Analysis of SNARE protein spatio-temporal dynamics during exocytosis in living cells

All cells in higher organisms have a requirement to move membranes and proteins around, whilst identifying and maintaining the compartments the various factors are in. A final step in this process may be secretion, or exocytosis, where specialised cells can export hormones (like insulin) or neurotransmitters (like serotonin) to the outside of the cells, thus enabling inter-cell communication. It has been known for some time that all these processes are mediated by specialised proteins in the cell; this was defined in the test-tube, and the protein players identified. Until recently, however, it has not been possible to identify where and when these proteins interact
with one another inside cells. Microscopy has been limited to the simple observational process of seeing where things are. My laboratory, and many others, have helped develop functional imaging , where microscopes can be used not only for seeing things, but looking at their functions. In particular, I have techniques for visualising proteins as they interact with partners inside living cells. These approaches will be used to bridge the gap between our biochemical knowledge from the test-tube, and what actually happens in the cell during secretion.

Mammalian regulated exocytosis requires an essential set of proteins; the plasma membrane tSNAREs syntaxin and SNAP-25 and the vesicle resident vSNARE synaptobrevin. The interactions between these proteins have been well characterised in vitro. However, the cellular temporal and spatial organisation of these interactions remains unknown.
In order to address this, we will determine the following, using a variety of complimentary techniques, in living neuroendocrine cells in real time:

1. What is the spatio-temporal organisation of the binary tSNARE complex in cells?
2. Do SNARE cluster heterogeneities influence docking site selection?
3. How are the SNARE proteins arranged when an LDCV is present?
4. How are SNARE spatio-temporal interactions influenced by accessory proteins?

In this project we aim to deliver a detailed description of tSNARE protein dynamic interactions in living cells, before and after the moment of vesicle fusion, and examine any effects accessory proteins may have on these interactions. These data are essential to further our understanding of exocytosis.