Advertisement
Journal Article
Fa1p is a 171 kDa protein essential for axonemal microtubule severing in Chlamydomonas
R.J. Finst, P.J. Kim, E.R. Griffis, L.M. Quarmby
Journal of Cell Science 2000 113: 1963-1971;

Summary

A key event in deflagellation or deciliation is the severing of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. Previous genetic analysis revealed three genes that are essential for deflagellation in Chlamydomonas. We have now identified the first of these products, Fa1p, a protein required for Ca(2+)-dependent, axonemal microtubule severing. Genetic mapping and the availability of a tagged allele allowed us to physically map the gene to the centromere-proximal domain of the mating-type locus. We identified clones of Chlamydomonas genomic DNA that rescued the Ca(2+)-dependent axonemal microtubule severing defect of fa1 mutants. The FA1 cDNA, obtained by RT-PCR, encodes a novel protein of 171 kDa, which is predicted to contain an amino-terminal coiled-coil domain and three Ca(2+)/calmodulin binding domains. By western analysis and subcellular fractionation, the FA1 product is enriched in flagellar-basal body complexes. Based on these observations and previous studies, we hypothesize that a Ca(2+)-activated, Ca(2+)-binding protein binds Fa1p leading ultimately to the activation of axonemal microtubule severing.

REFERENCES

    1. Altschul, S. F.,
    2. Madden, T. L.,
    3. Schäffer, A. A.,
    4. Zhang, J.,
    5. Zhang, Z.,
    6. Miller, W. and
    7. Lipman, D. J.
    (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res 25, 3389–3402
    OpenUrlAbstract/FREE Full Text
    1. Anderson, R. G. W.
    (1974). Isolation of ciliated or unciliated basal bodies from the rabbit oviduct. J. Cell Biol 60, 393–404
    OpenUrlAbstract/FREE Full Text
    1. Auclair, W. and
    2. Siegel, B. W.
    (1966). Cilia regeneration in the sea urchin embryo: evidence for a pool of ciliary proteins. Science 154, 913–915
    OpenUrlAbstract/FREE Full Text
    1. Beck, K. and
    2. Brodsky, B.
    (1998). Supercoiled protein motifs: the collagen triple-helix and alpha-helical coiled-coil. J. Struct. Biol 122, 17–29
    OpenUrlCrossRefPubMedWeb of Science
    1. Debuchy, R.,
    2. Purton, S. and
    3. Rochaix, J. D.
    (1989). 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 8, 2803–2809
    OpenUrlPubMedWeb of Science
    1. Eyal, Y.,
    2. Neumann, H.,
    3. Or, E. and
    4. Frydman, A.
    (1999). Inverse single-strand RACE: An adaptor-independent method of 5RACE. BioTechniques 27, 656–658
    OpenUrlPubMedWeb of Science
    1. Fernandez, E.,
    2. Schnell, R.,
    3. Ranum, L. P. W.,
    4. Hussey, S. C.,
    5. Silflow, C. D. and
    6. Lefebvre, P. A.
    (1989). Isolation and characterization of the nitrate reductase structural gene of Chlamydomonasreinhardtii. Proc. Nat. Acad. Sci. USA 86, 6449–6453
    OpenUrlAbstract/FREE Full Text
    1. Ferris, P. J. and
    2. Goodenough, U. W.
    (1994). The mating-type locus of Chlamydomonas reinhardtii contains highly rearranged DNA sequences. Cell 76, 1135–1145
    OpenUrlCrossRefPubMedWeb of Science
    1. Finst, R. J.,
    2. Kim, P. J. and
    3. Quarmby, L. M.
    (1998). Genetics of the deflagellation pathway in Chlamydomonas. Genetics 149, 927–936
    OpenUrlAbstract/FREE Full Text
    1. Gitelman, S. E. and
    2. Witman, G. B.
    (1980). Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella. J. Cell Biol 87, 764–70
    OpenUrlAbstract/FREE Full Text
    1. Hartman, J. J.,
    2. Mahr, J.,
    3. McNally, K.,
    4. Okawa, K.,
    5. Iwamatsu, A.,
    6. Thomas, S.,
    7. Cheesman, S.,
    8. Heuser, J.,
    9. Vale, R. D. and
    10. McNally, F. J.
    (1998). Katanin, a microtubule-severing protein, is a novel AAA ATPase that target to the centrosome using a WD40-containing subunit. Cell 93, 277–287
    OpenUrlCrossRefPubMedWeb of Science
    1. Hastie, A. T.,
    2. Dicker, D. T.,
    3. Hingley, S. T.,
    4. Kueppers, F.,
    5. Higgins, M. L. and
    6. Weinbaum, G.
    (1986). Isolation of cilia from porcine tracheal epithelium and extraction of dynein arms. Cell Motil. Cytoskel 6, 25–34
    OpenUrlCrossRefPubMedWeb of Science
    1. Jarvik, J. W. and
    2. Suhan, J. P.
    (1991). The role of the flagellar transition region: Inferences from the analysis of a Chlamydomonas mutant with defective transition region structures. J. Cell Sci 99, 731–740
    OpenUrlAbstract/FREE Full Text
    1. Heimburg, T.,
    2. Schunemann, J.,
    3. Weber, K. and
    4. Geisler, N.
    (1999). FTIR-spectroscopy of multistranded coiled coil proteins. Biochemistry 38, 12727–12734
    OpenUrlCrossRefPubMed
    1. King, S. M.,
    2. Otter, T. and
    3. Witman, G. B.
    (1986). Purification and characterization of Chlamydomonas flagellar dyneins. Meth. Enzymol 134, 291–305
    OpenUrlCrossRefPubMed
    1. Kozminski, K. G.,
    2. Diener, D. R. and
    3. Rosenbaum, J. L.
