Quantitative Tools for Fluorescence Microscopy Applied to the Study of Single Vesicle Docking

PhD defence by Andreas H. Kunding.

Abstract

A trafficking organelle, also known as a vesicle, is a nanoscopic spherical membrane enclosing an aqueous lumen. Vesicles are of utmost importance for living cells as tools to mediate intra- and extra-cellular transport of biomaterial. Cells display several biochemically and morphologically distinct populations of vesicles, each evolved to handle specific biological tasks. Here, I will show how membrane size, an important morphological feature, can influence transport properties of a vesicle.

Applying confocal fluorecence microscopy we studied the transport dynamics of artificial phospholipid vesicles in the biological relevant size range, i.e. radii ranging between 15-150 nm. We studied in detail the binding kinetics of single vesicles undergoing docking, i.e. protein-mediated tethering of apposing membranes. This was enabled by development of several fluorescence imaging-based protocols for online quantification of docking determinants, e.g. tether-molecule membrane density and most importantly the vesicle size. In this way we found that intermembrane geometry had a profound impact on the docking efficiency, and could vary more than two orders of magnitude depending on the size of the apposing membranes. We believe this mechanism is of biological significance and might help to explain, why the cell has evolved several vesicle populations of distinct size and purpose. 

Supervisor: Dimitrios Stamou