RT Journal Article
SR Electronic
T1 Local Protein Dynamics during Microvesicle Exocytosis in Neuroendocrine Cells
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 245647
DO 10.1101/245647
A1 Agila Somasundaram
A1 Justin W. Taraska
YR 2018
UL http://biorxiv.org/content/early/2018/01/10/245647.abstract
AB Calcium triggered exocytosis is key to many physiological processes, including neurotransmitter and hormone release by neurons and endocrine cells. Dozens of proteins regulate exocytosis, yet the temporal and spatial dynamics of these factors during vesicle fusion remain unclear. Here we use total internal reflection fluorescence microscopy to visualize local protein dynamics at single sites of exocytosis of small synaptic-like microvesicles in live cultured neuroendocrine PC12 cells. We employ two-color imaging to simultaneously observe membrane fusion (using vesicular acetylcholine transporter (VAChT) tagged to pHluorin) and the dynamics of accessory proteins at the moments surrounding exocytosis. Our experiments reveal that many proteins, including the SNAREs syntaxin1 and VAMP2, the SNARE modulator tomosyn, and Rab proteins, are pre-clustered at fusion sites and rapidly lost at fusion. The ATPase NSF is recruited at fusion. Interestingly, the endocytic BAR domain-containing proteins amphiphysin1, syndapin2, and endophilins are dynamically recruited to fusion sites, and slow down the release of vesicle membrane-bound cargo. A similar effect on cargo release was seen with the over-expression of the GTPases dynamin1 and dynamin2. These results suggest that proteins involved in classical clathrin-mediated endocytosis regulate exocytosis of synaptic-like microvesicles, possibly by modulating the highly curved neck of the vesicle fusion pore. Our findings provide insights into the dynamics, assembly, and mechanistic roles of many key modulators of exocytosis and endocytosis at single sites of microvesicle fusion in live cells.AbbreviationsSVsynaptic vesicleSLMVsynaptic-like microvesicleLDCVlarge dense core vesicleTIRFtotal internal reflection fluorescence microscopyCMEclathrin-mediated endocytosis