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34 Thomas Söllner Goal To reveal how the molecular fusion machinery assembles and controls regulated exocytosis and fusion pore dynamics. Mechanistic and structural insights are obtained by reconstituting membrane fusion in vitro using recombinant proteins, liposomes, and purified synaptic vesicles. In vitro data are validated by exocytosis studies in vivo. Background Physiological processes such as neurotransmission and the regulation of the blood glucose level require the release of neurotransmitters and insulin in a spatially and temporally controlled manner. A cascade of reactions and a series of protein interactions control the stepwise assembly of the fusion machinery. The first step is the interaction of secretory vesicles with the plasma membrane, which is mediated by compartment-specific tethering proteins. These proteins determine where fusion occurs. The tethers together with other regulatory components control the subsequent assembly of the fusion machinery. The fusion machinery consists of compartment-specific v- SNAREs localized on secretory vesicles, which pair with cognate t-SNAREs on the plasma membrane. SNARE complexes assemble in a stepwise fashion and distinct regulators either accelerate or arrest the formation of intermediates. This assembly line creates distinct pools of vesicles 1991 Ph.D. from Ludwig-Maximilians University Munich 1991-1993 Postdocoral Fellow at the Sloan-Kettering Institute, New York, USA 1994-1997 Assistant Laboratory Member at the Sloan-Kettering Institute 1998-2004 Assistant Professor at the Sloan-Kettering Institute 2004-2005 Associate Professor at the Sloan-Kettering Institute since 2005 Full Professor, Heidelberg University Biochemistry Center (BZH) Regulated Membrane Fusion: Molecular Mechanisms and Machinery Thomas Söllner with different release probabilities. Finally, a sensor that is directly coupled to the fusion machinery recognizes the incoming signal and triggers membrane fusion/fusion pore opening. This signal transduction event determines when vesicle fusion occurs. Research Highlights After having established that SNAREs contribute specificity to membrane trafficking and are the minimal fusion machinery, we have now developed additional in vitro assays that allow us to study regulated exocytosis. These assays include a single vesicle docking/fusion assay, a biochemical content mixing assay, a synaptic vesicle fusion assay, and an in vivo exocytosis assay using pHluorin - a pH-sensitive GFP, targeted into the lumen of secretory vesicles. Our recent analyses of the regulatory components showed that Rim1, which is localized at the active zone of neuronal synapses, contributes to the orderly assembly of the fusion machinery. Rim1 binds specifically the neuronal t-SNAREs syntaxin 1 and SNAP-25. It promotes t-SNARE complex assembly, but blocks a-SNAP binding. Thus Rim1 inhibits the dissociation of t-SNARE complexes by a-SNAP and NSF and per mits the subsequent assembly of SNAREpins (trans v-/t-SNARE complexes). Rim1-SNARE complexes bind complexins, which are required for calcium-regulated exocytosis. Out of the 4 mam-
Fig. 1: Model of regulated exocytosis at the neuronal synapse. Fig. 2: v-SNARE GUV (green channel) with docked small unilamellar t-SNARE vesicles (SUV, red channel). Fig. 3: Single vesicle exocytosis measured in PC12 cells by a transient increase of pHluorin-fluorescence. (pHluorin, a pH-sensitive GFP, was targeted into the lumen of secretory vesicles.) Fusion was triggered by addition of KCl. malian complexins, complexin I and II selec tively promote SNAREpin formation. In addition, complexins can arrest membrane fusion until the calcium sensor synaptotagmin releases the clamp. We are presently testing which functions rab proteins, Munc13, and Munc18 play in the overall assembly pathway. In addition, we are analyzing the role of lipids in the fusion process. In collabora- tive studies, we plan to obtain structural informa- tion of distinct fusion machinery assembly intermediates. Selected Publications 2004 - 2007 Söllner, T.H. (2007). Lipid droplets highjack SNAREs. Nat. Cell Biol. 9, 1219-20. Moelleken, J., Malsam, J., Betts, M., Movafeghi, A., Reckmann, I., Meissner, I., Hellwig, A., Russel, R., Söllner, T.H., Brügger, B., Wieland, F. (2007). Differential localization of isoformic coatomer complexes within the Golgi apparatus. Proc. Natl. Acad. Sci. USA 104, 4425-30. Cosson, P., Ravazzola, M., Varlamov, O., Söllner, T.H., Di Liberto, M., Volchuk, A., Rothman, J.E., Orci, L. (2005). Dynamic transport of SNARE proteins in the Golgi apparatus. Proc. Natl. Acad. Sci. USA 102, 14647-52. Cheng, Y. Sequeira, S.M., Malinina, L., Tereshko, V., Söllner, T.H., and Patel, D.J. (2004). Crystallographic identification of Ca2+ and Sr2+ coordination sites in synaptotagmin I C2B domain. Protein Science 13, 2665-2672. Söllner, T.H. (2004) Intracellular and viral membrane fusion: a uniting mechanism. Curr. Opinion Cell Biol 16, 429-435. Fukasawa, M., Varlamov, O., Eng, W.S., Söllner, T.H., and Rothman, J.E. (2004). Localization and activity of the SNARE Ykt6 determined by its regulatory domain and palmitoylation. Proc. Natl. Acad. Sci. USA 101, 4815-1820. Fix, M., Melia, T.J., Jaiswal, J.K., Rappoport, J.Z., You, D., Söllner, T.H., Rothman, J.E., and Simon, S.M. (2004). Imaging single membrane fusion events mediated by SNARE proteins. Proc. Natl. Acad. Sci. USA 101, 7311-7316. Thomas Söllner Biochemie-Zentrum der Universität Heidelberg (BZH) Im Neuenheimer Feld 345 D-69120 Heidelberg Phone: +49 (0)6221-54 5342 Fax: +49 (0)6221-54 5341 E-mail: thomas.soellner@bzh.uni-heidelberg.de Thomas Söllner 35
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