Pore-spanning membranes – a versatile tool to analyze SNARE-mediated single vesicle fusion
von Raphael Hubrich
Datum der mündl. Prüfung:2018-03-29
Betreuer:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Reinhard Jahn
EnglischSignal transmission in neurons and hormone secretion in neuroendocrine cells such as chromaffin cells are fundamental biological processes including SNARE- (soluble N-ethylmaleimide-sensitive-factor attachment receptor) mediated membrane fusion as the key step of exocytosis. The exact mechanism of this process, and in particular how a transient rise of the intracellular Ca2+-concentration triggers or even accelerates fusion remains unclear until today. In this work, an established in vitro fusion assay based on planar pore-spanning membranes (PSMs) was employed to analyze SNARE-mediated single vesicle fusion. The investigation focused on the impact of PI(4,5)P2 and synaptotagmin-1 (syt-1) on SNARE-mediated fusion. It was found that a PI(4,5)P2 content of 2 mol% in the t-SNAREs (syx-1A, SNAP25) harboring PSM resulted in a remarkable fusion efficiency of ~92 % of the detected v-SNARE (syb 2) containing vesicles. Moreover, the co-reconstitution of the Ca2+-sensor syt-1 into the vesicular membrane aside syb 2 resulted in a significant increase in fusion kinetics in the presence of 100 µM Ca2+. This was concluded from the finding that, under equal conditions, the docking lifetime in the absence of syt-1 was determined to be twice as long (+syt-1: τ_docking^max = 22 ± 2 s vs. –syt-1: τ_docking^max = 44 ± 1 s). Aside from synthetic vesicle fusion, SNARE-mediated fusion of natural dense core vesicles, the chromaffin granules (CGs), was investigated. CGs were isolated from bovine glands to monitor and analyze single CG fusion behavior on artificial PSMs. Interestingly, CGs showed similar fusion kinetics on PSMs as observed for synthetic vesicles. This was revealed by highly comparable distributions of the docking lifetime. However, after the onset of fusion CGs exhibited a much more diverse fusion behavior in comparison to that of synthetic vesicles. Moreover, a detailed mobility analysis of SNARE-bound CGs on PSMs was performed. CGs were found to be mobile on both parts of the PSM, the free-standing (f-PSM) and the solid supported one (s PSM), with mean diffusion coefficients of 0.34 µm2/s (f-PSM) and 0.12 µm2/s (s PSM). The model system of PSMs therefore constitutes the first planar artificial membrane on which mobility of SNARE-bound CGs was monitored and analyzed. In general, a very diverse diffusion behavior of CGs on PSMs was observed, which is in good agreement with that monitored in live chromaffin cells.
Keywords: SNAREs; PSMs; Chromaffin Granules