Abstract
Tetrahymena cilia contain a three-headed 22S (outer arm) dynein and a single-headed 14S dynein. In this study, we have employed an in vitro assay of microtubule translocation along dynein-coated glass surfaces to characterize the motile properties of 14S dynein, 22S dynein, and proteolytic fragments of 22S dynein. Microtubule translocation produced by intact 22S dynein and 14S dynein differ in a number of respects including (a) the maximal velocities of movement; (b) the ability of 22S dynein but not 14S dynein to utilize ATP gamma S to induce movement; (c) the optimal pH and ionic conditions for movement; and (d) the effects of Triton X-100 on the velocity of movement. These results indicate that 22S and 14S dyneins have distinct microtubule translocating properties and suggest that these dyneins may have specialized roles in ciliary beating. We have also explored the function of the multiple ATPase heads of 22S dynein by preparing one- and two-headed proteolytic fragments of this three-headed molecule and examining their motile activity in vitro. Unlike the single-headed 14S dynein, the single-headed fragment of 22S dynein did not induce movement, even though it was capable of binding to microtubules. The two-headed fragment, on the other hand, translocated microtubules at velocities similar to those measured for intact 22S dynein (10 microns/sec). This finding indicates that the intact three-headed structure of 22S dynein is not essential for generating microtubule movement, which raises the possibility that multiple heads may serve some regulatory function or may be required for maximal force production in the beating cilium.
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- Chantler P. D., Szent-Györgyi A. G. Regulatory light-chains and scallop myosin. Full dissociation, reversibility and co-operative effects. J Mol Biol. 1980 Apr 15;138(3):473–492. doi: 10.1016/s0022-2836(80)80013-1. [DOI] [PubMed] [Google Scholar]
- Dentler W. L., Pratt M. M., Stephens R. E. Microtubule-membrane interactions in cilia. II. Photochemical cross-linking of bridge structures and the identification of a membrane-associated dynein-like ATPase. J Cell Biol. 1980 Feb;84(2):381–403. doi: 10.1083/jcb.84.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gibbons I. R. Cilia and flagella of eukaryotes. J Cell Biol. 1981 Dec;91(3 Pt 2):107s–124s. doi: 10.1083/jcb.91.3.107s. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gibbons I. R., Evans J. A., Gibbons B. H. Acetate anions stabilize the latency of dynein 1 ATPase and increase the velocity of tubule sliding in reactivated sperm flagella. Prog Clin Biol Res. 1982;80:181–184. doi: 10.1002/cm.970020734. [DOI] [PubMed] [Google Scholar]
- Gibbons I. R., Fronk E. A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella. J Biol Chem. 1979 Jan 10;254(1):187–196. [PubMed] [Google Scholar]
- Gibbons I. R., Rowe A. J. Dynein: A Protein with Adenosine Triphosphatase Activity from Cilia. Science. 1965 Jul 23;149(3682):424–426. doi: 10.1126/science.149.3682.424. [DOI] [PubMed] [Google Scholar]
- Gibbons I. R. Studies on the adenosine triphosphatase activity of 14 S and 30 S dynein from cilia of Tetrahymena. J Biol Chem. 1966 Dec 10;241(23):5590–5596. [PubMed] [Google Scholar]
- Goodenough U. W., Gebhart B., Mermall V., Mitchell D. R., Heuser J. E. High-pressure liquid chromatography fractionation of Chlamydomonas dynein extracts and characterization of inner-arm dynein subunits. J Mol Biol. 1987 Apr 5;194(3):481–494. doi: 10.1016/0022-2836(87)90676-0. [DOI] [PubMed] [Google Scholar]
- Goodenough U. W., Heuser J. E. Substructure of inner dynein arms, radial spokes, and the central pair/projection complex of cilia and flagella. J Cell Biol. 1985 Jun;100(6):2008–2018. doi: 10.1083/jcb.100.6.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goodenough U., Heuser J. Structural comparison of purified dynein proteins with in situ dynein arms. J Mol Biol. 1984 Dec 25;180(4):1083–1118. doi: 10.1016/0022-2836(84)90272-9. [DOI] [PubMed] [Google Scholar]
