Skip to main content
Genetics logoLink to Genetics
. 1998 Jun;149(2):927–936. doi: 10.1093/genetics/149.2.927

Genetics of the deflagellation pathway in Chlamydomonas.

R J Finst 1, P J Kim 1, L M Quarmby 1
PMCID: PMC1460167  PMID: 9611203

Abstract

Signal-induced deflagellation in Chlamydomonas involves Ca2+-activated breakage of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. In this study, we isolated 13 new deflagellation mutants that can be divided into two phenotypic classes, the Adf class and the Fa class. Cells with the Adf deflagellation phenotype are defective in acid-stimulated Ca2+ influx, but can be induced to deflagellate by treatment with nonionic detergent and Ca2+. Genetic analyses show that the five new Adf mutations, as well as the previously identified adf1 mutation, are alleles of the ADF1 gene. Mutants in the second phenotypic class, the Fa mutants, fail to deflagellate in response to any known chemical stimulus and are defective in Ca2+-activated microtubule severing. Genetic analysis of these eight new Fa strains demonstrated that they define two complementation groups, and one of these contains the previously identified fa1 mutation. Diploid analysis showed that five alleles map to the FA1 gene, whereas four alleles define a novel gene that we have named FA2. The isolation of multiple mutant alleles of each gene, generated by either ultraviolet irradiation or insertional mutagenesis, indicates that ADF1, FA1, and FA2 may be the only genes that can be identified in a loss-of-function screen. These alleles should provide a better understanding of the regulation of microtubule severing by Ca2+.

Full Text

The Full Text of this article is available as a PDF (230.1 KB).

