Master thesis by Søren Andersen – Niels Bohr Institute - University of Copenhagen

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Master thesis by Søren Andersen

Cells are surrounded by membranes and contain various intracellular membranous structures such as the golgi apparatus, the nucleus and vesicles, all of which are spatially defined by a membrane. These membranes have a characteristic curvature, i.e. rather flat for the extracellular membrane and highly curved for smaller vesicles, like synaptic vesicles. The cell contains proteins involved in regulating the curvature of membranous structures. Studies on membrane curvature sensing proteins, such as the BAR (Bin/Amphiphysin/RVS) superfamily, have been performed with artificial liposomes and mostly by bulk assays. However, the artificial liposomes lack the membrane proteins associated with cellular membranes. Clearly, it would be of interest to study curvature sensing proteins with biological membranes.

Here we describe a new assay using single native PC12 cell vesicles. Curvature sensing and fractional binding of the neuronal F-BAR-domain containing syndapin I are demonstrated. To demonstrate curvature sensing, a method is developed whereby the native vesicle sizes are determined from their gaussian width by application of a standard curve derived from fluorescent nm-sized beads. It is demonstrated that the SH3 domain of syndapin I negatively regulates membrane interaction. An amphipatic helix shows comparable curvature sensing as compared to the syndapin constructs. Native vesicles show low fractional binding. In comparison, artificial liposomes bind 10-15 times more syndapin I than native vesicles indicating that few sites are available for binding on native vesicles.

Master thesis by Søren Andersen