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Reconstitution of SNARE Mediated Membrane Fusion

Kreutzberger, Alex
Thesis/Dissertation; Online
Kreutzberger, Alex
Tamm, Lukas
Regulated exocytosis is a process by which cells release neurotransmitters, peptides, proteins, and small molecules in response to a stimulus. This process is necessary for cell-cell communication in multi-cellular organisms. The final step of regulated exocytosis is membrane fusion, where the membrane of a secretory vesicle merges with the plasma membrane, opening a pore releasing soluble cargo. The molecular machinery that controls fusion is coupled to respond to intracellular calcium. The proteins involved include the SNARE proteins (the fusion machinery), the calcium sensor synaptotagmin, and the regulatory proteins complexin, Munc18, Munc13, and CAPS. I will show the use of planar supported bilayers as a model system to study membrane fusion of single vesicles (Chapter 3). This assay was used to investigate the activity of different plasma membrane target SNARE complexes (Chapter 4), the effect of membrane curvature (Chapter 5) and cholesterol (Chapter 6) on the fusion process, how complexin-1 interacts with the plasma membrane SNARE proteins (Chapter 7), and how the fusion process is coupled to calcium (Chapter 8). These results have led to a new model of membrane fusion. In the absence of calcium, secretory vesicle fusion is arrested in the presence of the SNARE proteins, Munc18, and complexin, but will fuse readily in the presence of calcium. Meanwhile, the protein CAPS or Munc13 act as a kinetic factors controlling the rate of fusion in response to a calcium stimulus.
University of Virginia, Department of Biophysics, PHD (Doctor of Philosophy), 2017
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PHD (Doctor of Philosophy)
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