Super-resolution molecular microscopy of hormonal secretion

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. However, due to the inherent limits in the resolution of the optical techniques used in these studies, the molecular organisation of the SNARE proteins in the plasma membrane have remained elusive.
We have employed 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 have developed and applied new processing and analysis techniques to quantify the molecular organisation. Using a combination of GSDIM (ground state depletion followed by individual molecule return) and PALM (photoactivation localisation microscopy), under TIRF illumination, we were able to determine the location of typically 50,000 SNARE proteins in each cell, with a precision of between 5 to 10 nm. For both GSDIM 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 SNARE PALM data using Ripley’s K function demonstrated a non-random distribution, characteristic of clustering. Performing PALM experiments in live cells, followed by automated denoising and particle tracking (sptPALM), allowed us to quantify the movement of tens of thousands of individual SNARE proteins with a lateral precision of ~20 nm. We have analysed this data in a number of ways, both as a population examining spatial trends and at the level of individual tracks, to uncover the properties governing the molecular movement of the SNARE proteins. Spatial analysis of track density over the plasma membrane demonstrated a non-random distribution, with hot-spots of SNARE protein movement. At the individual track level, the SNARE proteins exhibited a caged motion, indicative of a preference, or exclusion, from distinct areas of the plasma membrane. Together these data provide a molecular map of the organisation of the SNARE secretory machinery at the plasma membrane.