SNARE-Mediated Fusion of Single Liposomes
Intra-, inter- and extracellular processes of membrane fusion are of vital importance for all biological organisms. Most intracellular fusion events in eukaryotic cells are mediated by SNARE proteins. However, despite intensive investigations important aspects of the biophysical mechanisms governing these regulating processes are still poorly understood. In this project we collaborate with the research group of Professor Reinhard Jahn to investigate the mechanism of SNARE-mediated fusion of liposomes in a systematic manner. SNAREs can be reconstituted into liposomes and it has been shown that these liposomes undergo fusion in a manner that shares characteristics with biological fusion reactions. By combining confocal single particle detection of differently labelled liposomes with fluorescence life-time measurements of single liposomes we are able to distinguish and determine the absolute population of free as well as docked, hemifused and completely fused liposomes. Additionally, an analysis of the burst brightness allows determining the number of fusion rounds (fusion of more than two liposomes) as well as potential docking or fusion reactions of exclusively red- or green-labelled liposomes. The thorough information obtained from the single particle analysis will provide important details about mechanistic details and the time course of fusion reactions.
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Left: confocal set-up for the detection and fluorescence life-time analysis of single liposomes diffusing through the focal detection volume by pulsed two-photon excitation. Right: schematic representation of fluorescence bursts originating from single liposomes in the case of a) free liposomes, b) docked liposomes, c) hemi-fused liposomes and c) completely fused liposomes.
For details see:
D. Zwilling, A. Cypionka, W. Pohl, D. Fasshauer, P. J. Walla, M. Wahl, R. Jahn, „Early endosomal SNAREs form a structurally conserved SNARE complex and fuse liposomes with multiple topologies”, EMBO Journal 26 (1), 9-18, (2007).