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. 1994 Dec 1;127(5):1311–1325. doi: 10.1083/jcb.127.5.1311

Components of a "dynein regulatory complex" are located at the junction between the radial spokes and the dynein arms in Chlamydomonas flagella

PMCID: PMC2120243  PMID: 7962092

Abstract

Previous studies of flagellar mutants have identified six axonemal polypeptides as components of a "dynein regulatory complex" (DRC). The DRC is though to coordinate the activity of the multiple flagellar dyneins, but its location within the axoneme has been unknown (Huang et al., 1982; Piperno et al., 1992). We have used improved chromatographic procedures (Kagami and Kamiya, 1992) and computer averaging of EM images (Mastronarde et al., 1992) to analyze the relationship between the DRC and the dynein arms. Our results suggest that some of the DRC components are located at the base of the second radial spoke in close association with the inner dynein arms. (a) Averages of axoneme cross- sections indicate that inner arm structures are significantly reduced in three DRC mutants (pf3 < pf2 < sup-pf-3 < wt). (b) These defects are more pronounced in distal/medial regions of the axoneme than in proximal regions. (c) Analysis of flagellar extracts by fast protein liquid chromatography and SDS-PAGE indicates that a specific dynein I2 isoform is missing in pf3 and reduced in pf2 and sup-pf-3. Comparison with ida4 and pf3ida4 extracts reveals that this isoform differs from those missing in ida4. (d) When viewed in longitudinal section, all three DRC mutants lack a crescent-shaped density above the second radial spoke, and pf3 axonemes lack additional structures adjacent to the crescent. We propose that the crescent corresponds in part to the location of the DRC, and that this structure is also directly associated with a subset of the inner dynein arms. This position is appropriate for a complex that is thought to mediate signals between the radial spokes and the dynein arms.

