The long-term objective of these studies is to elucidate the mechanisms by which exocyst recruitment and assembly are involved in vesicle tethering, docking and fusion. The exocyst is an octomeric protein complex (Sec3/5/6/8/10/15/Exo70/84) that spatially targets vesicles to specific sites on the plasma membrane, such as the bud tip in yeast, where it was identified in a series of seminal studies. In mammalian cells it is responsible for a range of polarized cellular processes such as cell division, migration, stabilization of junctions and formation of a primary cilium. Despite the ubiquitous and essential role of the exocyst, there are critical gaps in understanding how the exocyst 'tethering'complex and other putative tethers function to anchor vesicles and coordinate with downstream SNARE-mediated fusion machinery. While tethers are believed to involve long-range interactions, be reversible and regulate the fidelity of vesicle fusion, these concepts are generally untested and there are few assays to monitor the dynamic changes in discrete biophysical and molecular states of the exocyst. Resolving these topics will include addressing the stoichiometry of the mammalian complex, assembly and disassembly of the exocyst holocomplex, and its coordination with the fusion machinery, all of which are key goals in the proposed studies.
The specific aims of this study are: 1) to critically test the longstanding "exocyst as a tether" hypothesis and to study the dynamics of its recruitment to vesicles;2) to address how the exocyst is able to biophysically promote docking and fusion;3) to address how post-translational modifications of the exocyst affects its stability and regulates its function. These studies will provide new knowledge of how tethers function and establish a new platform to monitor their discrete states. Understanding the basic mechanisms of how the exocyst functions is highly relevant to human diseases, since its dysfunction is associated with metastasis of cancer cells, diabetes, and ciliopathies, and it is pathologically targeted by anthra toxin. A deeper understanding of how the exocyst functions as a tether and how it is regulated may help identify new molecular targets of intervention.

Public Health Relevance

The exocyst is a protein complex that targets vesicles to the cell surface and plays an essential role in cell migration, release of hormones, and cell division. As such, its dysfunction contributes to diseases including polycystic kidney disease, diabetes and cancer. Our studies will address key mechanisms of how the exocyst works and is regulated, which may ultimately lead to new therapeutic strategies for treatment of these diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM098498-01A1
Application #
8502796
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2013-09-01
Project End
2016-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
1
Fiscal Year
2013
Total Cost
$349,650
Indirect Cost
$139,650
Name
Yale University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520