Single Vesicle Investigation of Biological Membrane Fusion Reactions by Fluorescence Microscopy

PhD defence by Sune M. Christensen.

Abstract

Membrane fusion is an ubiquitous process in cellular biology. Detailed understanding of membrane fusion reactions is important both for understanding the principles of life and for preventing disease. This thesis is focused on membrane fusion in synapses, the process that facilitates nerve signaling. Vesicle and target membrane localised neuronal fusion promoters (SNAREs) were reconstituted in different vesicle populations together with the Ca²⁺ sensor synaptotagmin-1. One vesicle population was immobilised at a surface and allowed to react with a freely diffusing vesicle population thereby allowing the monitoring of docking and fusion between individual vesicle pairs by time resolved fluorescence microscopy. Quantification of docking probabilities revealed that target membrane and vesicle curvature regulates the efficiency of the docking reaction, thus hinting at a hitherto unidentified regulation mechanism for biological fusion reactions. Vesicle fusion was found to take place via several kinetically distinct modes distinguished by subsecond to minute scale time constants. Surprisingly, the results show that only about 60 % of the vesicles in the assay bound a partner during the experiments. Together with the fusion data this observation highlights pronounced heterogeneous behaviour of the model system for neuronal membrane fusion. To investigate the origin of this heterogeneity we studied the binding of synaptotagmin-1 (C2AB) to individual vesicles. This study showed that Ca²⁺, contrary to common consensus, does not change the density of C2AB on the vesicles membrane but changes the number of vesicles that admit protein. Since C2AB was found to be the major determinant for intermembrane tethering this result explains why not all vesicles bound a partner in the docking and fusion assay. Ca2+-triggered recruitment of C2AB to membranes is believed to be the trigger of membrane fusion in neurons and the data thus provides a possible molecular explanation for the well-documented heterogeneity in release probability among synaptic vesicles in a synapse. Finally, founded on the established single vesicle fusion assay we developed a platform for highly parallel mixing of selfenclosed zepto- to femto-litre (10⁻¹⁵ l - 10⁻²¹ l) aliquots, a regime inaccessible to present state of the art fluidic techniques. The platform, named Zeptofluidics, was systematically characterised using lipid mixing and content mixing assays and a proof of principle for its potential in biotechnological applications was achieved by demonstrating the triggering of an enzymatic reaction. We believe that Zeptofluidics exemplifies the coming of age of self-assembly based technologies and will provide solutions for future applications demanding extreme miniaturisation. 

Supervisor:
Dimitrios Stamou, stamou@nano.ku.dk, +45 35 32 04 79