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Selected References

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  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Brokaw C. J., Kamiya R. Bending patterns of Chlamydomonas flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function. Cell Motil Cytoskeleton. 1987;8(1):68–75. doi: 10.1002/cm.970080110. [DOI] [PubMed] [Google Scholar]
  3. Brokaw C. J., Luck D. J. Bending patterns of chlamydomonas flagella: III. A radial spoke head deficient mutant and a central pair deficient mutant. Cell Motil. 1985;5(3):195–208. doi: 10.1002/cm.970050303. [DOI] [PubMed] [Google Scholar]
  4. Brokaw C. J., Luck D. J., Huang B. Analysis of the movement of Chlamydomonas flagella:" the function of the radial-spoke system is revealed by comparison of wild-type and mutant flagella. J Cell Biol. 1982 Mar;92(3):722–732. doi: 10.1083/jcb.92.3.722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fox L. A., Sale W. S. Direction of force generated by the inner row of dynein arms on flagellar microtubules. J Cell Biol. 1987 Oct;105(4):1781–1787. doi: 10.1083/jcb.105.4.1781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Holmes J. A., Dutcher S. K. Cellular asymmetry in Chlamydomonas reinhardtii. J Cell Sci. 1989 Oct;94(Pt 2):273–285. doi: 10.1242/jcs.94.2.273. [DOI] [PubMed] [Google Scholar]
  9. Hoops H. J., Witman G. B. Outer doublet heterogeneity reveals structural polarity related to beat direction in Chlamydomonas flagella. J Cell Biol. 1983 Sep;97(3):902–908. doi: 10.1083/jcb.97.3.902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Huang B., Piperno G., Luck D. J. Paralyzed flagella mutants of Chlamydomonas reinhardtii. Defective for axonemal doublet microtubule arms. J Biol Chem. 1979 Apr 25;254(8):3091–3099. [PubMed] [Google Scholar]
  11. Huang B., Ramanis Z., Luck D. J. Suppressor mutations in Chlamydomonas reveal a regulatory mechanism for Flagellar function. Cell. 1982 Jan;28(1):115–124. doi: 10.1016/0092-8674(82)90381-6. [DOI] [PubMed] [Google Scholar]
  12. Kamiya R., Kurimoto E., Muto E. Two types of Chlamydomonas flagellar mutants missing different components of inner-arm dynein. J Cell Biol. 1991 Feb;112(3):441–447. doi: 10.1083/jcb.112.3.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kamiya R. Mutations at twelve independent loci result in absence of outer dynein arms in Chylamydomonas reinhardtii. J Cell Biol. 1988 Dec;107(6 Pt 1):2253–2258. doi: 10.1083/jcb.107.6.2253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kato T., Kagami O., Yagi T., Kamiya R. Isolation of two species of Chlamydomonas reinhardtii flagellar mutants, ida5 and ida6, that lack a newly identified heavy chain of the inner dynein arm. Cell Struct Funct. 1993 Dec;18(6):371–377. doi: 10.1247/csf.18.371. [DOI] [PubMed] [Google Scholar]
  15. King S. M., Otter T., Witman G. B. Purification and characterization of Chlamydomonas flagellar dyneins. Methods Enzymol. 1986;134:291–306. doi: 10.1016/0076-6879(86)34097-7. [DOI] [PubMed] [Google Scholar]
  16. Kurimoto E., Kamiya R. Microtubule sliding in flagellar axonemes of Chlamydomonas mutants missing inner- or outer-arm dynein: velocity measurements on new types of mutants by an improved method. Cell Motil Cytoskeleton. 1991;19(4):275–281. doi: 10.1002/cm.970190406. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Luck D., Piperno G., Ramanis Z., Huang B. Flagellar mutants of Chlamydomonas: studies of radial spoke-defective strains by dikaryon and revertant analysis. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3456–3460. doi: 10.1073/pnas.74.8.3456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mastronarde D. N., O'Toole E. T., McDonald K. L., McIntosh J. R., Porter M. E. Arrangement of inner dynein arms in wild-type and mutant flagella of Chlamydomonas. J Cell Biol. 1992 Sep;118(5):1145–1162. doi: 10.1083/jcb.118.5.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Piperno G., Luck D. J. Axonemal adenosine triphosphatases from flagella of Chlamydomonas reinhardtii. Purification of two dyneins. J Biol Chem. 1979 Apr 25;254(8):3084–3090. [PubMed] [Google Scholar]
  22. Piperno G., Mead K., LeDizet M., Moscatelli A. Mutations in the "dynein regulatory complex" alter the ATP-insensitive binding sites for inner arm dyneins in Chlamydomonas axonemes. J Cell Biol. 1994 Jun;125(5):1109–1117. doi: 10.1083/jcb.125.5.1109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Piperno G., Mead K., Shestak W. The inner dynein arms I2 interact with a "dynein regulatory complex" in Chlamydomonas flagella. J Cell Biol. 1992 Sep;118(6):1455–1463. doi: 10.1083/jcb.118.6.1455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Piperno G., Ramanis Z., Smith E. F., Sale W. S. Three distinct inner dynein arms in Chlamydomonas flagella: molecular composition and location in the axoneme. J Cell Biol. 1990 Feb;110(2):379–389. doi: 10.1083/jcb.110.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Piperno G., Ramanis Z. The proximal portion of Chlamydomonas flagella contains a distinct set of inner dynein arms. J Cell Biol. 1991 Feb;112(4):701–709. doi: 10.1083/jcb.112.4.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Porter M. E., Power J., Dutcher S. K. Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms. J Cell Biol. 1992 Sep;118(5):1163–1176. doi: 10.1083/jcb.118.5.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sale W. S., Satir P. Direction of active sliding of microtubules in Tetrahymena cilia. Proc Natl Acad Sci U S A. 1977 May;74(5):2045–2049. doi: 10.1073/pnas.74.5.2045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schroer T. A., Sheetz M. P. Two activators of microtubule-based vesicle transport. J Cell Biol. 1991 Dec;115(5):1309–1318. doi: 10.1083/jcb.115.5.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Smith E. F., Sale W. S. Microtubule binding and translocation by inner dynein arm subtype I1. Cell Motil Cytoskeleton. 1991;18(4):258–268. doi: 10.1002/cm.970180403. [DOI] [PubMed] [Google Scholar]
  30. Smith E. F., Sale W. S. Regulation of dynein-driven microtubule sliding by the radial spokes in flagella. Science. 1992 Sep 11;257(5076):1557–1559. doi: 10.1126/science.1387971. [DOI] [PubMed] [Google Scholar]
  31. Smith E. F., Sale W. S. Structural and functional reconstitution of inner dynein arms in Chlamydomonas flagellar axonemes. J Cell Biol. 1992 May;117(3):573–581. doi: 10.1083/jcb.117.3.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Takada S., Sakakibara H., Kamiya R. Three-headed outer arm dynein from Chlamydomonas that can functionally combine with outer-arm-missing axonemes. J Biochem. 1992 Jun;111(6):758–762. doi: 10.1093/oxfordjournals.jbchem.a123832. [DOI] [PubMed] [Google Scholar]
  33. Waxman L., Goldberg A. L. Protease La from Escherichia coli hydrolyzes ATP and proteins in a linked fashion. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4883–4887. doi: 10.1073/pnas.79.16.4883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Witman G. B. Isolation of Chlamydomonas flagella and flagellar axonemes. Methods Enzymol. 1986;134:280–290. doi: 10.1016/0076-6879(86)34096-5. [DOI] [PubMed] [Google Scholar]
  35. Witman G. B., Plummer J., Sander G. Chlamydomonas flagellar mutants lacking radial spokes and central tubules. Structure, composition, and function of specific axonemal components. J Cell Biol. 1978 Mar;76(3):729–747. doi: 10.1083/jcb.76.3.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  37. Zaugg-Vesti B. R., Franzeck U. K., von Ziegler C., Furrer J., Pfister G., Yanar A., Bollinger A. Skin capillary aneurysms detected by indocyanine green in type I diabetes with and without retinal microaneurysms. Int J Microcirc Clin Exp. 1995 Jul-Aug;15(4):193–198. doi: 10.1159/000178975. [DOI] [PubMed] [Google Scholar]

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