Molecular Analysis of Eukaryotic Membrane Secretion
The process of secretion (or exocytosis) involves the fusion of cargo-containing vesicles with the plasma membrane and is a fundamental property of all eukaryotic cells. In higher organisms this mechanism has evolved to provide the highly regulated release of neurotransmitters in the brain and hormones such as adrenaline and insulin. This process is targeted by many toxins, including clostridial neurotoxins, and is also deficient in a number of disease states. Our research is focused on understanding at the molecular level how this highly orchestrated process operates and what happens when this process goes wrong.
1. Spatial Organisation of the Fusion Machinery
The process of membrane fusion is catalysed by the SNARE proteins. This highly conserved protein family mediates the fusion of membrane-bound compartments in all eukaryotic cells. These proteins have been proposed to provide the energy to drive membrane merger in the final steps of membrane fusion. In humans, regulated secretion occurs in highly specialised regions of cells, epitomised by the localised fusion of synaptic vesicles at the active zone of a synapse. We are investigating the spatial organisation of the SNARE fusion machinery from the whole cell to the single molecule level. To examine this we are using advanced optical bio-imaging techniques including the super-resolution PALM technique, which allows the observation of thousands of single proteins at the plasma membrane. By examining the SNAREs and other components of the release machinery (ion channels and accessory proteins) we aim to generate a molecular map of these proteins and uncover the determinants of their spatial organisation.
Figure 1. Super-resolution microscopy of SNAREs. Standard resolution microscopy of the base of a secretory cell (left). The region highlighted is shown using the PALM technique revealing the position of individual SNARE proteins (right).
2. SNARE Protein Regulators
In neuronal and neuroendocrine cells, the release of cargo is highly regulated. We are interested in how SNARE accessory proteins (synaptotagmin, complexin, munc18 NSF and small GTPases) can regulate this process from the single molecule to the system-wide level. We are investigating the role of these proteins both in vitro, using highly purified protein components, and also in a cellular environment using advanced microscopic techniques. Many of these investigations utilise Förster resonance energy transfer (FRET) to report with high spatial sensitivity changes in protein interactions and conformations. Through these studies we aim to uncover the impact of these accessory proteins on SNARE protein interactions and the process of membrane fusion.
Figure 2. Model of SNARE and accessory protein interactions preceding membrane fusion.