting protein X), first identified through its calcium-dependent binding to the penta-EF-hand protein ALG-2 (apoptosis-linked gene 2) 11,12. Alix is a 95 kD cytoplasmic protein with multiple interacting partners. The N-terminal Bro1-like domain 13 , binds the endosome-resident lipid, lysobisphosphatidic acid (LBPA) 14,15 and the charged multivesicular body protein 4B (CHMP4B) component of the endosomal sorting complex III required for transport (ESCRT-III) 16 , while the C-terminal long proline-rich domain (PRD) binds endophilin-A 12 , and also contains distinct interaction sites for ALG-2, the tumor suppressor gene 101 (Tsg-101) component of ESCRT-I, and Cbl Interacting protein-85 (CIN85) 17. The central region of Alix consists of a V-shaped domain composed of two triple-helical bundles which mediate protein dimerization 18. Alix is

Published
2016

50. SNAP-tag Based Proteomics Approach for the Study of the Retrograde Route

Author

Christophe Lamaze, Michel Azoulay, Ludger Johannes, Getao Shi, Damarys Loew, Florent Dingli, and Jean-Claude Florent

Subjects
Endosome, Endocytic cycle, Chemical biology, Cell Biology, Golgi apparatus, Biology, Proteomics, Biochemistry, Fusion protein, Cell biology, SNAP-tag, symbols.namesake, Membrane protein, Structural Biology, Genetics, symbols, Molecular Biology
Abstract

Proteomics is a powerful technique for protein identification at large scales. A number of proteomics approaches have been developed to study the steady state composition of intracellular compartments. Here, we report a novel vectorial proteomics strategy to identify plasma membrane proteins that undergo retrograde transport to the trans-Golgi network (TGN). This strategy is based on the covalent modification of the plasma membrane proteome with a membrane impermeable benzylguanine derivative. Benzylguanine-tagged plasma membrane proteins that are subsequently targeted to the retrograde route are covalently captured by a TGN-localized SNAP-tagged fusion protein, which allows for their identification. The approach was validated step-by-step using a well explored retrograde cargo protein, the B-subunit of Shiga toxin. It was then extended to the proteomics format. Among other hits we found one of the historically first identified cargo proteins that undergo retrograde transport, which further validated our approach. Most of the other hits were kinases, receptors or transporters. In conclusion, we have pioneered a vectorial proteomics approach that complements traditional methods for the study of retrograde protein trafficking. This approach is of generic nature and could in principle be extended to other endocytic pathways.

Published
2012

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