Molecular mechanisms of exocytosis-endocytosis coupling in neuroendocrine cells : role of Scramblase-1 and Oligophrenin-1 proteins Catherine Estay Ahumada To cite this version: Catherine Estay Ahumada. Molecular mechanisms of exocytosis-endocytosis coupling in neuroen- docrine cells : role of Scramblase-1 and Oligophrenin-1 proteins. Neurobiology. Université de Stras- bourg, 2016. English. NNT : 2016STRAJ088. tel-01726966 HAL Id: tel-01726966 https://tel.archives-ouvertes.fr/tel-01726966 Submitted on 8 Mar 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE THÈSE présentée par : Catherine Estay Ahumada soutenue le : 2 Décembre 2016 pour obtenir le grade de : Docteur de l’Université de Strasbourg Discipline : Science du vivant Spécialité : Aspects moléculaires et cellulaires de la biologie Mécanismes moléculaires du couplage exocytose-endocytose dans les cellules neuroendocrines : rôle des protéines Scramblase-1 et Oligophrénine-1 THÈSE dirigée par : GASMAN Stéphane Directeur de recherche INSERM, Université de Strasbourg RAPPORTEURS : DANGLOT Lydia Chargé de recherche INSERM, Université de Paris Diderot BENHAMOU Marc Chargé de recherche INSERM, Université de Paris Diderot AUTRE MEMBRE DU JURY : LELIEVRE Vincent Professeur Université de Strasbourg INVITE : ORY Stéphane Chargé de recherche CNRS, Université de Strasbourg (membre invité) UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE THÈSE présentée par : Catherine Estay Ahumada soutenue le : 2 Décembre 2016 pour obtenir le grade de : Docteur de l’Université de Strasbourg Discipline : Science du vivant Spécialité : Aspects moléculaires et cellulaires de la biologie Mécanismes moléculaires du couplage exocytose-endocytose dans les cellules neuroendocrines : rôle des protéines Scramblase-1 et Oligophrénine-1 Soutenue publiquement le 2 Décembre 2016 devant le jury composé de : Dr. DANGLOT Lydia (Rapporteur externe) Dr. BENHAMOU Marc (Rapporteur externe) Dr. LELIEVRE Vincent (Examinateur interne) Dr. GASMAN Stéphane (Directeur de thèse) Dr. ORY Stéphane (membre invité) Acknowledgements First of all I would like to thank the jury members for accepting the proposition to judge my thesis. It is a pleasure for me tho know that my work is evaluated by such experts as Dr. Lydia Danglot specialist in cellular neurobiology and membrane trafficking; Dr. Marc Benhamou specialist in signaling and phospholipid scramblase 1 and Dr. Vincent Lelièvre specialist in neuroscience. I would like particularly to thank my supervisor, Dr. Stéphane Gasman for allowing me to make my thesis in this laboratory, give me the opportunity to perform science and help me in the writing of this manuscript thesis. Especially I would like to thank Dr. Stéphane Ory, for which I feel a great admiration, thank you for all your help during the realization of my thesis, design of experiments, analysis and also for all the advice on the writing of this manuscript thesis... it was not easy. For me it was a great honor to meet you and work with you, because you are a brilliant and creative professional, available at all times to discuss science. Thank you very much for everything Stheph. Thank you very much to all the Gasman–Vital group, all the present and the past member for very interesting Monday scientific discussions. Thank you to Dr. Nicolas Vitale, Dr. Sylvette Chasserot-Golaz, you were always in a good disposition to give me advice and explanations. Thank you to Dr. Nancy Grant it was very nice to share the office with you, I thank you for your scientific and personal advice they were always very helpful. For me it was a pleasure to work near a rigorous and methodical person like you. Many thanks to Tam Thahouly, for her assistance with molecular biology and of course all those beautiful cell cultures. I also want to thank you for those long conversations in the culture room and your joy. It was a pleasure working with such an efficient and effective professional thank you very much for everything. Special enormous thank at all my friends, Mili for all your help and support during the writing of the thesis (eres un sol),Nuria… mi guapa, the perfect woman Aurea-Maria, Juli.. tan linda,Marie… mi bella, Alvaro (mi querido marido de la semana), Fernado (bebote), Fer (chilango), Orkan, Romain, Ivan for being my chosen family, thank you for sharing incredible and unforgettable moments. A mega thank you to my Très belle famille, especially Nathalie and Jean Claude for all your help. They have been incredible all the time, without you this period would have been very difficult. Thank you for the support of my parents, siblings and nieces regardless independently the distance. And the most especial and and sincere thank to my guapos boys. Dr.Houy for his support, comprehension, and help during the thesis writing, the work we do and endless scientific discussions, thanks your Grace. And finally Hector (my mitochondria), my guapo bello thank you for all your comprehension, patience and sacrifices that you had to endure during my doctorate... Thank you very much bebé. Table of contents TABLE OF CONTENTS ........................................................................................................................................ 1 AVANT-PROPOS ............................................................................................................................................... 3 LIST OF ABBREVIATIONS ................................................................................................................................... 6 I. GENERAL INTRODUCTION ........................................................................................................................ 9 1. THE ADRENAL GLAND ....................................................................................................................................... 9 2. CALCIUM REGULATED EXOCYTOSIS IN CHROMAFFIN CELLS: FROM THE BIOGENESIS TO THE RELEASE ................................. 11 2.1 Biogenesis of secretory granules ..................................................................................................... 12 2.2 Transport of secretory granules ....................................................................................................... 14 2.3 Pools of secretory granules .............................................................................................................. 16 2.4 Late phases of exocytosis: from docking to fusion ........................................................................... 17 2.4.2 Priming ............................................................................................................................................ 21 2.4.3 Fusion .............................................................................................................................................. 23 3. ROLE OF CALCIUM SENSORS IN REGULATED EXOCYTOSIS ......................................................................................... 25 3.1 SYNAPTOTAGMIN ...................................................................................................................................... 25 3.2 MUNC PROTEINS ...................................................................................................................................... 26 3.3 DOC2 PROTEINS ....................................................................................................................................... 26 2+ 4. THE RHO-FAMILY PROTEINS IN CA -REGULATED EXOCYTOSIS ................................................................................. 27 2+ 5. ROLE OF LIPIDS IN CA -REGULATED EXOCYTOSIS .................................................................................................. 30 5.1 Anionic lipids ................................................................................................................................... 30 5.2 Cholesterol ....................................................................................................................................... 31 6. COMPENSATORY ENDOCYTOSIS IN NEUROENDOCRINE CELLS .................................................................................... 32 6.1 Clathrin-dependent endocytosis ...................................................................................................... 34 6.2 PROTEINS AND LIPIDS IN COMPENSATORY ENDOCYTOSIS .................................................................................... 36 6.2.1 SNARE proteins in endocytosis......................................................................................................... 36 6.2.2 BAR domain proteins in endocytosis ................................................................................................ 37 6.2.3 Role of lipids in endocytosis ............................................................................................................. 39 6.3 PROTEINS WITH DUAL FUNCTIONS ................................................................................................................