- Hosokawa Y., Miki-Noumura T. Bending motion of Chlamydomonas axonemes after extrusion of central-pair microtubules. J Cell Biol. 1987 Sep;105(3):1297–1301. doi: 10.1083/jcb.105.3.1297. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson K. A. Pathway of the microtubule-dynein ATPase and the structure of dynein: a comparison with actomyosin. Annu Rev Biophys Biophys Chem. 1985;14:161–188. doi: 10.1146/annurev.bb.14.060185.001113. [DOI] [PubMed] [Google Scholar]
- Johnson K. A. Preparation and properties of dynein from Tetrahymena cilia. Methods Enzymol. 1986;134:306–317. doi: 10.1016/0076-6879(86)34098-9. [DOI] [PubMed] [Google Scholar]
- Johnson K. A., Wall J. S. Structure and molecular weight of the dynein ATPase. J Cell Biol. 1983 Mar;96(3):669–678. doi: 10.1083/jcb.96.3.669. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kamiya R. Extrusion and Rotation of the central-pair microtubules in detergent-treated Chlamydomonas flagella. Prog Clin Biol Res. 1982;80:169–173. doi: 10.1002/cm.970020732. [DOI] [PubMed] [Google Scholar]
- King S. M., Witman G. B. Structure of the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Location of epitopes and protease-sensitive sites. J Biol Chem. 1988 Jul 5;263(19):9244–9255. [PubMed] [Google Scholar]
- Kishino A., Yanagida T. Force measurements by micromanipulation of a single actin filament by glass needles. Nature. 1988 Jul 7;334(6177):74–76. doi: 10.1038/334074a0. [DOI] [PubMed] [Google Scholar]
- Kodama T., Fukui K., Kometani K. The initial phosphate burst in ATP hydrolysis by myosin and subfragment-1 as studied by a modified malachite green method for determination of inorganic phosphate. J Biochem. 1986 May;99(5):1465–1472. doi: 10.1093/oxfordjournals.jbchem.a135616. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lye R. J., Porter M. E., Scholey J. M., McIntosh J. R. Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans. Cell. 1987 Oct 23;51(2):309–318. doi: 10.1016/0092-8674(87)90157-7. [DOI] [PubMed] [Google Scholar]
- Mabuchi I., Shimizu T. Electrophoretic studies on dyneins from Tetrahymena cilia. J Biochem. 1974 Nov;76(5):991–999. [PubMed] [Google Scholar]
- Marchese-Ragona S. P., Wall J. S., Johnson K. A. Structure and mass analysis of 14S dynein obtained from Tetrahymena cilia. J Cell Biol. 1988 Jan;106(1):127–132. doi: 10.1083/jcb.106.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mitchell D. R., Rosenbaum J. L. A motile Chlamydomonas flagellar mutant that lacks outer dynein arms. J Cell Biol. 1985 Apr;100(4):1228–1234. doi: 10.1083/jcb.100.4.1228. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mocz G., Tang W. J., Gibbons I. R. A map of photolytic and tryptic cleavage sites on the beta heavy chain of dynein ATPase from sea urchin sperm flagella. J Cell Biol. 1988 May;106(5):1607–1614. doi: 10.1083/jcb.106.5.1607. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mogami Y., Takahashi K. Calcium and microtubule sliding in ciliary axonemes isolated from Paramecium caudatum. J Cell Sci. 1983 May;61:107–121. doi: 10.1242/jcs.61.1.107. [DOI] [PubMed] [Google Scholar]
- Okagaki T., Kamiya R. Microtubule sliding in mutant Chlamydomonas axonemes devoid of outer or inner dynein arms. J Cell Biol. 1986 Nov;103(5):1895–1902. doi: 10.1083/jcb.103.5.1895. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Omoto C. K., Kung C. Rotation and twist of the central-pair microtubules in the cilia of Paramecium. J Cell Biol. 1980 Oct;87(1):33–46. doi: 10.1083/jcb.87.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Omoto C. K., Witman G. B. Functionally significant central-pair rotation in a primitive eukaryotic flagellum. Nature. 1981 Apr 23;290(5808):708–710. doi: 10.1038/290708a0. [DOI] [PubMed] [Google Scholar]
- Paschal B. M., King S. M., Moss A. G., Collins C. A., Vallee R. B., Witman G. B. Isolated flagellar outer arm dynein translocates brain microtubules in vitro. Nature. 1987 Dec 17;330(6149):672–674. doi: 10.1038/330672a0. [DOI] [PubMed] [Google Scholar]
- Paschal B. M., Shpetner H. S., Vallee R. B. MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties. J Cell Biol. 1987 Sep;105(3):1273–1282. doi: 10.1083/jcb.105.3.1273. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Piperno G. Isolation of a sixth dynein subunit adenosine triphosphatase of Chlamydomonas axonemes. J Cell Biol. 1988 Jan;106(1):133–140. doi: 10.1083/jcb.106.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Porter M. E., Scholey J. M., Stemple D. L., Vigers G. P., Vale R. D., Sheetz M. P., McIntosh J. R. Characterization of the microtubule movement produced by sea urchin egg kinesin. J Biol Chem. 1987 Feb 25;262(6):2794–2802. [PubMed] [Google Scholar]
