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Elucidating the molecular mechanisms of cellular membrane fusion machineries

Elucidating the molecular mechanisms of cellular membrane fusion machineries

By In Aiv Internship On November 15, 2019

Internship title:

Name: Membrane Traffic in Healthy and Diseased Brain
Affiliation: UMR_S1266 INSERM- Université Paris Descartes
Address: 102-108, rue de la santé 75014 Paris
E-mail: david.tareste@inserm.fr

LAB Director
Name: Thierry Galli
Phone number: 0140789226
E-mail: thierry.galli@inserm.fr

Name: David Tareste
Phone number: 0140789249
E-mail: david.tareste@inserm.fr

Subject Keywords: Mitochondria; Membrane fusion; Mitofusin proteins; Lipids
Tools and methodologies: In vitro reconstitution; liposome docking and fusion assays; fluorescence spectroscopy; optical and electron microscopy; in situ mitochondrial fusion assay
Summary of lab\’s interests: Our goal is to elucidate the molecular mechanisms of cellular membrane fusion machineries using a combination of in situ and in vitro approaches. Candidate fusion proteins are either studied within cells or reconstituted into artificial membrane systems with defined and tunable biophysical properties (e.g. supported lipid bilayers, liposomes of various sizes ranging from tens of nanometers to tens of micrometers). The capacity of these proteins to induce membrane docking, deformation and/or fusion is monitored through a combination of spectroscopy and fluorescence or electron microscopy techniques.
Project summary: Mitochondria constantly move, fuse and divide within cells. The balance between fusion and fission defines mitochondrial morphology and is crucial for normal mitochondrial and cellular function. Outer mitochondrial membrane fusion is mediated by Mitofusin proteins whose molecular architecture consists of an N-terminal GTPase domain, a first heptad repeat domain (HR1), a transmembrane (TM) region, and a second heptad repeat domain (HR2). Mutations in any of these functional domains impair Mitofusin function, but their exact role in mitochondrial fusion remains elusive. In vitro reconstitution studies by us and others suggest that the HR2 of Mitofusin mediates short distance (~10 nm) membrane docking by forming homotypic antiparallel dimers, while its HR1 – owing to its amphipathic nature – triggers fusion by perturbing the membrane structure. Mitochondrial fusion is also regulated by specific lipids such as cardiolipin (CL), phosphatidylethanolamine (PE) and phosphatidic acid (PA). Reduction in CL and/or PE level in mitochondrial membranes impairs mitochondrial fusion, and increase of PA level promotes close apposition of mitochondrial membranes, which is a necessary step of the fusion event. However, the exact mode of action of these lipids in mitochondrial fusion is not fully understood. The current project aims at elucidating how Mitofusin-mediated fusion is regulated by lipids.
Interdisciplinary aspect of the project: The main ambition of this project is to understand the molecular mechanisms of mitochondrial fusion using an integrated approach ranging from molecular biophysics to cell biology. This approach is intended to bridge the gap between physiological observations in vivo and biophysical assays with recombinant proteins in vitro, and thus to allow physiologically relevant conclusions to be drawn from in vitro observations. To this end, we will use a combination of approaches including cell-free in vitro liposome docking and fusion assays, as well as live cell imaging of mitochondrial fusion in situ, and morphological analysis of mitochondria by electron microscopy.