Professor Purdue University West Lafayette, Indiana
Body of Abstract: Exocytosis is the process for delivery of new membrane, membrane proteins, and extracellular matrix components to the plasma membrane (PM) and cell wall, and encompasses packaging of cargo into secretory vesicles as well as their transport, tethering, docking and fusion with the PM. The conserved exocyst complex facilitates the tethering of secretory vesicles to the PM, while also functioning in autophagy and defense response. Although much remains to be discovered about how this complex assembles and functions in plants, the lipid-binding subunit EXO70 was reported as a landmark for exocyst targeting to the PM. A small molecule, Endosidin2 or ES2, was previously identified as an exocyst complex inhibitor that binds to the EXO70A1 subunit and disrupts the polarized distribution of EXO70A1 in root epidermal cells. Despite this evidence, the molecular mode of action of ES2 remains ambiguous. ES2 could affect EXO70 recruitment to the PM by interfering with its lipid-binding activity. Alternatively, ES2 could alter assembly of the full exocyst complex. To inform the use of ES2 as an exocytosis inhibitor, we need to understand its mode of action at a molecular and cellular level. Using PM enrichment and proteomics approaches, we profiled the PM protein abundance change after short-term ES2 treatment. Using high spatiotemporal resolution live-cell imaging, we quantified the dynamic behavior of exocyst foci at the PM. Preliminary data suggest that the density, rate of appearance and lifetime of stationary foci of EXO70A1 were altered by short-term ES2 treatments. Additionally, we will test whether the dynamic behavior of other exocyst subunits is altered by ES2. Finally, we used in silico docking and protein-lipid overlays to investigate whether the lipid-binding activity of EXO70 is altered. Collectively, we expect to characterize the mode of action of ES2 as a useful exocytosis inhibitor and tool for chemical genetics.