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Structural and evolutionary studies on the vesicle fusion machinery

Applicant Fasshauer Dirk
Number 133055
Funding scheme Project funding (Div. I-III)
Research institution Département des neurosciences fondamentales Faculté de Biologie et de Médecine Université de Lausanne
Institution of higher education University of Lausanne - LA
Main discipline Biochemistry
Start/End 01.05.2011 - 30.04.2015
Approved amount 600'000.00
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All Disciplines (2)

Discipline
Biochemistry
Cellular Biology, Cytology

Keywords (8)

neurosecretion; SNARE proteins; vesicle trafficking; evolution; exocytosis; Eukaryotic cells; membrane fusion; Synaptic vesicles

Lay Summary (English)

Lead
Lay summary
This study aims at elucidating the workings of the molecular machinery involved in the release of neurotransmitters from synaptic vesicles.The cells of our bodies-as also those of other animals, plants, fungi and many unicellular organisms-have a much more complex structure than the cells of bacteria. In our eukaryotic cells there are many separate reaction compartments in which different tasks are performed. Exchange between compartments is mediated by cargo-loaded vesicles that bud from a donor organelle, are transported through the cytosol, and eventually fuse with the target compartment. As each of the internal compartments serves a specific and vital cellular function, it must maintain its identity during vesicle trafficking. This is achieved by specific protein machineries that tightly regulate each transport step. The molecular machinery that catalyzes the docking and fusion of vesicles consists of a complicated protein network. The machinery is best studied for the process of neuronal exocytosis. The core of the release machinery is composed of SNARE proteins, which are thought to zipper into a tight complex between the two membranes. Another important factor of the release machinery is the SM protein Munc18. Intriguingly, these core elements are highly conserved among all eukaryotes and throughout the cell. ZielWe want to establish a detailed evolutionary and functional description of the protein interaction network involved in neuronal exocytosis. For our biochemical studies, we almost exclusively use recombinant proteins expressed in bacteria (E. coli). Next to standard biochemical techniques, we employ spectroscopic (Circular Dichroism and Fluorescence Spectroscopy) and calorimetric (Isothermal Titration Calorimetry) methods. Where feasible, we also make use of high-resolution structural techniques. For our bioinformatic studies, we have developed an innovative database system that allows the management of protein families. The results can be looked at on http://bioinformatics.mpibpc.mpg.de/snare/.BedeutungAt chemical synapses, neurons communicate via rapid release of neurotransmitters from synaptic vesicles upon a transient influx of Ca2+ ions. This process is central to understand brain´s most remarkable feats: memory and learning. However, the comprehension of the molecular events is still in its infancy.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Novel cell types, neurosecretory cells, and body plan of the early-diverging metazoan Trichoplax adhaerens.
Smith Carolyn L, Varoqueaux Frédérique, Kittelmann Maike, Azzam Rita N, Cooper Benjamin, Winters Christine A, Eitel Michael, Fasshauer Dirk, Reese Thomas S (2014), Novel cell types, neurosecretory cells, and body plan of the early-diverging metazoan Trichoplax adhaerens., in Current biology : CB, 24(14), 1565-72.
The SM protein Sly1 accelerates assembly of the ER-Golgi SNARE complex.
Demircioglu F Esra, Burkhardt Pawel, Fasshauer Dirk (2014), The SM protein Sly1 accelerates assembly of the ER-Golgi SNARE complex., in Proceedings of the National Academy of Sciences of the United States of America, 111(38), 13828-33.
Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment.
Honigmann Alf, van den Bogaart Geert, Iraheta Emilio, Risselada H Jelger, Milovanovic Dragomir, Mueller Veronika, Müllar Stefan, Diederichsen Ulf, Fasshauer Dirk, Grubmüller Helmut, Hell Stefan W, Eggeling Christian, Kühnel Karin, Jahn Reinhard (2013), Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment., in Nature structural & molecular biology, 20(6), 679-86.
Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation.
Colbert Karen N, Hattendorf Douglas A, Weiss Thomas M, Burkhardt Pawel, Fasshauer Dirk, Weis William I (2013), Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation., in Proceedings of the National Academy of Sciences of the United States of America, 110(31), 12637-42.
Molecular machines governing exocytosis of synaptic vesicles.
Jahn Reinhard, Fasshauer Dirk (2012), Molecular machines governing exocytosis of synaptic vesicles., in Nature, 490(7419), 201-7.
Munc18-1 mutations that strongly impair SNARE-complex binding support normal synaptic transmission.
Meijer Marieke, Burkhardt Pawel, de Wit Heidi, Toonen Ruud F, Fasshauer Dirk, Verhage Matthijs (2012), Munc18-1 mutations that strongly impair SNARE-complex binding support normal synaptic transmission., in The EMBO journal, 31(9), 2156-68.
Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis.
Klöpper Tobias H, Kienle Nickias, Fasshauer Dirk, Munro Sean (2012), Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis., in BMC biology, 10, 71-71.
Primordial neurosecretory apparatus identified in the choanoflagellate Monosiga brevicollis.
Burkhardt Pawel, Stegmann Christian M, Cooper Benjamin, Kloepper Tobias H, Imig Cordelia, Varoqueaux Frédérique, Wahl Markus C, Fasshauer Dirk (2011), Primordial neurosecretory apparatus identified in the choanoflagellate Monosiga brevicollis., in Proceedings of the National Academy of Sciences of the United States of America, 108(37), 15264-9.

