Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 May 1;108(5):1751–1760. doi: 10.1083/jcb.108.5.1751

Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii

PMCID: PMC2115546  PMID: 2654141

Abstract

Chlamydomonas cells excise their flagella in response to a variety of experimental conditions (e.g., extremes of temperature or pH, alcohol or detergent treatment, and mechanical shear). Here, we show that flagellar excision is an active process whereby microtubules are severed at select sites within the transition zone. The transition zone is located between the flagellar axoneme and the basal body; it is characterized by a pair of central cylinders that have an H shape when viewed in longitudinal section. Both central cylinders are connected to the A tubule of each microtubule doublet of the transition zone by fibers (approximately 5 nm diam). When viewed in cross section, these fibers are seen to form a distinctive stellate pattern characteristic of the transition zone (Manton, I. 1964. J. R. Microsc. Soc. 82:279- 285; Ringo. D. L. 1967. J. Cell Biol. 33:543-571). We demonstrate that at the time of flagellar excision these fibers contract and displace the microtubule doublets of the axoneme inward. We believe that the resulting shear force and torsional load act to sever the axonemal microtubules immediately distal to the central cylinder. Structural alterations of the transition zone during flagellar excision occur both in living cells and detergent-extracted cell models, and are dependent on the presence of calcium (greater than or equal to 10(-6) M). Immunolocalization using monoclonal antibodies against the calcium- binding protein centrin demonstrate the presence of centrin in the fiber-based stellate structure of the transition zone of wild-type cells. Examination of the flagellar autotomy mutant, fa-1, which fails to excise its flagella (Lewin, R., and C. Burrascano. 1983. Experientia. 39:1397-1398), demonstrates that the fa-1 lacks the ability to completely contract the fibers of the stellate structure. We conclude that flagellar excision in Chlamydomonas involves microtubule severing that is mediated by the action of calcium-sensitive contractile fibers of the transition zone. These observations have led us to question whether microtubule severing may be a more general phenomenon than previously suspected and to suggest that microtubule severing may contribute to the dynamic behavior of cytoplasmic microtubules in other cells.

