Dr. Stephens found that axonemal precursor tubulin is conveyed to the tip of the growing cilium in associate with the membrane while the tektins with which it must co-assemble are conveyed in direct association with the axoneme.To further study the basic process of ciliogenesis as a model for programmed protein assembly and to address the relative importance of these two newly- discovered routes for protein transport, he proposes three specific aims: 1) to investigate the nature and dynamics of ciliary membrane- associate protein transport by studying the interactions of tubulin with lipids and with axoneme-bound transmembrane proteins; 2) to characterize the association and translocation of non-tubulin proteins in axoneme-associated transport by exploring conditions for the selective release of proteins in transit and by identifying potential molecular motors; and 3) to elucidate the structural role of tektin-A, an integral component of outer doublet microtubules, centrioles, and basal bodies, by further incorporation and localization studies. Based on recent findings that specific cAMP-dependent phosphorylations of dynein light and heavy chains, correlating respectively with the neuronal or hormonal activation of cilia or flagella, are distal from the ATPase site, he proposes to study the mechanochemical basis for the regulation of dynein- microtubule interaction by cAMP. To continue this exploration using a model system in which he can directly compare flagella with cilia, he proposes: 1) to domain-map the dynein molecules by localizing the flagellar heavy chain phosphorylation site and the ciliary dynein intermediate chain/phosphorylated light chain complex, testing its possible role as a regulatory element; 2) to determine the role of heavy or light chain phosphorylation by studying the in vitro activity of phosphorylated and non-phosphorylated dynein molecules or parts thereof; and 3) to explore differences in dynein-microtubule interactions in the activated versus non-activated state of both flagella and cilia by using chemical cross-linking techniques.
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