- Sale W. S., Fox L. A. Isolated beta-heavy chain subunit of dynein translocates microtubules in vitro. J Cell Biol. 1988 Nov;107(5):1793–1797. doi: 10.1083/jcb.107.5.1793. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sale W. S., Goodenough U. W., Heuser J. E. The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella. J Cell Biol. 1985 Oct;101(4):1400–1412. doi: 10.1083/jcb.101.4.1400. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sheetz M. P., Block S. M., Spudich J. A. Myosin movement in vitro: a quantitative assay using oriented actin cables from Nitella. Methods Enzymol. 1986;134:531–544. doi: 10.1016/0076-6879(86)34118-1. [DOI] [PubMed] [Google Scholar]
- Shimizu T., Johnson K. A. Kinetic evidence for multiple dynein ATPase sites. J Biol Chem. 1983 Nov 25;258(22):13841–13846. [PubMed] [Google Scholar]
- Shimizu T., Kimura I. Effects of N-ethylmaleimide on dynein adenosinetriphosphatase activity and its recombining ability with outer fibers. J Biochem. 1974 Nov;76(5):1001–1008. [PubMed] [Google Scholar]
- Shimizu T. The substrate specificity of dynein from Tetrahymena cilia. J Biochem. 1987 Nov;102(5):1159–1165. doi: 10.1093/oxfordjournals.jbchem.a122154. [DOI] [PubMed] [Google Scholar]
- Tash J. S., Krinks M., Patel J., Means R. L., Klee C. B., Means A. R. Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm. J Cell Biol. 1988 May;106(5):1625–1633. doi: 10.1083/jcb.106.5.1625. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Toyoshima Y. Y. Chymotryptic digestion of Tetrahymena 22S dynein. I. Decomposition of three-headed 22S dynein to one- and two-headed particles. J Cell Biol. 1987 Aug;105(2):887–895. doi: 10.1083/jcb.105.2.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Toyoshima Y. Y. Chymotryptic digestion of Tetrahymena ciliary dynein. II. Pathway of the degradation of 22S dynein heavy chains. J Cell Biol. 1987 Aug;105(2):897–901. doi: 10.1083/jcb.105.2.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Toyoshima Y. Y., Kron S. J., McNally E. M., Niebling K. R., Toyoshima C., Spudich J. A. Myosin subfragment-1 is sufficient to move actin filaments in vitro. Nature. 1987 Aug 6;328(6130):536–539. doi: 10.1038/328536a0. [DOI] [PubMed] [Google Scholar]
- Vale R. D., Reese T. S., Sheetz M. P. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell. 1985 Aug;42(1):39–50. doi: 10.1016/s0092-8674(85)80099-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vale R. D., Szent-Gyorgyi A. G., Sheetz M. P. Movement of scallop myosin on Nitella actin filaments: regulation by calcium. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6775–6778. doi: 10.1073/pnas.81.21.6775. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vale R. D., Toyoshima Y. Y. Rotation and translocation of microtubules in vitro induced by dyneins from Tetrahymena cilia. Cell. 1988 Feb 12;52(3):459–469. doi: 10.1016/s0092-8674(88)80038-2. [DOI] [PubMed] [Google Scholar]
- Vallee R. B., Wall J. S., Paschal B. M., Shpetner H. S. Microtubule-associated protein 1C from brain is a two-headed cytosolic dynein. Nature. 1988 Apr 7;332(6164):561–563. doi: 10.1038/332561a0. [DOI] [PubMed] [Google Scholar]
- Walter M. F., Satir P. Calcium does not inhibit active sliding of microtubules from mussel gill cilia. Nature. 1979 Mar 1;278(5699):69–70. doi: 10.1038/278069a0. [DOI] [PubMed] [Google Scholar]
- Warner F. D., Perreault J. G., McIlvain J. H. Rebinding of Tetrahymena 13 S and 21 S dynein ATPases to extracted doublet microtubules. The inner row and outer row dynein arms. J Cell Sci. 1985 Aug;77:263–287. doi: 10.1242/jcs.77.1.263. [DOI] [PubMed] [Google Scholar]
- Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yano Y., Miki-Noumura T. Sliding velocity between outer doublet microtubules of sea-urchin sperm axonemes. J Cell Sci. 1980 Aug;44:169–186. doi: 10.1242/jcs.44.1.169. [DOI] [PubMed] [Google Scholar]