Microtubules are major structural components of a diverse number of cellular organelles and events (mitosis, cilia, neuronal elongation, intracellular transport, and maintenance of cell shape and internal cytoplasmic composition). It is not known, however, how the microtubules of these varying functions are specified, nor in fact in what ways they are structurally distinct. The objective of the proposed research is to gain an understanding of how microtubule regulation is achieved in higher eukaryotes. Two lines of investigation will be pursued. In the first, each of the functional genes within a vertebrate genome for the two principle subunits of microtubules, Alpha butulin and Beta tubulin, will be isolated. Furthermore, the complete primary sequences of the encoded polypeptides will be determined. Recent data have already revealed surprising sequence heterogeneity among Beta tubulin polypeptides. Not only does such heterogeneity imply functional significance, it offers an exciting new opportunity for investigation of in vivo microtubule organization. Specifically, these sequence differences may be exploited for the synthesis of peptides unique to individual tubulin subunits (either Alpha or Beta). Using antibodies generated against each peptide, individual tubulin subunits will be localized in microtubules in situ both by light microscopic and electron microscopic methods. This analysis will be done in multiple cell and tissue types which utilize microtubules for divergent purposes and which express different tubulin gene sequences. A second line of inquiry will be the determination of the molecular events through which tubulin synthesis is regulated in mammalian cells by a novel and apparently autoregulatory mechanism. Although it is currently known that elevation of the pool size of depolymerized tubulin subunits in mammalian cells results in the rapid, specific loss of cytoplasmic tubulin RNAs, whether the ultimate regulatory event is exercised at the level of tubulin RNA transcription, RNA processing/transport, or cytoplasmic RNA degradation remains to be determined. This investigation will now be undertaken. In addition, the identification of in vitro conditions in which this autoregulatory control mechanism can be maintained or induced will be attempted.
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