Full Text

The Full Text of this article is available as a PDF (3.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baron A. T., Salisbury J. L. Identification and localization of a novel, cytoskeletal, centrosome-associated protein in PtK2 cells. J Cell Biol. 1988 Dec;107(6 Pt 2):2669–2678. doi: 10.1083/jcb.107.6.2669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bloodgood R. A. Resorption of organelles containing microtubules. Cytobios. 1974 Mar-Apr;9(35):142–161. [PubMed] [Google Scholar]
  3. Blum J. J. Existence of a breaking point in cilia and flagella. J Theor Biol. 1971 Nov;33(2):257–263. doi: 10.1016/0022-5193(71)90065-8. [DOI] [PubMed] [Google Scholar]
  4. Bretscher A., Weber K. Villin is a major protein of the microvillus cytoskeleton which binds both G and F actin in a calcium-dependent manner. Cell. 1980 Jul;20(3):839–847. doi: 10.1016/0092-8674(80)90330-x. [DOI] [PubMed] [Google Scholar]
  5. Brown S. S., Yamamoto K., Spudich J. A. A 40,000-dalton protein from Dictyostelium discoideum affects assembly properties of actin in a Ca2+-dependent manner. J Cell Biol. 1982 Apr;93(1):205–210. doi: 10.1083/jcb.93.1.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cavalier-Smith T. Basal body and flagellar development during the vegetative cell cycle and the sexual cycle of Chlamydomonas reinhardii. J Cell Sci. 1974 Dec;16(3):529–556. doi: 10.1242/jcs.16.3.529. [DOI] [PubMed] [Google Scholar]
  7. Dentler W. L. Microtubule-membrane interactions in cilia and flagella. Int Rev Cytol. 1981;72:1–47. doi: 10.1016/s0074-7696(08)61193-6. [DOI] [PubMed] [Google Scholar]
  8. Fabiato A., Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75(5):463–505. [PubMed] [Google Scholar]
  9. Fisher G., Kaneshiro E. S., Peters P. D. Divalent cation affinity sites in Paramecium aurelia. J Cell Biol. 1976 May;69(2):429–442. doi: 10.1083/jcb.69.2.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Gilula N. B., Satir P. The ciliary necklace. A ciliary membrane specialization. J Cell Biol. 1972 May;53(2):494–509. doi: 10.1083/jcb.53.2.494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goodenough U. W. Motile detergent-extracted cells of Tetrahymena and Chlamydomonas. J Cell Biol. 1983 Jun;96(6):1610–1621. doi: 10.1083/jcb.96.6.1610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hasegawa T., Takahashi S., Hayashi H., Hatano S. Fragmin: a calcium ion sensitive regulatory factor on the formation of actin filaments. Biochemistry. 1980 Jun 10;19(12):2677–2683. doi: 10.1021/bi00553a021. [DOI] [PubMed] [Google Scholar]
  14. Huang B., Mengersen A., Lee V. D. Molecular cloning of cDNA for caltractin, a basal body-associated Ca2+-binding protein: homology in its protein sequence with calmodulin and the yeast CDC31 gene product. J Cell Biol. 1988 Jul;107(1):133–140. doi: 10.1083/jcb.107.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Keates R. A., Hallett F. R. Dynamic instability of sheared microtubules observed by quasi-elastic light scattering. Science. 1988 Sep 23;241(4873):1642–1645. doi: 10.1126/science.241.4873.1642. [DOI] [PubMed] [Google Scholar]
  16. Kupfer A., Louvard D., Singer S. J. Polarization of the Golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental wound. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2603–2607. doi: 10.1073/pnas.79.8.2603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lefebvre P. A., Rosenbaum J. L. Regulation of the synthesis and assembly of ciliary and flagellar proteins during regeneration. Annu Rev Cell Biol. 1986;2:517–546. doi: 10.1146/annurev.cb.02.110186.002505. [DOI] [PubMed] [Google Scholar]
  18. Lewin R. A., Lee T. H., Fang L. S. Effects of various agents on flagellar activity, flagellar autotomy and cell viability in four species of Chlamydomonas (chlorophyta: volvocales). Symp Soc Exp Biol. 1982;35:421–437. [PubMed] [Google Scholar]
  19. McDonald K. Osmium ferricyanide fixation improves microfilament preservation and membrane visualization in a variety of animal cell types. J Ultrastruct Res. 1984 Feb;86(2):107–118. doi: 10.1016/s0022-5320(84)80051-9. [DOI] [PubMed] [Google Scholar]
  20. McFadden G. I., Schulze D., Surek B., Salisbury J. L., Melkonian M. Basal body reorientation mediated by a Ca2+-modulated contractile protein. J Cell Biol. 1987 Aug;105(2):903–912. doi: 10.1083/jcb.105.2.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Melkonian M., Robenek H. Eyespot membranes of Chlamydomonas reinhardii: a freeze-fracture study. J Ultrastruct Res. 1980 Jul;72(1):90–102. doi: 10.1016/s0022-5320(80)90138-0. [DOI] [PubMed] [Google Scholar]
