Study of SNARE-mediated membrane fusion with a novel single vesicle fusion assay
by Agata Witkowska
Date of Examination:2016-11-23
Date of issue:2018-06-12
Advisor:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Andreas Janshoff
Persistent Address:
http://dx.doi.org/10.53846/goediss-6921
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Description:PhD thesis
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Abstract
English
Membrane fusion in the secretory pathway of eukaryotic cells, including exocytosis of synaptic vesicles, is catalysed by SNARE proteins. In case of the synaptic vesicle fusion, two of the members of this protein family reside on the presynaptic plasma membrane and one on synaptic vesicles. When these three proteins come together they undergo an exergonic reaction of zippering, from membrane distal to membrane proximal regions, to form a coiled coil structure. This process pulls membranes towards each other and induces membrane fusion. Despite many years of research, the molecular mechanism of the SNARE-driven fusion is still not fully understood, and remaining questions concern the extraordinary synaptic vesicle exocytosis speed, or translation of the zippering force to fusing membranes. In vitro, SNAREs are sufficient to mediate effective fusion of both native and artificial membranes. Over the past years, reconstitution systems have been instrumental in characterising the basic features of the SNARE-mediated membrane fusion in combination with various accessory proteins (especially these involved in synaptic vesicle exocytosis). First, ensemble measurements of SNARE-mediated liposome fusion were performed, however they lacked the possibility of distinguishing of reaction steps. Therefore, more recently, microscopy assays were developed that allow observation of the fusion reaction on a single vesicle level. Nevertheless, these assays often lack temporal resolution for monitoring fast fusion reaction catalysed by neuronal SNAREs, and usually involve membrane immobilisation to a solid surface that may lead to various artefacts. In this study a novel single vesicle assay was developed for monitoring membrane fusion on the example of neuronal exocytosis. This assay includes giant unilamellar vesicles as presynaptic plasma membrane mimics and smaller vesicles (either liposomes or purified secretory granules). Membrane topology of giant liposomes ensures that only a small portion of the membrane is involved in interactions with the surface. Additionally, this free-standing membrane is largely free of curvature stress, similarly as presynaptic plasma membrane. Assay allows monitoring ms-kinetics of secretory vesicle fusion, that is close to fusion rates observed in vivo in neuroendocrine cells. What is more, due to correct membrane topology, docked vesicles are very mobile and the mechanism of docking can be studied in great detail. The assay presented in this study is also very versatile as it can be adapted for other studies concerning for example endosomal membrane fusion or viral cell entry.
Keywords: SNARE; neurotransmission; GUV; iSCAT; microscopy; synaptic vesicle; liposome
