The long-term objective of this proposal is to elucidate mechanisms responsible for the establishment and maintenance of the polarized epithelial phenotype. Epithelial cells sort plasma membrane proteins and lipids into apical and basolateral domains in order to carry out vectorial functions for the organism. The experiments proposed will utilize the well-characterized MDCK cell system, established polarity assays and quantitative live imaging techniques to investigate fundamental aspects of the sorting of apical and basolateral plasma membrane proteins and the structure of the Trans Golgi Network (TGN).
Specific aim 1 is to characterize in detail the exit pathways from the TGN of plasma membrane proteins with different apical and basolateral sorting signals. GFP-, YFP-, CFP- tagged proteins will be used in double and triple imaging assays, together with similarly tagged Golgi markers to determine the number, kinetics of Golgi exit, type of transporting intermediates and microtubule motor requirements of individual exit pathways from the Golgi complex.
Specific aim 2 is to characterize in detail the role of adaptor protein complexes (AP1A, AP1 B, AP3, ? AP4, GGAs) and coat proteins in basolateral and apical sorting in TGN and endosomes. We will use ? biochemical targeting and recycling assays, antisense oligonucleotides, RNAi, specific inhibitors and imaging techniques to investigate the participation of these molecules in distinct apical and basolateral exit pathways from the TGN.
Specific aim 3 is to study the mechanisms involved in the selective regulation of Golgi exit pathways and surface polarity by cdc42 in MDCK cells, recently demonstrated by our laboratory. We will search for downstream effectors of cdc42 that mediate these selective trafficking and polarity alterations using mutant forms of cdc42 selectively impaired in their ability to interact with downstream effectors (IQGAP, WASP, etc). The experiments proposed will provide novel information on the molecular bases of normal and abnormal function of epithelial organs, such as kidney, liver, intestine, lung and exocrine / endocrine glands. ? ?
| Tanos, Barbara E; Yeaman, Charles; Rodriguez-Boulan, Enrique (2018) An emerging role for IQGAP1 in tight junction control. Small GTPases 9:375-383 |
| 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: |
| 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 |
| 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; 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 |
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