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