The molecular organisation of regulated secretion using super-resolution microscopy

Intercellular communication is commonly mediated by the regulated fusion, or exocytosis, of cargo-containing vesicles with the plasma membrane. Although this process occurs in a diverse range of specialised cell types, for example neurons and neuroendocrine cells, the underlying core protein machinery is highly conserved. SNARE proteins are known to actively mediate the fusion of the secretory vesicle and plasma membranes, and have been observed using fluorescence microscopy to form distinct clusters on the plasma membrane.

We are using super-resolution molecular imaging to observe the architecture and dynamics of large cohorts of SNARE proteins in intact cells. These approaches are high-content in nature and we develop and apply new processing and analysis techniques to quantify their molecular organisation. Using a combination of dSTORM (direct stochastic optical reconstruction microscopy) and PALM (photoactivation localisation microscopy), under TIRF illumination, we can determine the location of typically 50,000 SNARE proteins in each cell, with a precision of between 5 to 10 nm. For both dSTORM and PALM we observed that the SNARE proteins form a heterogeneous distribution across the bilayer plane, exhibiting areas of high and low molecular density. Quantitative analysis of this distribution indicates it is non-random in nature with a degree of clustering. Performing PALM experiments in live cells, followed by automated denoising and particle tracking (sptPALM), allows us to quantify the movement of tens of thousands of individual SNARE proteins with a lateral precision of ~20 nm. Together these data provide a molecular map of the organisation of the SNARE secretory machinery at the plasma membrane.