Proposed studies will continue the characterization of the cytoskeletal apparatus which underlies and supports the brush border (BB) membrane of the intestinal epithelial cell. There are 2 main objectives: 1. to continue the characterization of the molecular properties of key BB cytoskeletal proteins. Studies will continue ongoing analysis of 110K-calmodulin (110K-CM), the protein complex which links the microvillus (MV) actin bundle to the plasma membrane, and which has properties similar to the mechanoenzyme, myosin. Studies will investigate the functional properties of the calmodulin in the complex, assess the mechanochemical potential of 110K-CM and determine the molecular basis for the interaction of 110K-CM with the membrane. Collaborative studies to sequence the gene encoding human 110K will be continued. We will also continue ongoing studies on the molecular and functional characterization of spectrin (a major component of the terminal web region of the BB) and spectrin binding proteins present in the intestinal epithelial cells of both avian and mammalian species. This will include the characterization of proteins which regulate the interaction of avian BB spectrin with actin as well as the characterization of proteins of the mammalian BB which are immunologically related to protein 4.1 - a protein of the erythrocyte membrane skeleton which effects the association of actin with spectrin. 2. to continue studies on the characterization of BB assembly. This will include continued analysis of the expression and distribution of proteins of the MV core, including villin, fimbrin and the 110K-CM during embryogenesis in the chick. In addition we will initiate a collaborative study on the analysis of BB assembly in the HT-29 cell line. These cells, derived from a human colonic adenocarcinoma, can be induced to differentiate into a BB-containing enterocyte-like cell by simple manipulation of the culture medium. Initial studies will include the identification of BB proteins present in these cells, including immunochemical and localization studies to assess the changes in levels of expression and distribution of BB cytoskeletal proteins during induced BB assembly. Preliminary characterization of changes in the phosphorylation and fatty acylation state of BB proteins which may accompany BB assembly will also be conducted.
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