Over 100 different types of epithelial cells perform essential vectorial functions required for homeostasis and survival of the organism. A key property underlying these functions is polarity, i.e. the ability of epithelial cells to localize different transporters, channels and hormone receptors to opposite (apical and basolateral) domains of their plasma membrane (PM). The experiments proposed will utilize state of the art molecular, biochemical and microscopic techniques to elucidate fundamental mechanisms involved in epithelial polarization. The proposed experiments follow up on our group's recent finding that clathrin plays a broad role of clathrin in basolateral protein sorting and that some epithelia lack the basolateral sorting adaptor AP-1B.
Specific aim 1 will investigate in detail the roles of clathrin adaptors AP-1A and AP-1B, two major basolateral sorting adaptors, in carrier assembly by quantitative 4D 3-color live imaging analysis. We will search for trafficking machinery engaged by AP-1A and AP-1B using a proteomics approach developed by Bernard Hoflack (Dresden).
Specific aim 2 will investigate the role of clathrin adaptors AP-2, AP-3, AP-4 and GGAs in polarized trafficking, using siRNA and ad hoc trafficking assays. The experiments will focus both on MDCK cells and on kidney proximal tubule epithelium, which lacks AP-1B.
Specific aim 3 will investigate the role of Golgins and rabs in polarized trafficking, combining Golgin knock-down and inhibition of candidate rabs with trafficking assays for a panel of apical and basolateral cargos. We will test the hypothesis that TGN Golgins, elongated coil-coil molecules that bind membranes via rabs, mediate highly dynamic physical interactions between TGN and recycling endosomes that are required for efficient polarized sorting.
Specific aim 4 will characterize the specific steps regulated by rabs 6,8,10, 11,17,25 in polarized trafficking, using ad hoc assays to explore these steps. The studies proposed will contribute to a deeper understanding of epithelial trafficking mechanisms, which, in turn, is likely to help understand and develop cures for human diseases.

Public Health Relevance

Human organs contain over 100 different types of epithelial types, which perform vital functions for the organism that depend on a fundamental property, polarity. The long term objective of this proposal is to elucidate the molecular mechanisms responsible for the establishment and maintenance of epithelial cell polarity. Because loss of polarity may cause cancer and diseases of the kidney, liver and other epithelial organs, the experiments proposed are bound to generate information relevant to preventing and curing human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034107-30
Application #
8628837
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Ainsztein, Alexandra M
Project Start
1984-02-01
Project End
2016-02-29
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
30
Fiscal Year
2014
Total Cost
$445,315
Indirect Cost
$181,815
Name
Weill Medical College of Cornell University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Thuenauer, Roland; Hsu, Ya-Chu; Carvajal-Gonzalez, Jose Maria et al. (2014) Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates. Proc Natl Acad Sci U S A 111:4127-32
Perez Bay, Andres E; Schreiner, Ryan; Benedicto, Ignacio et al. (2014) Galectin-4-mediated transcytosis of transferrin receptor. J Cell Sci 127:4457-69
Rodriguez-Boulan, Enrique; Macara, Ian G (2014) Organization and execution of the epithelial polarity programme. Nat Rev Mol Cell Biol 15:225-42
Lehmann, Guillermo L; Benedicto, Ignacio; Philp, Nancy J et al. (2014) Plasma membrane protein polarity and trafficking in RPE cells: past, present and future. Exp Eye Res 126:5-15
Thuenauer, Roland; Rodriguez-Boulan, Enrique; Römer, Winfried (2014) Microfluidic approaches for epithelial cell layer culture and characterisation. Analyst 139:3206-18
Rodriguez-Boulan, Enrique; Perez-Bay, Andres; Schreiner, Ryan et al. (2013) Response: the "tail" of the twin adaptors. Dev Cell 27:247-8
Perez Bay, Andres E; Schreiner, Ryan; Mazzoni, Francesca et al. (2013) The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP-1B-deficient epithelia. EMBO J 32:2125-39
Carvajal-Gonzalez, Jose Maria; Gravotta, Diego; Mattera, Rafael et al. (2012) Basolateral sorting of the coxsackie and adenovirus receptor through interaction of a canonical YXXPhi motif with the clathrin adaptors AP-1A and AP-1B. Proc Natl Acad Sci U S A 109:3820-5
Lengfeld, Justin; Wang, Qiwei; Zohlman, Andrew et al. (2012) Protein kinase C ýý regulates the release of collagen type I from vascular smooth muscle cells via regulation of Cdc42. Mol Biol Cell 23:1955-63
Castorino, John J; Deborde, Sylvie; Deora, Ami et al. (2011) Basolateral sorting signals regulating tissue-specific polarity of heteromeric monocarboxylate transporters in epithelia. Traffic 12:483-98

Showing the most recent 10 out of 98 publications