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. 1996 Feb 1;132(3):359–370. doi: 10.1083/jcb.132.3.359

PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella

PMCID: PMC2120723  PMID: 8636214

Abstract

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

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Selected References

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  1. Adams G. M., Huang B., Piperno G., Luck D. J. Central-pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants. J Cell Biol. 1981 Oct;91(1):69–76. doi: 10.1083/jcb.91.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allen C., Borisy G. G. Structural polarity and directional growth of microtubules of Chlamydomonas flagella. J Mol Biol. 1974 Dec 5;90(2):381–402. doi: 10.1016/0022-2836(74)90381-7. [DOI] [PubMed] [Google Scholar]
  3. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  4. Behnke O., Forer A. Evidence for four classes of microtubules in individual cells. J Cell Sci. 1967 Jun;2(2):169–192. doi: 10.1242/jcs.2.2.169. [DOI] [PubMed] [Google Scholar]
  5. Bernstein M., Beech P. L., Katz S. G., Rosenbaum J. L. A new kinesin-like protein (Klp1) localized to a single microtubule of the Chlamydomonas flagellum. J Cell Biol. 1994 Jun;125(6):1313–1326. doi: 10.1083/jcb.125.6.1313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Binder L. I., Dentler W. L., Rosenbaum J. L. Assembly of chick brain tubulin onto flagellar microtubules from Chlamydomonas and sea urchin sperm. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1122–1126. doi: 10.1073/pnas.72.3.1122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Brokaw C. J., Nagayama S. M. Modulation of the asymmetry of sea urchin sperm flagellar bending by calmodulin. J Cell Biol. 1985 Jun;100(6):1875–1883. doi: 10.1083/jcb.100.6.1875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chisholm D. A convenient moderate-scale procedure for obtaining DNA from bacteriophage lambda. Biotechniques. 1989 Jan;7(1):21–23. [PubMed] [Google Scholar]
  10. Cuomo C. A., Kirch S. A., Gyuris J., Brent R., Oettinger M. A. Rch1, a protein that specifically interacts with the RAG-1 recombination-activating protein. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6156–6160. doi: 10.1073/pnas.91.13.6156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Debuchy R., Purton S., Rochaix J. D. The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus. EMBO J. 1989 Oct;8(10):2803–2809. doi: 10.1002/j.1460-2075.1989.tb08426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dutcher S. K. Flagellar assembly in two hundred and fifty easy-to-follow steps. Trends Genet. 1995 Oct;11(10):398–404. doi: 10.1016/s0168-9525(00)89123-4. [DOI] [PubMed] [Google Scholar]
  14. Dutcher S. K., Huang B., Luck D. J. Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii. J Cell Biol. 1984 Jan;98(1):229–236. doi: 10.1083/jcb.98.1.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Euteneuer U., McIntosh J. R. Polarity of some motility-related microtubules. Proc Natl Acad Sci U S A. 1981 Jan;78(1):372–376. doi: 10.1073/pnas.78.1.372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Fox L. A., Sawin K. E., Sale W. S. Kinesin-related proteins in eukaryotic flagella. J Cell Sci. 1994 Jun;107(Pt 6):1545–1550. doi: 10.1242/jcs.107.6.1545. [DOI] [PubMed] [Google Scholar]
  19. Franke W. W., Goldschmidt M. D., Zimbelmann R., Mueller H. M., Schiller D. L., Cowin P. Molecular cloning and amino acid sequence of human plakoglobin, the common junctional plaque protein. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4027–4031. doi: 10.1073/pnas.86.11.4027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Görlich D., Prehn S., Laskey R. A., Hartmann E. Isolation of a protein that is essential for the first step of nuclear protein import. Cell. 1994 Dec 2;79(5):767–778. doi: 10.1016/0092-8674(94)90067-1. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Howard D. R., Habermacher G., Glass D. B., Smith E. F., Sale W. S. Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase. J Cell Biol. 1994 Dec;127(6 Pt 1):1683–1692. doi: 10.1083/jcb.127.6.1683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. James S. W., Ranum L. P., Silflow C. D., Lefebvre P. A. Mutants resistant to anti-microtubule herbicides map to a locus on the uni linkage group in Chlamydomonas reinhardtii. Genetics. 1988 Jan;118(1):141–147. doi: 10.1093/genetics/118.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Johnson K. A., Haas M. A., Rosenbaum J. L. Localization of a kinesin-related protein to the central pair apparatus of the Chlamydomonas reinhardtii flagellum. J Cell Sci. 1994 Jun;107(Pt 6):1551–1556. doi: 10.1242/jcs.107.6.1551. [DOI] [PubMed] [Google Scholar]
