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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM020644-21A2
Application #
2173610
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1978-09-01
Project End
1999-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
21
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Boston University
Department
Physiology
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Stephens, R E; Lemieux, N A (1999) Molecular chaperones in cilia and flagella: implications for protein turnover. Cell Motil Cytoskeleton 44:274-83
Stephens, R E (1994) Rapid induction of a hyperciliated phenotype in zinc-arrested sea urchin embryos by theophylline. J Exp Zool 269:106-15
Stephens, R E (1994) Tubulin and tektin in sea urchin embryonic cilia: pathways of protein incorporation during turnover and regeneration. J Cell Sci 107 ( Pt 2):683-92
Stephens, R E; Prior, G (1992) Dynein from serotonin-activated cilia and flagella: extraction characteristics and distinct sites for cAMP-dependent protein phosphorylation. J Cell Sci 103 ( Pt 4):999-1012
Stephens, R E (1991) Tubulin in sea urchin embryonic cilia: characterization of the membrane-periaxonemal matrix. J Cell Sci 100 ( Pt 3):521-31
Stephens, R E; Good, M J (1990) Filipin-sterol complexes in molluscan gill ciliated epithelial cell membranes: intercalation into ciliary necklaces and induction of gap junctional particle arrays. Cell Tissue Res 262:301-6
Stephens, R E (1989) Quantal tektin synthesis and ciliary length in sea-urchin embryos. J Cell Sci 92 ( Pt 3):403-13
Tilney, M S; Tilney, L G; Stephens, R E et al. (1989) Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea. J Cell Biol 109:1711-23
Stephens, R E; Oleszko-Szuts, S; Linck, R W (1989) Retention of ciliary ninefold structure after removal of microtubules. J Cell Sci 92 ( Pt 3):391-402
Stephens, R E (1988) Separation of tubulin subunits by reversed-phase high-performance liquid chromatography. J Chromatogr 448:371-86

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