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.

Public Health Relevance

The exocyst complex is needed for cell morphology changes, growth, neuronal development, primary cilia formation, and cell communication. Specific mutations of exocyst subunits have been associated with certain diseases, most notably, ciliopathies such as Joubert Syndrome and Meckel-Gruber syndrome (Exo84 and Sec8 respectively), while overexpression of exocyst subunits has been linked to certain types of cancers. By elucidating exocyst structural information and mechanisms of action, therapeutic strategies can be devised to alleviate overactive exocyst or rescue exocyst defects resulting from mutations.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM123704-01
Application #
9328745
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Sakalian, Michael
Project Start
2017-03-12
Project End
2020-03-11
Budget Start
2017-03-12
Budget End
2018-03-11
Support Year
1
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Lepore, Dante M; Martínez-Núñez, Leonora; Munson, Mary (2018) Exposing the Elusive Exocyst Structure. Trends Biochem Sci 43:714-725