Protein trafficking and epithelial adaptation to physiological stimuli are well-developed in the kidney collecting duct, where intercalated cells (IC) and principal cells (PC) show chronic and acute responses that include: a) regulation of gene and protein expression; b) alteration of the composition of the plasma membrane by vesicle trafficking. We will use novel transgenic mice that express EGFP or Beta-galactosidase in PC or IC, and new technology including laser capture microdissection and mass spectrometry to dissect epithehal remodeling (in PC and IC) and V-ATPase trafficking (in IC) at three levels.
Our aims are: 1) To examine cell-specific gene expression, protein expression and cell """"""""plasticity"""""""" in rodent collecting ducts during epithelial remodeling. Remodeling, induced by acetazolamide treatment (carbonic anhydrase inhibition) and acid/base manipulation, could occur by phenotypic switching among epithelial cells and/or by apoptotic cell loss and/or cell division. We will explore these mechanisms using immunocytochemistry, laser capture microdissection, real-time PCR, Western blotting and in situ hybridization to follow changes in the expression and localization of cell-specific proteins and mRNA (V-ATPase subunit, AE1, AE4, NHERF, AQP2, AQP4, V2R) during cell remodeling. 2) To examine vesicular pathways involved in the acute regulation of V-ATPase trafficking in IC. We propose that different pathways of protein targeting exist in A-IC and B-IC, that transcytosis is responsible for the heterogeneous distribution of V-ATPase in B-IC, and that VATPase endocytosis occurs via a novel molecular mechanism. We will: a) identify vesicle trafficking pathways in subtypes of IC using specific cell markers coupled with quantification of FITC-dextran and HRP endocytosis, b) determine the effect of acute acid-base manipulations on exocytosis, endocytosis and transcytosis, c) examine the proteome of purified V-ATPase transporting vesicles from rat kidney using 2D Gel electrophoresis and mass spectrometry; d) examine the function of identified """"""""accessory"""""""" proteins on V-ATPase trafficking by transfection of cultured IMCD cells. 3) To elucidate the role of the PDZ protein NHERF in V-ATPase trafficking and function: Based on our finding that the 56 kD B 1 V-ATPase subunit binds to the PDZ protein NHERF, which is concentrated in B-IC, we will: a) characterize the site of interaction of NHERF-GST fusion proteins with the VATPase complex; b) examine the expression, localization and rearrangement of NHERF, ezrin, actin and the VATPase in IC under different acid/base conditions in vivo; c) express mutant constructs of NHERF in IMCD cells in culture by transfection and TAT-mediated protein transfer to determine their effect on polarized trafficking and function of the V-ATPase, using immunocytochemistry, membrane fractionation and self-referencing, proton-selective microelectrodes; d) use isolated cortical and medullary endosomes to determine the effect of PDZ proteins on V-ATPase function (by fluorimetry) and V-ATPase structure (by freeze-fracture electron microscopy). Together, these studies represent a multidisciplinary approach to examine epithelial acute and chronic adaptive responses of epithelia that will be relevant not only to renal physiology, but also to cell physiology in general and pathophysiology as more and more """"""""diseases of protein trafficking"""""""" are uncovered.
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