Temporally Resolving Protein and Lipid Colocalization at Exocytic Sites in INS-1 Cells

Abstract

The controlled release of material from cells, called exocytosis, is critical for many life processes ranging from synaptic transmission to hormone secretion. Exocytosis is mediated by the assembly of SNARE proteins into a complex that links vesicular and plasma membranes and promotes membrane fusion. Despite a detailed understanding of SNARE complex assembly, a comprehensive description of the arrival and departure of the myriad of other proteins involved in exocytosis is lacking. Here, we describe the localization and dynamics of over a dozen proteins at sites of dense core vesicle exocytosis inside living INS-1 cells. We use two color total internal reflectance fluorescence microscopy to visualize transiently transfected, fluorescently tagged proteins. NPY-GFP, a dense core vesicle cargo protein, is used to localize vesicles and temporally align many exocytic events across several cells. mCherry is used to localize a particular exocytic protein of interest relative to the dense core vesicle and determine its temporal behavior relative to the moment of fusion. Intriguingly, we find that both positive and negative regulators of SNARE complex assembly appear to be present at exocytic sites and diffuse away after fusion occurs. This data suggests that effectors acting on the SNARE complex must be biochemically or conformationally regulated; they are not spatially regulated by exclusion or inclusion at exocytic sites. We are currently examining one exocytic protein, tomosyn, to determine how this protein might be regulated. We are also investigating lipid dynamics at the site of exocytosis using fluorescently tagged lipid binding domains. Our imaging approach should show whether critical lipids are locally synthesized or laterally diffuse to the site of exocytosis. With our data we seek to build a thorough description of protein and lipid behavior at the moment of exocytosis.