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Time-resolved structural study of calcium-dependent membrane fusion

English title Time-resolved structural study of calcium-dependent membrane fusion
Applicant Zuber Benoît
Number 163761
Funding scheme SNSF Professorships
Research institution Institut für Anatomie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Molecular Biology
Start/End 01.07.2016 - 30.06.2018
Approved amount 618'033.00
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All Disciplines (2)

Discipline
Molecular Biology
Biochemistry

Keywords (8)

liposomes; cryo-electron tomography; membrane fusion; neurotransmitter release; SNARE; synapse; PC12 cells; synaptosome

Lay Summary (French)

Lead
La communication entre cellules est essentielle au bon fonctionnement du corps. De nombreux processus de communication, par exemple dans le cadre de la transmission nerveuse ou des signaux hormonaux, se font via des substances chimiques qui sont sécrétées par une cellule et détectées par une ou plusieurs autres cellules. Ces substances sont stockées dans des vésicules (des sphères délimitées par une membrane) au sein de la cellule. En réponse à un stimulus provocant l’élévation intracellulaire de la concentration en calcium, ces vésicules fusionnent avec la membrane plasmique, qui délimite la cellule, libérant ainsi leur contenu hors de la cellule. Même si ce processus, l’exocytose, a été très étudié, nombre de ses aspects restent mal compris.
Lay summary

Nous sommes partis de l’idée que pour mieux la comprendre nous pourrions visualiser l’exocytose à l’œuvre. Pour cela nous utilisons la cryo-tomographie électronique (CTE), une méthode de microscopie rendant possible la visualisation directe des membranes et de certaines protéines, et ce avec une préservation presque parfaite de l’échantillon. Nous avons dans notre précédent projet établi ou optimisé trois systèmes expérimentaux permettant la visualisation de l’exocytose par CTE. Nous avons notamment mis au jour des déformations des membranes intervenant juste avant la fusion. Nous allons étudier diverses questions ; par exemple à quel point le réseau de filaments qui maintient ensemble les vésicules présentes dans une synapse neuronale ainsi que le cortex d’actine dans les cellules neuroendocrines sont perturbés au moment même de l’exocytose. Nous allons également nous focaliser sur les protéines présentes dans les vésicules et la membrane plasmique afin de tenter de quantifier leur nombre et les changements structurels majeurs intervenant lors de l’exocytose.

Notre projet s’inscrit dans la recherche fondamentale sur les système nerveux et hormonal. Une meilleure compréhension pourrait contribuer à l’établissement de nouveaux traitements contre des maladies tels le syndrome de Parkinson, la dépression, ou le diabète.

Direct link to Lay Summary Last update: 29.04.2016

Responsible applicant and co-applicants

Employees

Publications

Publication
Molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei
Hoffmann Anneliese, Käser Sandro, Jakob Martin, Amodeo Simona, Peitsch Camille, Týč Jiří, Vaughan Sue, Zuber Benoît, Schneider André, Ochsenreiter Torsten (2018), Molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei, in Proceedings of the National Academy of Sciences, 115(8), E1809-E1818.
A Plasmodium plasma membrane reporter reveals membrane dynamics by live-cell microscopy
Burda Paul-Christian, Schaffner Marco, Kaiser Gesine, Roques Magali, Zuber Benoît, Heussler Volker T. (2017), A Plasmodium plasma membrane reporter reveals membrane dynamics by live-cell microscopy, in Scientific Reports, 7(1), 9740-9740.
Biogenesis of the mitochondrial DNA inheritance machinery in the mitochondrial outer membrane of Trypanosoma brucei
Käser Sandro, Willemin Mathilde, Schnarwiler Felix, Schimanski Bernd, Poveda-Huertes Daniel, Oeljeklaus Silke, Haenni Beat, Zuber Benoît, Warscheid Bettina, Meisinger Chris, Schneider André (2017), Biogenesis of the mitochondrial DNA inheritance machinery in the mitochondrial outer membrane of Trypanosoma brucei, in PLOS Pathogens, 13(12), e1006808-e1006808.
Cell-free reconstitution reveals centriole cartwheel assembly mechanisms.
Guichard P, Hamel V, Le Guennec M, Banterle N, Iacovache I, Nemčíková V, Flückiger I, Goldie K N, Stahlberg H, Lévy D, Zuber B, Gönczy P (2017), Cell-free reconstitution reveals centriole cartwheel assembly mechanisms., in Nature communications, 8, 14813-14813.
High resolution microscopy reveals an unusual architecture of the Plasmodium berghei endoplasmic reticulum.
Kaiser Gesine, De Niz Mariana, Zuber Benoît, Burda Paul-Christian, Kornmann Benoît, Heussler Volker T, Stanway Rebecca R (2016), High resolution microscopy reveals an unusual architecture of the Plasmodium berghei endoplasmic reticulum., in Molecular microbiology, 102(5), 775-791.
Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins.
Braga-Lagache Sophie, Buchs Natasha, Iacovache Mircea-Ioan, Zuber Benoît, Jackson Christopher Benjamin, Heller Manfred (2016), Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins., in Molecular & cellular proteomics : MCP, 15(12), 3640-3652.
iMEM: Isolation of Plasma Membrane for Cryoelectron Microscopy.
Peitsch Camille Françoise, Beckmann Sven, Zuber Benoît (2016), iMEM: Isolation of Plasma Membrane for Cryoelectron Microscopy., in Structure (London, England : 1993), 24(12), 2198-2206.
Mitochondrial growth during the cell cycle of Trypanosoma brucei bloodstream forms.
Jakob Martin, Hoffmann Anneliese, Amodeo Simona, Peitsch Camille, Zuber Benoît, Ochsenreiter Torsten (2016), Mitochondrial growth during the cell cycle of Trypanosoma brucei bloodstream forms., in Scientific reports, 6, 36565-36565.
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Collaboration

Group / person Country
Types of collaboration
Dr Harvey McMahon lab / MRC Laboratory of Molecular Biology, Cambridge Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof Smita Saxena lab / Institue of Cell Biology, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof Henning Stahlberg, C-CINA / Biozentrum University of Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof Dimitrios Fotiadis lab / Institute of Biochemistry and Molecular Medicine, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof Gisou van der Goot / EPFL, Lausanne Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof Annette Draeger lab / Institute of Anatomy, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Dr Vladan Lucic, Max Planck Institute for Biochemistry Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
8th International Conference on Electron Tomography Talk given at a conference Rapid Modifications of Synapses during Neurotransmitter Release 16.09.2018 Les Diablerets, Switzerland Zuber Benoît;
Gordon Research Conference on Three dimensional electron microscopy Talk given at a conference Synaptic vesicle exocytosis and rapid modifications of the presynaptic cytomatrix trapped by time-resolved cryo-electron tomography 11.06.2017 Les Diablerets, Switzerland Zuber Benoît;


Communication with the public

Communication Title Media Place Year
Talks/events/exhibitions Présentation du CEMOVIS à la société de physique valaisanne. Western Switzerland 2017
Talks/events/exhibitions Visite du laboratoire par des élèves du "freies Gymnasium" de Berne German-speaking Switzerland 2017
Talks/events/exhibitions Visite du laboratoire par des élèves du gymnase de Schüpfheim German-speaking Switzerland 2017
Media relations: radio, television Wissenschaftsmagazin - Nobelpreisverleihungen SRF 2 German-speaking Switzerland 2017

Associated projects

Number Title Start Funding scheme
139098 Time-resolved structural study of calcium-dependent membrane fusion 01.07.2012 SNSF Professorships
139098 Time-resolved structural study of calcium-dependent membrane fusion 01.07.2012 SNSF Professorships
179520 Synaptic cytomatrix and exocytosis architecture 01.03.2019 Project funding (Div. I-III)

Abstract

Ca2+-dependent membrane fusion is a cellular mechanism leading to the secretion of the content of a membrane-bound vesicle into the extracellular space. An elevation of intracellular Ca2+ concentration triggers the fusion of the vesicle membrane with the plasma membrane and thereby connects the vesicle lumen with the extracellular space. Exocytosis is necessary for a wide range of physiological processes such as neuronal communication, hormone release, and inflammatory response. In spite of intensive research efforts the molecular mechanism of Ca2+-dependent membrane fusion is still not understood in detail. We reasoned that a direct visualization with molecular resolution of the membrane fusion protein machinery in action and of fusing membranes could resolve some of the long standing controversies. We have developed a workflow to that end: micrometre-sized droplets of a solution eliciting membrane fusion are sprayed; the sample is then plunge-frozen after a defined time delay. This delay ranges from a couple of milliseconds to tens of seconds. Since the sprayed solution and the sample are fluorescently labelled, the positions where the sample was stimulated can be identified by cryo-fluorescence microscopy. The same positions can be then investigated by cryo-electron tomography. We optimised 3 experimental systems for this application: (i) isolated and functional nerve terminals (synaptosomes), (ii) cell-free membrane patches with docked secretory vesicles and associated cytoplasmic elements derived from neuroendocrine cells, and (iii) an in vitro proteoliposome reconstitution system of Ca2+-dependent membrane fusion. We have reconstructed tomograms of fusing vesicles. In the proposed project we will collect data at different time delays and combine the strengths of each experimental system. In particular we will investigate structural changes during the course of exocytosis at the level of the fusion protein machinery and at the level of the membranes. Does synaptotagmin insert in the target membrane and does it correlate with membrane bending? How many SNARE complexes are present in fusing vesicles? Having structural hints that under our experimental conditions kiss-and-run exocytosis may take place in synaptosomes, we will further investigate this mechanism. Synaptic vesicles are highly connected by filaments in resting synapses. This network of filaments is profoundly modified by long exposure to exocytosis-stimulating solution (30-60 s), which suggests a role in the mobility of synaptic vesicles and in their recruitment for exocytosis (Fernandez-Busnadiego et al. J Cell Biol 2010). We will analyse the state of this network milliseconds after stimulation in order to better understand how reactive it is and if it could be involved in the early replenishment of the readily releasable vesicle pool. The role of actin cortex in neuroendocrine secretion is debated: does it form a barrier between secretory vesicles and the plasma membrane, thereby inhibiting fusion, or is it a scaffold for the vesicles, which promotes fusion by keeping them in contact with the plasma membrane? Our cell-free system resolves the actin cortex and its connections to secretory vesicles. We will investigate how the cortex and the connections evolve after stimulation. An "ultrafast" mode of endocytosis taking place in synapses 50 ms after stimulation was recently discovered (Watanabe et al. Nature 2013). It was shown to depend on actin but the used EM method could not resolve actin filaments. With our method we should be able to catch ultrafast endocytosis events and visualize actin filaments. Thereby we could better characterise the role of actin in ultrafast endocytosis. Overall, the original approach followed in our project will shed new light on the molecular mechanism of Ca2+-regulated exocytosis.
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