    (1993). High level expression of nonacetylatable-tubulin in Chlamydomonas reinhardtii. Cell Motil. Cytoskel 25, 158–170
    OpenUrlCrossRefPubMedWeb of Science
    1. LeDizet, M. and
    2. Piperno, G.
    (1995). 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 6, 713–723
    OpenUrlAbstract/FREE Full Text
    1. Lefebvre, P. A. and
    2. Rosenbaum, J. L.
    (1986). Regulation of the synthesis and assembly of ciliary and flagellar proteins during regeneration. Annu. Rev. Cell Biol 2, 517–546
    OpenUrlCrossRef
    1. Lewin, R. A.
    (1953). Studies on the flagella of algae. II. Formation of flagellaby Chlamydomonas in light and darkness. Ann. NY Acad. Sci 56, 1091–1093
    OpenUrlCrossRefPubMed
    1. Lohret, T. A.,
    2. McNally, F. J. and
    3. Quarmby, L. M.
    (1998). A role for katanin-mediated axonemal severing during Chlamydomonas deflagellation. Mol. Biol. Cell 9, 421–437
    OpenUrlAbstract/FREE Full Text
    1. Lohret, T. A.,
    2. Zhao, L. and
    3. Quarmby, L. M.
    (1999). Cloning of Chlamydomonas p60 katanin and localization to the site of outer doublet severing during deflagellation. Cell Motil. Cytoskel 43, 221–231
    OpenUrlCrossRefPubMedWeb of Science
    1. Lupas, A.,
    2. Van Dyke, M. and
    3. Stock, J.
    (1991). Predicting coiled coils from protein sequences. Science 252, 1162–1164
    OpenUrlFREE Full Text
    1. McNally, F. J. and
    2. Vale, R. D.
    (1993). Identification of katanin, an ATPase that severs and disassembles stable microtubules. Cell 75, 419–429
    OpenUrlCrossRefPubMedWeb of Science
    1. Mohammed, B. J.,
    2. Mitchell, T. J.,
    3. Andrew, P. W.,
    4. Hirst, R. A. and
    5. O'Callaghan, C.
    (1999). The effect of the pneumococcal toxin, pheumolysin on brain ependymal cilia. Microb. Pathog 27, 303–309
    OpenUrlCrossRefPubMedWeb of Science
    1. Pazour, G. J.,
    2. Sineshchekov, O. A. and
    3. Witman, G. B.
    (1995). Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii. J. Cell Biol 131, 427–440
    OpenUrlAbstract/FREE Full Text
    1. Quarmby, L. M. and
    2. Hartzell, H. C.
    (1994). Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii. J. Cell Biol 124, 807–815
    OpenUrlAbstract/FREE Full Text
    1. Quarmby, L. M.
    (1996). Ca2+influx activated by low pH in Chlamydomonas. J. Gen. Physiol 108, 351–361
    OpenUrlAbstract/FREE Full Text
    1. Quarmby, L. M. and
    2. Lohret, T. A.
    (1999). Microtubule severing. Cell Motil. Cytoskel 43, 1–9
    OpenUrlCrossRefPubMedWeb of Science
    1. Rhoads, A. R. and
    2. Friedberg, F.
    (1997). Sequence motifs for calmodulin recognition. FASEBJ 11, 331–340
    OpenUrlAbstract
    1. Romero, M. F.,
    2. Kanai, Y.,
    3. Gunshin, H. and
    4. Hediger, M. A.
    (1998). Expression cloning using Xenopus laevis oocytes. Meth. Enzymol 296, 17–52
    OpenUrlCrossRefPubMedWeb of Science
    1. Sager, R. and
    2. Granick, S.
    (1953). Nutritional studies with Chlamydomonas reinhardtii. Ann. NY Acad. Sci 56, 831–838
    OpenUrlCrossRefPubMedWeb of Science
    1. Sanders, M. A. and
    2. Salisbury, J. L.
    (1989). Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii. J. Cell Biol 108, 1751–1760
    OpenUrlAbstract/FREE Full Text
    1. Sanders, M. A. and
    2. Salisbury, J. L.
    (1994). Centrin plays an essential role in microtubule severing during flagellar excision in Chlamydomonas reinhardtii. J. Cell Biol 108, 795–806
    OpenUrl
    1. Shoeman, R. L. and
    2. Traub, P.
    (1993). Assembly of intermediate filaments. BioEssays 15, 605–611
    OpenUrlCrossRefPubMedWeb of Science
    1. Smith, E. F. and
    2. Sale, W. S.
    (1991). Microtubule binding and translocation by inner dynein arm subtype I1. Cell Motil. Cytoskel 18, 258–268
    OpenUrlCrossRefPubMedWeb of Science
    1. Stephens, R. E.
    (1975). The basal apparatus. Mass isolation from the molluscan ciliated gill epithelium and a preliminary characterization of striated rootlets. J. Cell Biol 64, 408–420
    OpenUrlAbstract/FREE Full Text
    1. Taillon, B. E.,
    2. Adler, S. A.,
    3. Suhan, J. P. and
    4. Jarvik, J. W.
    (1992). Mutational analysis of centrin: an EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas. J. Cell Biol 119, 1613–1624
    OpenUrlAbstract/FREE Full Text
    1. Vale, R.
    (1991). Severing of stable microtubules by a mitotically activated protein in Xenopus egg extracts. Cell 64, 827–839
    OpenUrlCrossRefPubMedWeb of Science
    1. Watson, M. R. and
    2. Hopkins, J. M.
    (1962). Isolated cilia from Tetrahymena pyriformis. Exp. Cell Res 28, 280–295
    OpenUrlCrossRefPubMedWeb of Science
    1. Wilkerson, C. G.,
    2. King, S. M. and
    3. Witman, G. B.
    (1994). Molecular analysis of theheavy chain of Chlamydomonas flagellar outer-arm dynein. J. Cell Sci 107, 497–506
    OpenUrlAbstract/FREE Full Text
    1. Witman, G. B.
    (1986). Isolation of Chlamydomonas flagella and flagellar axomenes. Meth. Enzymol 134, 280–290
    OpenUrlCrossRefPubMedWeb of Science
Back to top