  22. Mitchison T., Kirschner M. Dynamic instability of microtubule growth. Nature. 1984 Nov 15;312(5991):237–242. doi: 10.1038/312237a0. [DOI] [PubMed] [Google Scholar]
  23. Mitchison T., Kirschner M. Microtubule assembly nucleated by isolated centrosomes. Nature. 1984 Nov 15;312(5991):232–237. doi: 10.1038/312232a0. [DOI] [PubMed] [Google Scholar]
  24. Ringo D. L. Flagellar motion and fine structure of the flagellar apparatus in Chlamydomonas. J Cell Biol. 1967 Jun;33(3):543–571. doi: 10.1083/jcb.33.3.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rosenbaum J. L., Carlson K. Cilia regeneration in Tetrahymena and its inhibition by colchicine. J Cell Biol. 1969 Feb;40(2):415–425. doi: 10.1083/jcb.40.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. SAGER R., GRANICK S. Nutritional control of sexuality in Chlamydomonas reinhardi. J Gen Physiol. 1954 Jul 20;37(6):729–742. doi: 10.1085/jgp.37.6.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Salisbury J. L., Baron A. T., Coling D. E., Martindale V. E., Sanders M. A. Calcium-modulated contractile proteins associated with the eucaryotic centrosome. Cell Motil Cytoskeleton. 1986;6(2):193–197. doi: 10.1002/cm.970060218. [DOI] [PubMed] [Google Scholar]
  28. Salisbury J. L., Baron A. T., Sanders M. A. The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells. J Cell Biol. 1988 Aug;107(2):635–641. doi: 10.1083/jcb.107.2.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Salisbury J. L., Baron A., Surek B., Melkonian M. Striated flagellar roots: isolation and partial characterization of a calcium-modulated contractile organelle. J Cell Biol. 1984 Sep;99(3):962–970. doi: 10.1083/jcb.99.3.962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Salisbury J. L., Sanders M. A., Harpst L. Flagellar root contraction and nuclear movement during flagellar regeneration in Chlamydomonas reinhardtii. J Cell Biol. 1987 Oct;105(4):1799–1805. doi: 10.1083/jcb.105.4.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stendahl O. I., Stossel T. P. Actin-binding protein amplifies actomyosin contraction, and gelsolin confers calcium control on the direction of contraction. Biochem Biophys Res Commun. 1980 Jan 29;92(2):675–681. doi: 10.1016/0006-291x(80)90386-1. [DOI] [PubMed] [Google Scholar]
  32. Stossel T. P., Chaponnier C., Ezzell R. M., Hartwig J. H., Janmey P. A., Kwiatkowski D. J., Lind S. E., Smith D. B., Southwick F. S., Yin H. L. Nonmuscle actin-binding proteins. Annu Rev Cell Biol. 1985;1:353–402. doi: 10.1146/annurev.cb.01.110185.002033. [DOI] [PubMed] [Google Scholar]
  33. Tamm S. L. Iontophoretic localization of Ca-sensitive sites controlling activation of ciliary beating in macrocilia of Beroë: the ciliary rete. Cell Motil Cytoskeleton. 1988;11(2):126–138. doi: 10.1002/cm.970110206. [DOI] [PubMed] [Google Scholar]
  34. Tao W., Walter R. J., Berns M. W. Laser-transected microtubules exhibit individuality of regrowth, however most free new ends of the microtubules are stable. J Cell Biol. 1988 Sep;107(3):1025–1035. doi: 10.1083/jcb.107.3.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thompson G. A., Jr, Baugh L. C., Walker L. F. Nonlethal deciliation of Tetrahymena by a local anesthetic and its utility as a tool for studying cilia regeneration. J Cell Biol. 1974 Apr;61(1):253–257. doi: 10.1083/jcb.61.1.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tilney L. G., Bryan J., Bush D. J., Fujiwara K., Mooseker M. S., Murphy D. B., Snyder D. H. Microtubules: evidence for 13 protofilaments. J Cell Biol. 1973 Nov;59(2 Pt 1):267–275. doi: 10.1083/jcb.59.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vigers G. P., Coue M., McIntosh J. R. Fluorescent microtubules break up under illumination. J Cell Biol. 1988 Sep;107(3):1011–1024. doi: 10.1083/jcb.107.3.1011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Weiss R. L., Goodenough D. A., Goodenough U. W. Membrane particle arrays associated with the basal body and with contractile vacuole secretion in Chlamydomonas. J Cell Biol. 1977 Jan;72(1):133–143. doi: 10.1083/jcb.72.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Witman G. B., Carlson K., Berliner J., Rosenbaum J. L. Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes. J Cell Biol. 1972 Sep;54(3):507–539. doi: 10.1083/jcb.54.3.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wright R. L., Salisbury J., Jarvik J. W. A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation. J Cell Biol. 1985 Nov;101(5 Pt 1):1903–1912. doi: 10.1083/jcb.101.5.1903. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES