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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 139098
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 01.07.2012 - 30.06.2016
Approved amount 1'506'736.00
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All Disciplines (2)

Discipline
Molecular Biology
Biochemistry

Keywords (10)

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

Lay Summary (English)

Lead
Lay summary

Ca2+-dependent membrane fusion is an essential cellular process. Nerve transmission, hormone release by neuroendocrine cells, and insulin release by pancreatic  cells, to name a few, are all dependent on this process. It consists of the fusion of a membrane-bound vesicle with another membrane, typically the plasma membrane, in response to a rise in cytoplasmic Ca2+ concentration. Many diseases have been linked to abnormalities in a particular type of Ca2+-dependent membrane fusion. A few examples are neurodegenerative diseases, such as Parkinson’s disease and Huntington’s disease, diabetes, botulism, and tetanus. Despite its high biological and medical importance, the molecular mechanism of Ca2+-dependent membrane fusion is not understood in detail.

We will use cryo-electron microscopy (cryoEM) to directly visualize membrane fusion with a high spatial and temporal resolution. In contrast to all other electron microscopy techniques, cryoEM allows the observation of biological specimens in their fully-hydrated, and hence native, state. We expect that our new approach will provide a better understanding of Ca2+-dependent membrane fusion molecular mechanism.

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process.
Iacovache Ioan, De Carlo Sacha, Cirauqui Nuria, Dal Peraro Matteo, van der Goot F Gisou, Zuber Benoît (2016), Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process., in Nature communications, 7, 12062.
Impaired mTORC1-Dependent Expression of Homer-3 Influences SCA1 Pathophysiology.
Ruegsegger Céline, Stucki David M, Steiner Silvio, Angliker Nico, Radecke Julika, Keller Eva, Zuber Benoît, Rüegg Markus A, Saxena Smita (2016), Impaired mTORC1-Dependent Expression of Homer-3 Influences SCA1 Pathophysiology., in Neuron, 89(1), 129-46.
TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes.
Trikin Roman, Doiron Nicholas, Hoffmann Anneliese, Haenni Beat, Jakob Martin, Schnaufer Achim, Schimanski Bernd, Zuber Benoît, Ochsenreiter Torsten (2016), TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes., in PLoS pathogens, 12(5), 1005586-1005586.
A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons.
Studer Daniel, Klein Alycia, Iacovache Ioan, Gnaegi Helmut, Zuber Benoît (2014), A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons., in Journal of structural biology, 185(1), 125-8.
Direct observation of liquid crystals using cryo-TEM: Specimen preparation and low-dose imaging.
Gao Min, Kim Young-Ki, Zhang Cuiyu, Borshch Volodymyr, Zhou Shuang, Park Heung-Shik, Jákli Antal, Lavrentovich Oleg D, Tamba Maria-Gabriela, Kohlmeier Alexandra, Mehl Georg H, Weissflog Wolfgang, Studer Daniel, Zuber Benoît, Gnägi Helmut, Lin Fang (2014), Direct observation of liquid crystals using cryo-TEM: Specimen preparation and low-dose imaging., in Microscopy research and technique, 1.
The Fascinating Coat Surrounding Mycobacteria
Daffe Mamadou, Zuber Benoît (2014), The Fascinating Coat Surrounding Mycobacteria, in Remaut Han (ed.), Caister Academic Press, Norfolk, 179.
Implementation of a virtual correlative light and transmission electron microscope.
Voigt Tilman, Zuber Benoît, Gawatz Gerlinde, Herrmann Gudrun (2013), Implementation of a virtual correlative light and transmission electron microscope., in Microscopy research and technique, 1-8.
Structure and superorganization of acetylcholine receptor-rapsyn complexes
Zuber Benoît, Unwin Nigel (2013), Structure and superorganization of acetylcholine receptor-rapsyn complexes, in PNAS, 1-6.
Active release of pneumolysin prepores and pores by mammalian cells undergoing a Streptococcus pneumoniae attack
Wolfmeier Heidi, Radecke Julika, Schönauer Roman, Köffel René, Babiychuk Victoria, Drücker Patrick, Hathaway Lucy, Mitchell Tim, Zuber Benoît, Draeger Annette, Babiychuk Eduard, Active release of pneumolysin prepores and pores by mammalian cells undergoing a Streptococcus pneumoniae attack, in BBA - General Subjects, 0.
Mitochondrial impairments contribute to Spinocerebellar ataxia type 1 progression and can be ameliorated by the mitochondria-targeted antioxidant MitoQ.
Stucki David M, Ruegsegger Céline, Steiner Silvio, Radecke Julika, Murphy Michael P, Zuber Benoît, Saxena Smita, Mitochondrial impairments contribute to Spinocerebellar ataxia type 1 progression and can be ameliorated by the mitochondria-targeted antioxidant MitoQ., in Free radical biology & medicine, -.

