Untersuchung einzelner SNARE-vermittelter Membranfusionsereignisse auf planaren porenüberspannenden Membranen

Investigation of Single SNARE-mediated Membrane Fusion Events on Planar Pore-spanning Membranes

by Lando Lantbert Gregor Schwenen
Doctoral thesis
Date of Examination:2015-06-04
Date of issue:2015-10-20
Advisor:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Tim Salditt
Persistent Address: http://dx.doi.org/10.53846/goediss-5308

 

 

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Abstract

English

Even though there are a number of different in vitro fusion assays to analyze neuronal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) mediated fusion, still the essential features of the in vivo situation are only partially captured. In this thesis an in vitro fusion assay that provides a planar solvent-free pore-spanning membrane containing a ternary acceptor SNARE complex, which mimics the presynaptic membrane, to monitor fusion of single synaptobrevin containing vesicles is presented. Protein-containing pore-spanning membranes were obtained by spreading of giant unilamellar vesicles on gold covered6-mercapto-1-hexanol functionalized porous siliconnitride substrates with pore diameters of 1.2µm. The mobility of Lipids and the SNAREs in the pore-spanning membranes was shown by FCS-experiments, which is prerequisite for the formation of active fusion complexes. The fusion process was highly specific with an efficiency of 50 % and was analyzed by two color confocal laser scanning fluorescence microscopy in a timeresolved manner allowing to distinguish between vesicle docking, hemifusion and full fusion. A kinetics analysis revealed that two reaction steps need to take place to progress to fusion with a lifetime of the docked vesicles of about 50s.
Keywords: SNARE; soluble N-ethylmaleimide-sensitive factor attachment protein receptor; fusion; fusion assay; pore-spanning membranes; mobility of Lipids; mobility of SNAREs; porous substrates; vesicle docking; hemifusion; confocal laser scanning fluorescence microscopy; lipid membranes; surface functionalization; membrane fusion; membrane diffusion
Schlagwörter: SNARE; Membranfusion; Fusionsassay; porenüberspannende Membran; Diffusion in Lipidmembranen; konfokale Laserrastermikroskopie; Hemifusion; Oberflächenfunktionalisierung; poröse Substrate; Lipidmembranen; Vesikelfusion; Mobilität von SNARE-Proteinen
 
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