  28. Kindle K. L. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1228–1232. doi: 10.1073/pnas.87.3.1228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Küssel P., Frasch M. Pendulin, a Drosophila protein with cell cycle-dependent nuclear localization, is required for normal cell proliferation. J Cell Biol. 1995 Jun;129(6):1491–1507. doi: 10.1083/jcb.129.6.1491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. LeDizet M., Piperno G. The light chain p28 associates with a subset of inner dynein arm heavy chains in Chlamydomonas axonemes. Mol Biol Cell. 1995 Jun;6(6):697–711. doi: 10.1091/mbc.6.6.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. LeDizet M., Piperno G. ida4-1, ida4-2, and ida4-3 are intron splicing mutations affecting the locus encoding p28, a light chain of Chlamydomonas axonemal inner dynein arms. Mol Biol Cell. 1995 Jun;6(6):713–723. doi: 10.1091/mbc.6.6.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Lux F. G., 3rd, Dutcher S. K. Genetic interactions at the FLA10 locus: suppressors and synthetic phenotypes that affect the cell cycle and flagellar function in Chlamydomonas reinhardtii. Genetics. 1991 Jul;128(3):549–561. doi: 10.1093/genetics/128.3.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McCrea P. D., Turck C. W., Gumbiner B. A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin. Science. 1991 Nov 29;254(5036):1359–1361. doi: 10.1126/science.1962194. [DOI] [PubMed] [Google Scholar]
  37. Miller J. M., Wang W., Balczon R., Dentler W. L. Ciliary microtubule capping structures contain a mammalian kinetochore antigen. J Cell Biol. 1990 Mar;110(3):703–714. doi: 10.1083/jcb.110.3.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Omoto C. K., Kung C. The pair of central tubules rotates during ciliary beat in Paramecium. Nature. 1979 Jun 7;279(5713):532–534. doi: 10.1038/279532a0. [DOI] [PubMed] [Google Scholar]
  41. Peifer M., Berg S., Reynolds A. B. A repeating amino acid motif shared by proteins with diverse cellular roles. Cell. 1994 Mar 11;76(5):789–791. doi: 10.1016/0092-8674(94)90353-0. [DOI] [PubMed] [Google Scholar]
  42. Peifer M., Wieschaus E. The segment polarity gene armadillo encodes a functionally modular protein that is the Drosophila homolog of human plakoglobin. Cell. 1990 Dec 21;63(6):1167–1176. doi: 10.1016/0092-8674(90)90413-9. [DOI] [PubMed] [Google Scholar]
  43. Piperno G., Huang B., Ramanis Z., Luck D. J. Radial spokes of Chlamydomonas flagella: polypeptide composition and phosphorylation of stalk components. J Cell Biol. 1981 Jan;88(1):73–79. doi: 10.1083/jcb.88.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. Porter M. E., Knott J. A., Gardner L. C., Mitchell D. R., Dutcher S. K. Mutations in the SUP-PF-1 locus of Chlamydomonas reinhardtii identify a regulatory domain in the beta-dynein heavy chain. J Cell Biol. 1994 Sep;126(6):1495–1507. doi: 10.1083/jcb.126.6.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. RANDALL J., WARR J. R., HOPKINS J. M., MCVITTIE A. A SINGLE-GENE MUTATION OF CHLAMYDOMONAS REINHARDII AFFECTING MOTILITY: A GENETIC AND ELECTRON MICROSCOPE STUDY. Nature. 1964 Aug 29;203:912–914. doi: 10.1038/203912a0. [DOI] [PubMed] [Google Scholar]
  48. Riggleman B., Wieschaus E., Schedl P. Molecular analysis of the armadillo locus: uniformly distributed transcripts and a protein with novel internal repeats are associated with a Drosophila segment polarity gene. Genes Dev. 1989 Jan;3(1):96–113. doi: 10.1101/gad.3.1.96. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Sanders M. A., Salisbury J. L. Immunofluorescence microscopy of cilia and flagella. Methods Cell Biol. 1995;47:163–169. doi: 10.1016/s0091-679x(08)60805-5. [DOI] [PubMed] [Google Scholar]
  51. Schnell R. A., Lefebvre P. A. Isolation of the Chlamydomonas regulatory gene NIT2 by transposon tagging. Genetics. 1993 Jul;134(3):737–747. doi: 10.1093/genetics/134.3.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Shelanski M. L., Taylor E. W. Isolation of a protein subunit from microtubules. J Cell Biol. 1967 Aug;34(2):549–554. doi: 10.1083/jcb.34.2.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. Talian J. C., Olmsted J. B., Goldman R. D. A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture. J Cell Biol. 1983 Oct;97(4):1277–1282. doi: 10.1083/jcb.97.4.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. Tamm S. L., Tamm S. Ciliary reversal without rotation of axonemal structures in ctenophore comb plates. J Cell Biol. 1981 Jun;89(3):495–509. doi: 10.1083/jcb.89.3.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Török I., Strand D., Schmitt R., Tick G., Török T., Kiss I., Mechler B. M. The overgrown hematopoietic organs-31 tumor suppressor gene of Drosophila encodes an Importin-like protein accumulating in the nucleus at the onset of mitosis. J Cell Biol. 1995 Jun;129(6):1473–1489. doi: 10.1083/jcb.129.6.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Warner F. D., Satir P. The structural basis of ciliary bend formation. Radial spoke positional changes accompanying microtubule sliding. J Cell Biol. 1974 Oct;63(1):35–63. doi: 10.1083/jcb.63.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wilkerson C. G., King S. M., Witman G. B. Molecular analysis of the gamma heavy chain of Chlamydomonas flagellar outer-arm dynein. J Cell Sci. 1994 Mar;107(Pt 3):497–506. doi: 10.1242/jcs.107.3.497. [DOI] [PubMed] [Google Scholar]
  60. 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]
  61. 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]
  62. 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]
  63. Yano R., Oakes M., Yamaghishi M., Dodd J. A., Nomura M. Cloning and characterization of SRP1, a suppressor of temperature-sensitive RNA polymerase I mutations, in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Dec;12(12):5640–5651. doi: 10.1128/mcb.12.12.5640. [DOI] [PMC free article] [PubMed] [Google Scholar]

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