 Download PDF

Email
Journal Article
Fa1p is a 171 kDa protein essential for axonemal microtubule severing in Chlamydomonas
R.J. Finst, P.J. Kim, E.R. Griffis, L.M. Quarmby
Journal of Cell Science 2000 113: 1963-1971;
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
Alerts

Article navigation

Other journals from The Company of Biologists

Advertisement

Follow us on Instagram

Cell science is bursting with beautiful images and over on Instagram, we’re showing them off! Find both JCS and FocalPlane on Instagram for stories and techniques across cell biology.

An interview with Derek Walsh

Professor Derek Walsh is the guest editor of our new special issue Cell Biology of Host-Pathogen Interactions. In an interview, Derek tells us about his work in the field of DNA viruses, the impact of the pandemic on virology and what his role as Guest Editor taught him.

How to improve your scientific writing

"If you are a scientist and you want to succeed, you must become a writer."

How do scientists become master storytellers? We called on our journal Editors, proofreaders and contributors to our community sites for their advice on how to improve your scientific writing.

Meet the preLighters: Jennifer Ann Black

Following the theme of our latest special issue, postdoc Jennifer Ann Black studies replication stress and genome plasticity in Leishmania in Professor Luiz Tosi’s lab in Sao Paolo. We caught up with Jenn (virtually) to hear about her relocation to Brazil mid-pandemic, her research on parasites and what she enjoys about ‘preLighting’.

In our special issue, Chandrakar et al. and Rosazza et al. present their latest work on Leishmania.

Mole – The Corona Files

“There are millions of people around the world who continue to believe that the Terrible Pandemic is a hoax.”

Mole continues to offer his wise words to researchers on how to manage during the COVID-19 pandemic.

JCS and COVID-19

For more information on measures Journal of Cell Science is taking to support the community during the COVID-19 pandemic, please see here.

If you have any questions or concerns, please do not hestiate to contact the Editorial Office.