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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing (MBPP)
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Ainsztein, Alexandra M
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Yale University
Anatomy/Cell Biology
Schools of Medicine
New Haven
United States
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Huet-Calderwood, Clotilde; Rivera-Molina, Felix; Iwamoto, Daniel V et al. (2017) Novel ecto-tagged integrins reveal their trafficking in live cells. Nat Commun 8:570
Rauch, Liane; Hennings, Kirsten; Trasak, Claudia et al. (2016) Staphylococcus aureus recruits Cdc42GAP through recycling endosomes and the exocyst to invade human endothelial cells. J Cell Sci 129:2937-49
Xu, Yingke; Nan, Di; Fan, Jiannan et al. (2016) Optogenetic activation reveals distinct roles of PIP3 and Akt in adipocyte insulin action. J Cell Sci 129:2085-95
Deng, Yongqiang; Rivera-Molina, Felix E; Toomre, Derek K et al. (2016) Sphingomyelin is sorted at the trans Golgi network into a distinct class of secretory vesicle. Proc Natl Acad Sci U S A 113:6677-82
Rivera-Molina, Felix; Toomre, Derek (2013) Live-cell imaging of exocyst links its spatiotemporal dynamics to various stages of vesicle fusion. J Cell Biol 201:673-80