The trypanosomiases of equatorial Africa are of great medical and economic importance. Regulation of critical activities such as metabolism, motility, secretion, cell division and differentiation are not well understood in these organisms. In all eukaryotic cells studied in detail Ca regulates such critical events with calmodulin (CaM), acting as a broad spectrum regulatory molecule. We have isolated trypanosome calmodulin (TCaM) and characterized its structure in detail. We have determined its amino acid sequence, we have identified, isolated, sequenced and expressed the TCaM gene in E. coli. Using native and recombinant TCaM we have identified several TCaM binding proteins in trypanosome. We wish to continue these studies by characterizing TCaM expression and its interaction with response elements in African trypanosome during differentiation. We have reason to focus our attention on protein kinases, flagellar, microtubule and cytoskeletal networks, for they have been implicated in a variety of cellular function including secretion, axoplasmic transport, cell shape, motility and mitosis. In addition, drugs that interact with tubulin and microtubule (MT) systems appear to be effective antimitotic agents. Thus understanding the factors that regulate MT and cytoskeletal elements will provide important insights into cellular regulation and potentially provide clues for the control of trypanosomiasis. Calcium ions play a major role in regulating protein kinases, the polymerization of microtubules and their relationship to the cytoskeleton. TCaM appears to mediated some of the effects of calcium on protein kinases and tubulin dynamics. We have shown that the paraxial rod associated with the flagellum binds TCaM. In addition, trypanosome are girdled by a compact, single-layer of subpellicular microtubules beneath the plasma membrane. One of three TCaM binding proteins we have isolated is a calcium- calmodulin protein kinase that phosphorylates tubulin and microtubule associated proteins (MAPs). Experiments in this proposal are directed at examining trypanosome for MAPs and tubulin associated calmodulin kinase (TACK), purifying and characterizing the proteins and their genes, and determining in vitro and in vivo their roles in trypanosome biology.
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