This REVISED proposal seeks to explore the role of intracellular Ca++ in the development of cell-cell contact, junctions and apical-basolateral polarity in kidney epithelia. The """"""""Ca++ switch,"""""""" an MDCK cell model for developmental events in early nephrogenesis, will be employed. Preliminary studies (NEW DATA) suggest that mechanisms regulating intracellular Ca++, as well as signalling events activated by changes in intracellular Ca++, may be important in junction formation and polarization. Based on the preliminary data, it is hypothesized that global and/or local changes in intracellular Ca++ mediate the development of cell-cell contact, assembly of junctions (tight and desmosomal) and apical-basolateral polarization in the MDCK cell Ca++ switch model. Studies proposed seek to establish: 1) the mechanism of intracellular Ca++ rise, fall and stabilization during Ca++ switch (using digital imaging, single cell spectrofluorimetric measurements); 2) whether perturbation of intracellular Ca++ levels during the Ca++ switch affects the development of cell-cell contact, junctions and polarity (immunofluorescence); and 3) the role of Ca++ activated signalling events in the development of cell-cell contact, junctions and polarity (cell fractionation, binding assays). Additional experiments are proposed to continue studies aimed at elucidating the Ca++regulatory machinery which the PI has previously identified in the rough endoplasmic reticulum (RER): calcium binding proteins (CaBPs), the IP3 receptor (IP3R) and a small molecular mass GTP binding protein (SMG). These proteins regulate the IP3- sensitive intracellular Ca++ pool and may play important roles in the marked intracellular Ca++ changes observed during the MDCK cell Ca++ switch. Proposed studies will determine: 1)whether CaBPs exist as a Ca++- dependent complex in the RER (Western blots, column chromatography, isopycnic gradients); 2) whether the IP3Rs in RER and non-RER pools are functionally and structurally distinct (Cleveland digests, 2-D gels); and 3) whether a SMG in the RER is involved in Ca++ movement between the IP3- sensitive and insensitive intracellular Ca++ pools (ADP-ribosylation, Ca++ release assays). Preliminary data is provided for all sets of experiments proposed.
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