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Control of the terminal phase of SNARE-dependent membrane fusion

English title Control of the terminal phase of SNARE-dependent membrane fusion
Applicant Mayer Andreas
Number 179306
Funding scheme Project funding (Div. I-III)
Research institution Département de Biochimie Faculté de Biologie et Médecine Université de Lausanne
Institution of higher education University of Lausanne - LA
Main discipline Biochemistry
Start/End 01.04.2018 - 31.03.2022
Approved amount 1'224'000.00
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All Disciplines (2)

Discipline
Biochemistry
Cellular Biology, Cytology

Keywords (5)

Vesicular transport; Vacuole; Biochemistry; Yeast; Membrane fusion

Lay Summary (German)

Lead
Zellen müssen zwischen Ihren Organellen und ebenso mit Ihrer Umgebung Stoffe austauschen. Ein großer Teil dieses Austausches wird durch die Bildung von Transportvesikeln bewerkstelligt, die solches Material transportieren und an einem Zielorganell (oder der Zellmembran) abliefern. Diese "Lieferung" geschieht durch Fusion der Membran des Vesikels mit der Membran des Zielkompartiments. Das Projekt erforscht den molekularen Mechanismus dieser Fusionsprozesse.
Lay summary

Regulierte Membranfusion kontrolliert viele Aspekte der Physiologie eukaryotischer Zellen, da sie z.B. die Freisetzung von Neurotransmittern, Wachstumsfaktoren, Hormonen, Enzymen, Rezeptoren und Transportern bewerkstelligt. Diese Moleküle werden in intrazellulären Vesikeln gespeichert, die dann durch ein Signal zur Fusion mit der Zellmembran freigegeben werden, wobei sie ihren Inhalt in den extrazellulären Raum entlassen. Auch der Transport zwischen Organellen hängt von der Bildung und Fusion von Transportvesikeln ab. 

Solche Membranfusionsvorgänge werden von SNARE-Proteinen getrieben. Diese Proteine interagieren mit einer Vielzahl von Hilfsfaktoren (Rab-GTPasen und Tether-Faktoren), die die Bindung und Fusion der Membranen kontrollieren. Während der Fusion gehen die Membranen von einem gedockten Zustand in eine Hemifusion, auf die schließlich die Öffnung und Expansion der Fusionspore folgen, wodurch die beiden Membranen dann verschmelzen. Die Fusionsporenöffnung erfordert den größten Energieaufwand. Wie SNARE Proteine diese Energie liefern und auf die Membran übertragen, ist Gegenstand aktueller Forschung. 

Wir studieren die Frage mit Organellen (Vakuolen) aus der Bäckerhefe, da die relevanten Intermediate ihrer Fusion sehr gut messbar sind. Wir kombinieren dieses System mit einer Reihe synthetischer Werkzeuge, um die SNARE-Proteine sowie ihre Kontrollfaktoren zu modifizieren und die Konsequenzen für die Fusionsporen zu studieren. 

Diese Experimente sollten uns neue Einblicke in die Energetik und die Kontrolle intrazellulärer Membranfusionen erlauben, die für einfache Einzeller ebenso relevant sind wie für  pflanzliche und humane Zellen. 

 

 

Direct link to Lay Summary Last update: 07.04.2018

Responsible applicant and co-applicants

Employees

Publications

Publication
Fusion Pores Live on the Edge
Blokhuis Edgar M., D’Agostino Massimo, Mayer Andreas, Risselada H. Jelger (2020), Fusion Pores Live on the Edge, in The Journal of Physical Chemistry Letters, 11(4), 1204-1208.
SNAREs, tethers and SM proteins: how to overcome the final barriers to membrane fusion?
Risselada Herre Jelger, Mayer Andreas (2020), SNAREs, tethers and SM proteins: how to overcome the final barriers to membrane fusion?, in Biochemical Journal, 477(1), 243-258.
Assay of lipid mixing and fusion pore formation in the fusion of yeast vacuoles
D’Agostino Massimo, Mayer Andreas (2019), Assay of lipid mixing and fusion pore formation in the fusion of yeast vacuoles, Springer New York, New York, NY, 253-262.
SNARE ‐mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle
D'Agostino Massimo, Risselada Herre Jelger, Endter Laura J, Comte‐Miserez Véronique, Mayer Andreas (2018), SNARE ‐mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle, in The EMBO Journal, 37(19), 1-22.