Associated projects

Number Title Start Funding scheme
160343 Structural and evolutionary studies on the vesicle fusion machinery 01.05.2015 Project funding (Div. I-III)
145002 Microscale thermophoresis for the Faculty of Bology and Medicine in Lausanne 01.12.2012 R'EQUIP
133807 Introducing nanoscale cell biology: STED microscope 01.12.2010 R'EQUIP
160343 Structural and evolutionary studies on the vesicle fusion machinery 01.05.2015 Project funding (Div. I-III)

Abstract

Structural and evolutionary studies on the vesicle fusion machineryCalcium-dependent secretion of neurotransmitter-loaded synaptic vesicles is at the heart of synaptic transmission. The underlying molecular machinery that catalyzes the tethering-docking-priming-fusion of vesicles consists of a complicated protein network. Its core is composed of SNARE proteins and the SM protein Munc18, yet their mode of interaction is not understood. In addition, tethering factors like Munc13 play an important role. These core elements are highly conserved among all eukaryotes and throughout the cell from the endoplasmic reticulum to the plasma membrane. To come to a better understanding of the molecular events during neuronal exocytosis, we focus on a detailed structural, kinetic, thermodynamic, and phylogenetic characterization of the underlying protein-protein interactions. However, the large diversity of proteins involved in different secretion processes in animals renders the description of the molecular processes challenging. In fact, the evolutionary transition to animals, and to vertebrates in particular, saw a signi?cant increase in many of the protein families involved in secretion. This suggests that these lineages possess more finely tuned regulation mechanisms, but also tissue-speci?c specialization of the core traf?cking machinery. Moreover, novel factors seem to have been added during animal evolution. Nevertheless, it is often unclear whether such factors indeed represent special adaptations of certain trafficking steps or certain eukaryotic lineages. Based on a detailed description of the evolutionary history of the protein machinery involved in the vesicle fusion process, we therefore want to compare the underlying mechanisms at different stages of evolution.Our research will be organized in 6 interrelated projects:•Overall aim of the projects 1 to 4 is to understand the basic mechanism of the interaction between the SNAREs and SM proteins, i.e. the core vesicle fusion machinery, by comparing the machineries of different trafficking steps and of different organisms.-In project 1, we will continue our work on the interaction of the neuronal SM protein Munc18a/Unc18 with the neuronal SNARE proteins. These studies are already relatively far advanced and thus can serve as guide for the other biochemical projects.-In project 2, comparative investigations on the homologous machinery in baker´s yeast will be carried out. -In project 3, comparative investigations on the interaction of the SM protein Sly1 with the SNAREs involved in Golgi and ER trafficking are planned. -In project 4, we want to elucidate the function of a novel SM protein family member.•Aim of project 5 is to investigate the interaction of tethering-docking factor Munc13/Unc13 with the core secretion machinery. In part, this project can build on the groundwork carried out on the interaction of Munc18 with the SNAREs (see project 1). In addition, as the other SM proteins studied interact with homologous tethering machineries, the insights gained by project 5 will help the scientists working on projects 2 to 4.•Aim of project 6 is to carry out morphological and functional studies on the organization and evolution of the secretion apparatus of animals.
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