Collaboration

Group / person Country
Types of collaboration
Prof. Annette Draeger lab / Institute of Anatomy, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Prof Henning Stahlberg, C-CINA / Biozentrum University of Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Prof. Roger Kornberg Lab / Stanford University School of Medicine United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
Prof Dimitrios Fotiadis lab / Institute of Biochemistry and Molecular Medicine, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Prof. Gisou van der Goot, EPFL Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Dr Richard Henderson / MRC Laboratory of Molecular Biology Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Prof. Nigel Unwin / MRC Laboratory of Molecular Biology Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Dr Harvey McMahon lab / MRC Laboratory of Molecular Biology Great Britain and Northern Ireland (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
Gordon Research Conference on 3-dimensional electron microscopy Poster iMEM: isolation of plasma Membrane for cryo-Electron Microscopy 19.06.2016 Hongkong, Hongkong Camille Peitsch;
Gordon Research Conference on 3-dimensional electron microscopy Talk given at a conference Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore formation process (presented by postdoc Ioan Iacovache) 19.06.2016 Hongkong, Hongkong Zuber Benoît;
EMBO Meeting Poster Isolation of plasma membrane patches for cryo-electron microscopy 05.09.2015 Birmingham, Great Britain and Northern Ireland Camille Peitsch;
Annual Congress of the French Society of Microscopy Talk given at a conference Cryo-electron tomography of synapses 30.06.2015 Nice, France Zuber Benoît;
EMBO Workshop: Cryo-Electron Microscopy and 3D Image Processing Talk given at a conference Neuroendocrine cells membrane patches for cryo-electron tomography 31.08.2014 Heidelberg, Germany Camille Peitsch;
Gordon Research Conference on 3-dimensional electron microscopy Talk given at a conference A new tool facilitating vitreous cryosectioning (CEMOVIS) 23.06.2014 Girona, Spain Zuber Benoît;
LS2 meeting Poster Structural Changes During Exocytosis In A Cell-Free System Using Cryo-Electron Tomography 04.02.2014 Lausanne, Switzerland Camille Peitsch; Zuber Benoît;
4th European Synapse Meeting 2013 Poster Structure and superorganization of acetylcholine receptor-rapsyn complexes 28.08.2013 Bordeaux, France Zuber Benoît;
European Microscopy Conference Talk given at a conference architecture of acetylcholine receptors in the postsynaptic membrane 16.09.2012 Manchester, United Kingdom, Great Britain and Northern Ireland Zuber Benoît;
NCCR Workshop on Subtomogram Averaging Talk given at a conference architecture of acetylcholine receptors in the postsynaptic membrane 07.09.2012 Basel, Switzerland Zuber Benoît;


Self-organised

Title Date Place
LS2 meeting, session chairman 29.01.2015 Zurich, Switzerland
4th UniBe International Practical Course on Cryo-Electron Microscopy of Vitreous Sections 11.08.2014 Berne, Switzerland
3rd UB Practical Course on Cryo-Electron Microscopy of Vitreous Sections 02.07.2013 Institut f. Anatomie, Uni Bern, Switzerland

Communication with the public

Communication Title Media Place Year
New media (web, blogs, podcasts, news feeds etc.) Descripción atómica de un asesinato Facebook public page "Cienciorama" International 2016
Media relations: print media, online media Researchers find answer to countering bio-weapons Swissinfo.ch Rhaeto-Romanic Switzerland Western Switzerland Italian-speaking Switzerland German-speaking Switzerland 2016
Media relations: print media, online media Wie ein Bakterium Wundinfektionen und heftige Durchfälle auslöst der Standard International 2016

Awards

Title Year
Morphology Prize of the Swiss Society of Anatomy, Histology, and Embryology 2014

Associated projects

Number Title Start Funding scheme
150823 Serial block face SEM 01.12.2013 R'EQUIP
163761 Time-resolved structural study of calcium-dependent membrane fusion 01.07.2016 SNSF Professorships
157704 Direct electron detector and phase plate for cryo-transmission electron microscopy of biological samples 01.12.2014 R'EQUIP
163761 Time-resolved structural study of calcium-dependent membrane fusion 01.07.2016 SNSF Professorships
157748 Microscopy Equipment for Organ-on-Chips and Perfused Microfluidic Systems with High Speed Camera 01.12.2014 R'EQUIP

Abstract

The objective of my laboratory is to understand the mechanisms underlying Ca2+-dependent membrane fusion, a process at the heart of critical biological processes such as synaptic transmission and hormone release. Cytoplasmic vesicles fuse with the plasma membrane in response to an increased cytoplasmic Ca2+ concentration and release their content outside the cell; a process also known as exocytosis. Defects in this process have been linked to severe diseases such as Parkinson’s disease, tetanus, and diabetes. Ca2+-dependent membrane fusion has been extensively studied by a variety of approaches both in vivo and in vitro but several steps remain unclear. Our structural analysis of synapses both by cryo-electron microscopy of vitreous sections (CEMOVIS) and, in collaboration with V. Lucic and W. Baumeister’s group, by cryo-electron tomography (cryoET) has demonstrated the superiority of these techniques over conventional electron microscopy techniques. My laboratory will develop new methods for studying the fast process of Ca2+-dependent membrane fusion. First, we will use a novel cryoET approach to describe the sequence of structural changes occurring milliseconds after Ca2+ influx in isolated synapses with nanometre-resolution. Second, we will adapt a cell-free system of Ca2+-dependent exocytosis to cryoET. In this setup, the plasma membrane of neuroendocrine cells is purified along with docked, fusion-competent, secretory vesicles. There we will not only observe structural changes induced by Ca2+ rise but also localise the proteins involved in membrane fusion by specifically labelling them. Third, we will use an in vitro reconstitution system of Ca2+-dependent membrane fusion and combine it with cryoET. This will give us new ways of assessing the role of specific proteins in the process of bringing membranes together. Finally, we will study Ca2+-dependent membrane fusion in intact cells by cryoET of vitreous sections after improving high pressure freezing of adherent cells. Overall, our work will provide a better understanding of Ca2+-dependent membrane fusion and will serve as a structural framework for refinement of its model.
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