Collaboration

Group / person Country
Types of collaboration
Herre Jelger Risselada Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
XXIX. INTERNATIONAL CONFERENCE ON YEAST GENETICS AND MOLECULAR BIOLOGY Talk given at a conference Vacuolar fusion pores adapt organelle volume to phosphate storage requirements 18.08.2020 Gothenborg, Sweden Mayer Andreas;
EMBO Workshop "The Physics and Chemistry of Endocytosis at Multiple Scales" Poster Retromer regulates the fusion-fission equilibrium of vacuoles 01.09.2019 Ischia, Italy Courtellemont Thibault;
GRC conference "Organellar Channels" Talk given at a conference Phoshate homeostasis and vacuolar fusion pores 04.08.2019 West Dover, United States of America Mayer Andreas;
Membrane Biophysics of Exo-Endocytosis: From Model Systems to Cells Talk given at a conference Control of fusion after SNARE complex formation: Tethers in the driver’s seat 03.04.2019 Mandelieu-la-Napoule, France Mayer Andreas;


Associated projects

Number Title Start Funding scheme
163477 Bonus-of-Excellence: Regulation of SNARE-dependent membrane fusion 01.04.2016 Project funding (Div. I-III)

Abstract

Membrane fusion in eukaryotic cells mediates the biogenesis of organelles, vesicular traffic between them, and exo- and endocytosis of important signaling molecules, such as hormones and neurotransmitters. Conserved protein systems perform distinct tasks in intracellular membrane fusion. Whereas tether proteins have been believed to mediate only initial recognition and attachment of membranes (Baker & Hughson, 2016), SNARE protein complexes are considered as the core fusion engine (Weber et al, 1998). They provide mechanical energy to distort membranes and drive them through a hemifusion intermediate towards the formation of a fusion pore (Gao et al, 2012; Zorman et al, 2013; Zhang et al, 2016; Reese et al, 2005). Our recent findings indicated, however, that additional factors, such as tether protein complexes and SM proteins, participate in fusion up to its very last step and provide critical driving force. Thus, the actual SNARE-containing fusion machinery is much bigger and more complex than hitherto thought (D'Agostino et al., in press). This poses novel questions concerning the sequence of events in fusion. For the large majority of fusion reactions, the "constitutive" ones that do not use a calcium-dependent trigger mechanism acting in regulated exocytosis, it is unknown at which molecular step fusion is controlled. I want to address the following aspects of this critical issue: •What is the in vivo control point of vacuole fusion? SNARE activation, trans-SNARE pairing, SNARE-disassembly, hemifusion, pore-opening or pore-expansion?•What are the roles of SM and tether proteins in controlling SNARE function?•Is the GTPase cycle of Rab-proteins, which bind and probably regulate tether proteins, integrated into the reaction sequence, and if so, how?•What regulates the interaction between SNAREs and tether proteins at different phases of a fusion reaction?We will address these questions using lysosome-like yeast vacuoles as a highly versatile model system for SNARE-dependent fusion. I intend to develop assays that shall permit us - in living cells - to monitor lipid mixing and fusion pore opening and to engineer an artificial membrane tethering system to trigger membrane contact at will. The functions of the vacuolar SM protein, Rab-GTPase, and tether complex will be dissociated from each other to analyse their functions in the various reaction stages of membrane fusion individually. A combination of in vivo analyses, in vitro work with purified organelles or reconstituted lipid systems, and molecular dynamics simulations will permit us to gain fundamental novel insights into the core mechanism of the eukaryotic membrane fusion machinery.
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