Selected References

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

  1. Baker E. J., Diener D. R., Rosenbaum J. L. Accelerated poly(A) loss on alpha-tubulin mRNAs during protein synthesis inhibition in Chlamydomonas. J Mol Biol. 1989 Jun 20;207(4):771–781. doi: 10.1016/0022-2836(89)90243-x. [DOI] [PubMed] [Google Scholar]
  2. Baron A. T., Greenwood T. M., Bazinet C. W., Salisbury J. L. Centrin is a component of the pericentriolar lattice. Biol Cell. 1992;76(3):383–388. doi: 10.1016/0248-4900(92)90442-4. [DOI] [PubMed] [Google Scholar]
  3. Ebersold W. T. Chlamydomonas reinhardi: heterozygous diploid strains. Science. 1967 Jul 28;157(3787):447–449. doi: 10.1126/science.157.3787.447. [DOI] [PubMed] [Google Scholar]
  4. Evans J. H., Smith J. L., Keller L. R. Ion selectivity in the Chlamydomonas reinhardtii flagellar regeneration system. Exp Cell Res. 1997 Jan 10;230(1):94–102. doi: 10.1006/excr.1996.3410. [DOI] [PubMed] [Google Scholar]
  5. Fernández E., Schnell R., Ranum L. P., Hussey S. C., Silflow C. D., Lefebvre P. A. Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6449–6453. doi: 10.1073/pnas.86.17.6449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ferris P. J., Goodenough U. W. The mating-type locus of Chlamydomonas reinhardtii contains highly rearranged DNA sequences. Cell. 1994 Mar 25;76(6):1135–1145. doi: 10.1016/0092-8674(94)90389-1. [DOI] [PubMed] [Google Scholar]
  7. Ferris P. J. Localization of the nic-7, ac-29 and thi-10 genes within the mating-type locus of Chlamydomonas reinhardtii. Genetics. 1995 Oct;141(2):543–549. doi: 10.1093/genetics/141.2.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goodenough U. W., Hwang C., Martin H. Isolation and genetic analysis of mutant strains of Chlamydomonas reinhardi defective in gametic differentiation. Genetics. 1976 Feb;82(2):169–186. doi: 10.1093/genetics/82.2.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goodenough U. W., Hwang C., Warren A. J. Sex-limited expression of gene Loci controlling flagellar membrane agglutination in the chlamydomonas mating reaction. Genetics. 1978 Jun;89(2):235–243. doi: 10.1093/genetics/89.2.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goodenough U. W., Shames B., Small L., Saito T., Crain R. C., Sanders M. A., Salisbury J. L. The role of calcium in the Chlamydomonas reinhardtii mating reaction. J Cell Biol. 1993 Apr;121(2):365–374. doi: 10.1083/jcb.121.2.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hartzell L. B., Hartzell H. C., Quarmby L. M. Mechanisms of flagellar excision. I. The role of intracellular acidification. Exp Cell Res. 1993 Sep;208(1):148–153. doi: 10.1006/excr.1993.1232. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Huang B., Watterson D. M., Lee V. D., Schibler M. J. Purification and characterization of a basal body-associated Ca2+-binding protein. J Cell Biol. 1988 Jul;107(1):121–131. doi: 10.1083/jcb.107.1.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Karsenti E. Microtubule dynamics: severing microtubules in mitosis. Curr Biol. 1993 Apr 1;3(4):208–210. doi: 10.1016/0960-9822(93)90334-k. [DOI] [PubMed] [Google Scholar]
  15. Kindle K. L., Schnell R. A., Fernández E., Lefebvre P. A. Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J Cell Biol. 1989 Dec;109(6 Pt 1):2589–2601. doi: 10.1083/jcb.109.6.2589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Levy Y. Y., Lai E. Y., Remillard S. P., Heintzelman M. B., Fulton C. Centrin is a conserved protein that forms diverse associations with centrioles and MTOCs in Naegleria and other organisms. Cell Motil Cytoskeleton. 1996;33(4):298–323. doi: 10.1002/(SICI)1097-0169(1996)33:4<298::AID-CM6>3.0.CO;2-5. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Lohret T. A., McNally F. J., Quarmby L. M. A role for katanin-mediated axonemal severing during Chlamydomonas deflagellation. Mol Biol Cell. 1998 May;9(5):1195–1207. doi: 10.1091/mbc.9.5.1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. MINTZ R. H., LEWIN R. A. Studies on the flagella of algae. V. Serology of paralyzed mutants of Chlamydomonas. Can J Microbiol. 1954 Aug;1(1):65–67. doi: 10.1139/m55-009. [DOI] [PubMed] [Google Scholar]
  20. McNally F. J., Okawa K., Iwamatsu A., Vale R. D. Katanin, the microtubule-severing ATPase, is concentrated at centrosomes. J Cell Sci. 1996 Mar;109(Pt 3):561–567. doi: 10.1242/jcs.109.3.561. [DOI] [PubMed] [Google Scholar]
  21. McNally F. J., Vale R. D. Identification of katanin, an ATPase that severs and disassembles stable microtubules. Cell. 1993 Nov 5;75(3):419–429. doi: 10.1016/0092-8674(93)90377-3. [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. Nelson J. A., Savereide P. B., Lefebvre P. A. The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation. Mol Cell Biol. 1994 Jun;14(6):4011–4019. doi: 10.1128/mcb.14.6.4011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Paoletti A., Moudjou M., Paintrand M., Salisbury J. L., Bornens M. Most of centrin in animal cells is not centrosome-associated and centrosomal centrin is confined to the distal lumen of centrioles. J Cell Sci. 1996 Dec;109(Pt 13):3089–3102. doi: 10.1242/jcs.109.13.3089. [DOI] [PubMed] [Google Scholar]
  25. Pazour G. J., Sineshchekov O. A., Witman G. B. Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii. J Cell Biol. 1995 Oct;131(2):427–440. doi: 10.1083/jcb.131.2.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Quarmby L. M., Hartzell H. C. Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii. J Cell Biol. 1994 Mar;124(5):807–815. doi: 10.1083/jcb.124.5.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Quarmby L. M., Yueh Y. G., Cheshire J. L., Keller L. R., Snell W. J., Crain R. C. Inositol phospholipid metabolism may trigger flagellar excision in Chlamydomonas reinhardtii. J Cell Biol. 1992 Feb;116(3):737–744. doi: 10.1083/jcb.116.3.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rosenbaum J. L., Child F. M. Flagellar regeneration in protozoan flagellates. J Cell Biol. 1967 Jul;34(1):345–364. doi: 10.1083/jcb.34.1.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. SAGER R., GRANICK S. Nutritional studies with Chlamydomonas reinhardi. Ann N Y Acad Sci. 1953 Oct 14;56(5):831–838. doi: 10.1111/j.1749-6632.1953.tb30261.x. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Sanders M. A., Salisbury J. L. Centrin plays an essential role in microtubule severing during flagellar excision in Chlamydomonas reinhardtii. J Cell Biol. 1994 Mar;124(5):795–805. doi: 10.1083/jcb.124.5.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sanders M. A., Salisbury J. L. Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii. J Cell Biol. 1989 May;108(5):1751–1760. doi: 10.1083/jcb.108.5.1751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Satir B., Sale W. S., Satir P. Membrane renewal after dibucaine deciliation of Tetrahymena. Freeze-fracture technique, cilia, membrane structure. Exp Cell Res. 1976 Jan;97:83–91. doi: 10.1016/0014-4827(76)90657-1. [DOI] [PubMed] [Google Scholar]
  34. Stearns T., Evans L., Kirschner M. Gamma-tubulin is a highly conserved component of the centrosome. Cell. 1991 May 31;65(5):825–836. doi: 10.1016/0092-8674(91)90390-k. [DOI] [PubMed] [Google Scholar]
  35. Stearns T., Winey M. The cell center at 100. Cell. 1997 Oct 31;91(3):303–309. doi: 10.1016/s0092-8674(00)80414-6. [DOI] [PubMed] [Google Scholar]
  36. Taillon B. E., Adler S. A., Suhan J. P., Jarvik J. W. Mutational analysis of centrin: an EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas. J Cell Biol. 1992 Dec;119(6):1613–1624. doi: 10.1083/jcb.119.6.1613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tam L. W., Lefebvre P. A. Cloning of flagellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis. Genetics. 1993 Oct;135(2):375–384. doi: 10.1093/genetics/135.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Vale R. D. Severing of stable microtubules by a mitotically activated protein in Xenopus egg extracts. Cell. 1991 Feb 22;64(4):827–839. doi: 10.1016/0092-8674(91)90511-v. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Yueh Y. G., Crain R. C. Deflagellation of Chlamydomonas reinhardtii follows a rapid transitory accumulation of inositol 1,4,5-trisphosphate and requires Ca2+ entry. J Cell Biol. 1993 Nov;123(4):869–875. doi: 10.1083/jcb.123.4.869. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES