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. 1986 Nov 1;103(5):1895–1902. doi: 10.1083/jcb.103.5.1895

Microtubule sliding in mutant Chlamydomonas axonemes devoid of outer or inner dynein arms

PMCID: PMC2114376  PMID: 2946702

Abstract

To clarify the functional differentiation between the outer and inner dynein arms in eukaryotic flagella, their mechanochemical properties were assessed by measuring the sliding velocities of outer-doublet microtubules in disintegrating axonemes of Chlamydomonas, using wild- type and mutant strains that lack either of the arms. A special procedure was developed to induce sliding disintegration in Chlamydomonas axonemes which is difficult to achieve by ordinary methods. The flagella were first fragmented by sonication, demembranated by Nonidet P-40, and then perfused under a microscope with Mg-ATP and nagarse, a bacterial protease with broad substrate specificity. The sliding velocity varied with the Mg-ATP concentration in a Michaelis-Menten manner in the axonemes from the wild type and a motile mutant lacking the outer dynein arm (oda38). The maximal sliding velocity and apparent Michaelis constant for Mg-ATP were measured to be 13.2 +/- 1.0 micron/s and 158 +/- 36 microM for the wild type and 2.0 +/- 0.1 micron/s and 64 +/- 18 microM for oda38. These maximal sliding velocities were significantly smaller than those estimated in beating axonemes; the reason is not clear. The velocities in the presence or absence of 10(-5) M Ca2+ did not differ noticeably. The axonemes of nonmotile mutants lacking either outer arms (pf13A, pf22) or inner arms (pf23) were examined for their ability to undergo sliding disintegration in the presence of 0.1 mM Mg-ATP. Whereas pf13A axonemes underwent normal sliding disintegration, the other two species displayed it only very poorly. The poor ability of pf23 axonemes to undergo sliding disintegration raises the possibility that the outer dynein arm cannot function well in the absence of the inner arm.

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Selected References

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  1. Allen R. D. A reinvestigation of cross-sections of cilia. J Cell Biol. 1968 Jun;37(3):825–831. doi: 10.1083/jcb.37.3.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bessen M., Fay R. B., Witman G. B. Calcium control of waveform in isolated flagellar axonemes of Chlamydomonas. J Cell Biol. 1980 Aug;86(2):446–455. doi: 10.1083/jcb.86.2.446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Brokaw C. J. Elastase digestion of demembranated sperm flagella. Science. 1980 Mar 21;207(4437):1365–1367. doi: 10.1126/science.6898364. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Dirksen E. R., Zeira M. Microtubule sliding in cilia of the rabbit trachea and oviduct. Cell Motil. 1981;1(2):247–260. doi: 10.1002/cm.970010207. [DOI] [PubMed] [Google Scholar]
  7. Gibbons B. H., Gibbons I. R. The effect of partial extraction of dynein arms on the movement of reactivated sea-urchin sperm. J Cell Sci. 1973 Sep;13(2):337–357. doi: 10.1242/jcs.13.2.337. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Gorman D. S., Levine R. P. Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci U S A. 1965 Dec;54(6):1665–1669. doi: 10.1073/pnas.54.6.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hata H., Yano Y., Miki-Noumura T. ATP concentration dependency of the tubule-extrusion velocity from the axonemes. Exp Cell Res. 1979 Sep;122(2):416–419. doi: 10.1016/0014-4827(79)90322-7. [DOI] [PubMed] [Google Scholar]
  12. Hata H., Yano Y., Mohri T., Mohri H., Miki-Noumura T. ATP-driven tubule extrusion from axonemes without outer dynein arms of sea-urchin sperm flagella. J Cell Sci. 1980 Feb;41:331–340. doi: 10.1242/jcs.41.1.331. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Hyams J. S., Borisy G. G. Isolated flagellar apparatus of Chlamydomonas: characterization of forward swimming and alteration of waveform and reversal of motion by calcium ions in vitro. J Cell Sci. 1978 Oct;33:235–253. doi: 10.1242/jcs.33.1.235. [DOI] [PubMed] [Google Scholar]
  15. Kamimura S., Takahashi K. Direct measurement of the force of microtubule sliding in flagella. Nature. 1981 Oct 15;293(5833):566–568. doi: 10.1038/293566a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Kamiya R., Okamoto M. A mutant of Chlamydomonas reinhardtii that lacks the flagellar outer dynein arm but can swim. J Cell Sci. 1985 Mar;74:181–191. doi: 10.1242/jcs.74.1.181. [DOI] [PubMed] [Google Scholar]
  18. Kamiya R., Witman G. B. Submicromolar levels of calcium control the balance of beating between the two flagella in demembranated models of Chlamydomonas. J Cell Biol. 1984 Jan;98(1):97–107. doi: 10.1083/jcb.98.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lindemann C. B., Gibbons I. R. Adenosine triphosphate-induced motility and sliding of filaments in mammalian sperm extracted with Triton X-100. J Cell Biol. 1975 Apr;65(1):147–162. doi: 10.1083/jcb.65.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. 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]
  23. Omoto C. K., Brokaw C. J. Bending patterns of Chlamydomonas flagella: II. Calcium effects on reactivated Chlamydomonas flagella. Cell Motil. 1985;5(1):53–60. doi: 10.1002/cm.970050105. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Piperno G., Luck D. J. Inner arm dyneins from flagella of Chlamydomonas reinhardtii. Cell. 1981 Dec;27(2 Pt 1):331–340. doi: 10.1016/0092-8674(81)90416-5. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Summers K. E., Gibbons I. R. Adenosine triphosphate-induced sliding of tubules in trypsin-treated flagella of sea-urchin sperm. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3092–3096. doi: 10.1073/pnas.68.12.3092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Summers K. ATP-induced sliding of microtubules in bull sperm flagella. J Cell Biol. 1974 Jan;60(1):321–324. doi: 10.1083/jcb.60.1.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Takahashi K., Shingyoji C., Kamimura S. Microtubule sliding in reactivated flagella. Symp Soc Exp Biol. 1982;35:159–177. [PubMed] [Google Scholar]
  30. Tamm S. L., Tamm S. Alternate patterns of doublet microtubule sliding in ATP-disintegrated macrocilia of the ctenophore Beroë. J Cell Biol. 1984 Oct;99(4 Pt 1):1364–1371. doi: 10.1083/jcb.99.4.1364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Warner F. D., Mitchell D. R. Structural conformation of ciliary dynein arms and the generation of sliding forces in Tetrahymena cilia. J Cell Biol. 1978 Feb;76(2):261–277. doi: 10.1083/jcb.76.2.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Warner F. D. New observations on flagellar fine structure. The relationship between matrix structure and the microtubule component of the axoneme. J Cell Biol. 1970 Oct;47(1):159–182. doi: 10.1083/jcb.47.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Warner F. D., Zanetti N. C. Properties of microtubule sliding disintegration in isolated Tetrahymena cilia. J Cell Biol. 1980 Aug;86(2):436–445. doi: 10.1083/jcb.86.2.436. [DOI] [PMC free article] [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. Yano Y., Miki-Noumura T. Recovery of sliding ability in arm-depleted flagellar axonemes after recombination with extracted dynein I. J Cell Sci. 1981 Apr;48:223–239. doi: 10.1242/jcs.48.1.223. [DOI] [PubMed] [Google Scholar]
  37. 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]

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