Protein 4.1 R (4.1 R) was originally identified as an essential component of the red cell membrane cytoskeleton. We have shown that 4.1R plays a role in epithelial morphogenesis and endothelial cell-to-cell junction assemblies. We now propose to characterize the contributions of 4.1 R to tight junction (TJ) biogenesis and membrane polarity in epithelial cells, as well as to adherens junction (AJ) assembly and integrity in endothelial cells. The localization of 4.1 R during epithelial morphogenesis shifts from the nucleus and cytoplasm of non-confluent cells to the lateral membrane of confluent cells, in conjunction with a switch from 4.1R 135 kD, lacking exon 17b, to 4.1 R 160 kD containing exon 17b. In non-confluent nuclei, 4.1R associates with ZO-2. In confluent cells, 4.1R associates with the polarity protein Par3, TJs, and AJs. Dominant negative mutant 4.1R's disrupt TJ integrity and produce an enlarged and flattened morphology. The behavior of 4.1 R during epithelial morphogenesis will be studied in both a model MDCK line and primary epithelial cultures. We shall dissect the mechanisms by which 4.1R associates with partner proteins to form polarized membranes and TJ's using in vitro and in vivo biochemical assays, and fluorescence-based imaging methods. Functional perturbations will be achieved by utilizing siRNA, dominant-negative mutants, and a Tet-off expression system. In AJs, 4.1R co-localizes and associates with the AJ proteins E-cadherin and beta-catenin. Overexpression of 4.1 R reduces E-cadherin localization to the AJs. In order to study AJ's in isolation, we shall use a well-characterized human umbilical vein endothelial cell (HUVEC) primary cell culture. Since HUVEC rarely express TJs, they are more suitable for analyzing the expression and sub-cellular distribution of 4.1 R and proteins with which it associates as they assemble into AJs. We shall delineate the impact of 4.1 R on AJs assembly, using approaches similar to those just described. We shall also test the hypothesis that complementarity may exist between 4.1R and its paralogues (4.1G, N, B), allowing them to compensate for the absence of 4.1 R in knock-out cells. Finally, we shall investigate the relationship between the shift in localization of 4.1 R from nucleus to peripheral membranes and the establishment of contact inhibition in terminally differentiating epithelial cells. These studies should yield new insights about the function of 4.1Rin cellular architecture and achievement of the post-mitotic contact inhibited state. ? ? ?
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