The long-range goal of this research is to understand the purpose and mechanism of a cytoskeletal differentiation, that is, modifications in the cytoskeleton which alter its role in cellular events. In particular, a reversible post-translational modification of a tubulin involves the addition or removal of a tyrosine residue at the C terminus. Previous research in the laboratory has determined that tyrosinated and nontyrosinated tubulin are contained in different populations of macrotubules in cultured cells.
The aims of this project are three-fold: (1) to determine whether asymmetry of distribution of nontyrosinated (Glu) and tyrosinated (Tyr) tubulins in microtubules is of widespread occurrence; (2) to ascertain whether or not the segregation of the Glu and Tyr tubulins into separate microtubules appears to be involved in mitosis or cell transformation; and (3) to determine the mechanism by which these two post-translationally modified tubulins become differentially distributed in vivo. Several types of cultured cells will be examined using peptide antibodies specific for Tyr and for Glu tubulin. Cells engaged in mitosis will be examined with light and electron microscopic immunolocalization in order to determine which population of mitotic microtubules contain which species of alpha-tubulin. Transformation by SV40 virus in two cell lines will be used as an example of cytoskeletal abnormalities induced by transformation. In each case in which asymmetry of Tyr and Glu tubulin is observed, the location and any putative functions of microtubules containing each will be noted. To learn about the significance and the mechanism of obtaining the observed differential distribution of Tyr and Glu tubulin, microtubules will be depolymerized in vivo and in lysed cell models. Repolymerization under several conditions will result in differential or equivalent distributions of Glu and Tyr tubulin. Several possible mechanisms for the segregation of Glu and Tyr tubulins into separate microtubules can thus be tested. Since the abnormal function of microtubules is an integral part of cancer and other diseases, attempts to discover the control of their localization and function in cell division, differentiation, and transformation is of obvious importance. (L)
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