Elucidation of exocyst tethering function by single molecule assays and EM
Dante Lepore | Munson Lab | F32 Award
The packaging, transport, recognition, and fusion of vesicle-enclosed cargo is an essential hallmark of eukaryotes, and must be tightly regulated for basic cellular organization. A key step in vesicular trafficking is a tethering event where vesicles are attached to their pre-determined target membrane prior to SNARE mediated fusion by tethering complexes. Multi-subunit tethering complexes (MTCs) are one such categorization of conserved tethering proteins, and are required for a majority a membrane trafficking steps within the cell. However, despite their name, there is a dearth of biochemical evidence showing the capacity of these MTCs to recruit and hold vesicles to a target membrane prior to fusion. Thus, I propose a series of experiments to reconstitute vesicle tethering in vitro using post-Golgi secretory vesicles and the MTC exocyst to definitively observe the proposed tethering activity of the complex and to gain structural and mechanistic insights into tethering events. To accurately and sensitively observe vesicle tethering, we seek to employ a single molecule assay TIRF microscopy based assay to observed individual tethering events and changes to exocyst conformations in real-time. Furthermore, we seek to gain structural insights into the overall architecture of the exocyst complex and the binding sites for various proteins that are thought to participate in the tethering event using negative stain electron microscopy techniques. The information gained from these experiments will not only determine the proposed tethering activity of exocyst and serve as a platform for other MTCs to be tested, but reveal mechanism details of the poorly understood, but essential tethering set in membrane trafficking.
