The proposed studies will continue the investigation of the molecular characterization of membrane skeleton and tight junctions (TJs) of renal epithelial cells. Two specific experimental objectives will be pursued. The first will be to initiate studies on the molecular and functional characterization of the myosin family of actin-based molecular motors expressed in the proximal tubule epithelial (PTE) cells. Analysis will begin with the characterization of myosin-VI, a newly discovered class of unconventional myosin implicated in organelle transport that is expressed in relatively high levels in the PTE cell. A full length cDNA encoding pig myosins-VI during cell-contact induced differentiation of CL4 cells will be analyzed. Myosin-VI will be purified and biochemically characterized. Experiments are proposed to identify """"""""docking proteins"""""""" which may link myosin-VI to the membrane. Finally, in an effort to determine the function of myosin-VI in PTE cells, stable CL4 lines will be created which overexpress myosin VI tail truncates or lack myosin-VI due to anti-sense transcript expression.
Our second aim i nvestigates molecular mechanisms of renal TJ regulation. This structure and its podocyte variant, the slit diaphragm, form the paracellular barrier and maintains cell polarity. Barrier properties are regulated during normal physiology and are altered in renal pathology. Evidence suggests protein kinases regulate assembly, disassembly and barrier properties of renal TJs. We will test whether the locus of regulation is phosphorylation of TJ-specific proteins. MDCK cells, a renal tubular model, lack intercellular junctions and polarity when cultured at low Ca++; junctional assembly and establishment of transmonolayer electrical resistance is induced by raising extracellular Ca++. The phosphorylation sate of four TJ proteins will be correlated with changes in their localization during TJ assembly, as monitored by monolayer electrical resistance during this Ca++ switch. Effects of protein kinase inhibitors and agonists on phosphorylation, structural and physiologic parameters will be examined. We recently observed that different isoforms of the TJ protein ZO-1 are expressed in renal tubular epithelial cells vs. renal endothelial cells and podocyte junctions. This distribution correlates with structural and functional differences. Pilot studies will examine if ZO-1 isoform expression is aberrant in human renal diseases and contributes to altered glomerular and tubular barrier function.
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