Over 150 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 molecular sorting mechanisms that regulate the trafficking of molecules essential for epithelial polarity and kidney function/pathobiology. The proposed experiments follow up on our group's recent findings that clathrin plays a broad role of clathrin in basolateral protein sorting and that the ubiquitous (AP-1A) and epithelial-specific (AP-1B) clathrin adaptors carry out complementary basolateral sorting functions in MDCK cells. As we have shown that kidney proximal tubule (KPT) lacks the basolateral sorting adaptor AP-1B, Specific aim 1 will investigate in detail the roles of clathrin adaptors AP-1A, AP-3, AP-4 and GGAs in basolateral sorting in KPT using a domain-selective biotinylation, mass spectroscopy and biochemical and live-imaging trafficking assays.
Specific aim 2 will investigate a novel role of clathrin and clathrin adaptors in the apical sorting of Megalin. We have recently described the complete apical recycling pathway of Megalin and our preliminary data suggest that the apical localization of this protein requires clathrin. Specific am 3 will investigate the mechanisms involved in the basolateral sorting of E-cadherin, a fundamental epithelial polarity protein. We will follow up on preliminary data indicating that the basolateral sorting of E-cadherin depends on both AP-1A and AP-1B in the recycling but not in the biosynthetic route, to search for novel TGN and endosomal basolateral sorting mechanisms that depend on sorting signals in the tightly associated protein beta-catenin.
Specific aim 4 will characterize the sorting mechanisms of copper transporters A7P7A and ATP7B, highly similar molecules which reside in the TGN in low copper and are released to the PM in low copper. The studies proposed will contribute to a deeper understanding of fundamental epithelial trafficking mechanisms and human (including kidney) diseases associated with malfunction of the molecules under study in this proposal.

Public Health Relevance

Human organs contain over 150 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 #
5R01GM034107-34
Application #
9432520
Study Section
Kidney Molecular Biology and Genitourinary Organ Development (KMBD)
Program Officer
Flicker, Paula F
Project Start
1984-02-01
Project End
2020-02-28
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
34
Fiscal Year
2018
Total Cost
Indirect Cost
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
Benedicto, Ignacio; Lehmann, Guillermo L; Ginsberg, Michael et al. (2017) Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors. Nat Commun 8:15374
Caceres, Paulo S; Benedicto, Ignacio; Lehmann, Guillermo L et al. (2017) Directional Fluid Transport across Organ-Blood Barriers: Physiology and Cell Biology. Cold Spring Harb Perspect Biol 9:
Perez Bay, Andres E; Schreiner, Ryan; Benedicto, Ignacio et al. (2016) The fast-recycling receptor Megalin defines the apical recycling pathway of epithelial cells. Nat Commun 7:11550
Tanos, Barbara E; Yeaman, Charles; Rodriguez-Boulan, Enrique (2016) An emerging role for IQGAP1 in tight junction control. Small GTPases :1-9
Tanos, Barbara E; Perez Bay, Andres E; Salvarezza, Susana et al. (2015) IQGAP1 controls tight junction formation through differential regulation of claudin recruitment. J Cell Sci 128:853-62
Song, Minseok; Giza, Joanna; Proenca, Catia C et al. (2015) Slitrk5 Mediates BDNF-Dependent TrkB Receptor Trafficking and Signaling. Dev Cell 33:690-702
de la Fuente-Ortega, Erwin; Gravotta, Diego; Perez Bay, Andres et al. (2015) Basolateral sorting of chloride channel 2 is mediated by interactions between a dileucine motif and the clathrin adaptor AP-1. Mol Biol Cell 26:1728-42
Bay, Andres E Perez; Schreiner, Ryan; Rodriguez-Boulan, Enrique (2015) Structural and functional analysis of endosomal compartments in epithelial cells. Methods Cell Biol 130:271-88
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
Rodriguez-Boulan, Enrique; Macara, Ian G (2014) Organization and execution of the epithelial polarity programme. Nat Rev Mol Cell Biol 15:225-42

Showing the most recent 10 out of 110 publications