The long-term goal of this proposal is to understand the molecular mechanisms that regulate exocytosis in order to comprehend and develop therapies for devastating human diseases in which exocytosis is defective, such as diabetes and polycystic kidney disease. The overall objective of this work is to dissect the spatio-temporal regulation of exocytosis at a molecular level during development of epithelial cell polarity. This proposal focuses on three components of the trafficking machinery: the Exocyst (a putative vesicle tethering factor), Munc18c (a regulator of tethering/fusion machinery), and Syntaxin 4 (a component of the fusion machinery). Based upon results of preliminary studies, a working hypothesis has been developed for how polarized trafficking to the basal-lateral membrane is established. Cell-cell adhesion promotes assembly, movement and association of Exocyst with intercellular contact sites defined by E-cadherin-associated protein complexes; association of transport vesicles with these sites coincides with an interaction between an Exocyst subunit (Sec6) and Munc18c, and this in turn regulates the activity of Syntaxin 4, leading to membrane fusion. ? ? The specific aims of this study are to: 1) identify mechanisms that specify targeting patch assembly at sites of cell-cell adhesion; 2) determine the functional significance of Sec6 binding to Munc18c in basal-lateral membrane trafficking; and 3) determine whether conformational changes in Sec6 regulate its association with Munc18c. The research plan to achieve these aims involves a combination of cell biology, biochemistry and molecular biology approaches. The significance of these studies is that they will define signaling pathways and specific interactions that couple an extracellular spatial cue (cell adhesion) to organization of components involved in membrane trafficking (vesicle docking/fusion machinery) leading to establishment of cell polarity. In the long term these studies will provide new insights into understanding the basis for abnormalities in membrane protein organizations characteristic of epithelial diseases. Also, because this proposal focuses on how information is transferred from vesicle to plasma membrane to initiate exocytosis, it has broad implications for many processes including antigen presentation, neurotransmission and hormone secretion. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067002-05
Application #
7491032
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Flicker, Paula F
Project Start
2004-09-01
Project End
2010-08-31
Budget Start
2008-09-01
Budget End
2010-08-31
Support Year
5
Fiscal Year
2008
Total Cost
$244,750
Indirect Cost
Name
University of Iowa
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Hazelett, C Clayton; Yeaman, Charles (2012) Sec5 and Exo84 mediate distinct aspects of RalA-dependent cell polarization. PLoS One 7:e39602
Hazelett, C Clayton; Sheff, David; Yeaman, Charles (2011) RalA and RalB differentially regulate development of epithelial tight junctions. Mol Biol Cell 22:4787-800
Parrini, Maria Carla; Sadou-Dubourgnoux, Amel; Aoki, Kazuhiro et al. (2011) SH3BP1, an exocyst-associated RhoGAP, inactivates Rac1 at the front to drive cell motility. Mol Cell 42:650-61
Bodemann, Brian O; Orvedahl, Anthony; Cheng, Tzuling et al. (2011) RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly. Cell 144:253-67
Andersen, Nicholas J; Yeaman, Charles (2010) Sec3-containing exocyst complex is required for desmosome assembly in mammalian epithelial cells. Mol Biol Cell 21:152-64
Overgaard, Christian E; Sanzone, Kaitlin M; Spiczka, Krystle S et al. (2009) Deciliation is associated with dramatic remodeling of epithelial cell junctions and surface domains. Mol Biol Cell 20:102-13
Hase, Koji; Kimura, Shunsuke; Takatsu, Hiroyuki et al. (2009) M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex. Nat Cell Biol 11:1427-32
Spiczka, Krystle S; Yeaman, Charles (2008) Ral-regulated interaction between Sec5 and paxillin targets Exocyst to focal complexes during cell migration. J Cell Sci 121:2880-91
Oztan, Asli; Silvis, Mark; Weisz, Ora A et al. (2007) Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells. Mol Biol Cell 18:3978-92