Microtubules are major structural components of a diverse number of cellular organelles and events (mitosis, neuronal elongation, intracellular transport, and maintenance of cell shape and internal cytoplasmic architecture). The objective of the proposed research is to gain an improved understanding of how microtubule regulation is achieved in higher eukaryotes. Three lines of investigation will be pursued. In the first of these, the molecular pathway with which tubulin expression is autoregulated through selective mRNA instability will be further dissected. Preceding data have already determined that Beta-tubulin mRNA stability is established by co- translational recognition of the amino-terminal Beta-tubulin tetrapeptide (Met-Arg-Glu-I1e) just after it emerges from the ribosome. We now intend to use biochemical approaches to identify the cellular factors that bind to this amino-terminal domain, to develop an in vitro system in which tubulin mRNA stability can be mimicked, to use DNA transfection to test formally whether tubulin RNA stability is autoregulated by its own end product, and to determine whether this pathway of mRNA instability is a general one. In a second aspect, the functional significance of the polypeptides encoded by the tubulin multi-gene family will be analyzed. We have already demonstrated that some Beta tubulin isotypes are preferentially assembled during neurite outgrowth. We will now use immunoelectron microscopy to localize the assembly products of each Beta isotype. Further, the physiological consequence of altering the amounts of specific isotypes expressed in cultured cells will be examined by DNA transfection methods.
The final aim i s the identification of components of kinetochores/centromeres, the chromosomal structures which are the binding sites for microtubules of the mitotic spindle. Using existing antibodies to the only cloned human centromere/kinetochore protein (CENP-B), kinetochore complexes will be isolated. These will be used for production of monoclonal antibodies that recognize new kinetochore components. Such antibodies will then be used to clone cDNAs for the corresponding proteins, to determine their sequences, and to assess the function of those proteins using microinjection techniques and in vitro assays.
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