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. 1984 Sep 1;99(3):962–970. doi: 10.1083/jcb.99.3.962

Striated flagellar roots: isolation and partial characterization of a calcium-modulated contractile organelle

PMCID: PMC2113404  PMID: 6381510

Abstract

We report the isolation of striated flagellar roots from the Prasinophycean green alga Tetraselmis striata using sedimentation in gradients of sucrose and flotation on gradients of colloidal silica. PAGE in the presence of 0.1% SDS demonstrates that striated flagellar roots are composed of a number of polypeptides, the most predominant one being a protein of 20,000 Mr. The 20,000 Mr protein band represents approximately 63% of the Coomassie Brilliant Blue staining of gels of isolated flagellar roots. Two-dimensional gel electrophoresis (isoelectric focusing and SDS PAGE) resolves the major 20,000 Mr flagellar root protein into two components of nearly identical Mr, but of differing isoelectric points (i.e., pl's of 4.9 and 4.8), which we have designated 20,000-Mr-alpha and 20,000-Mr-beta, respectively. Densitometric scans of two-dimensional gels of cell extracts indicate that the 20,000-Mr-alpha and -beta polypeptides vary, in their stoichiometry, between 2:1 and 1:1. This variability appears to be related to the state of contraction or extension of the striated flagellar roots at the time of cell lysis. Incubation of cells with 32PO4 followed by analysis of cell extracts by two-dimensional gel electrophoresis and autoradiography reveals that the more acidic 20,000- Mr-beta component is phosphorylated and the 20,000-Mr-alpha component contains no detectable label. These results suggest that the 20,000-Mr- alpha component is converted to the more acidic 20,000-Mr-beta form by phosphorylation. Both the 20,000-Mr-alpha and -beta flagellar root components exhibit a calcium-induced reduction in relative electrophoretic mobilities in two-dimensional alkaline urea gels. Antiserum raised in rabbits against the 20,000-Mr protein binds to both the 20,000-Mr-alpha and 20,000-Mr-beta forms of the flagellar root protein when analyzed by electrophoretic immunoblot techniques. Indirect immunofluorescence on vegetative or interphase cells demonstrate that the antibodies bind to two cyclindrical organelles located in the anterior region of the cell. Immunocytochemical investigations at ultrastructural resolution using this antiserum and a colloidal gold-conjugated antirabbit-IgG reveals immunospecific labeling of striated flagellar roots and their extensions. We conclude that striated flagellar roots are simple ion-sensitive contractile organelles composed predominantly of a 20,000 Mr calcium-binding phosphoprotein, and that this protein is largely responsible for the motile behavior of these organelles.

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Selected References

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  1. Adelstein R. S., Eisenberg E. Regulation and kinetics of the actin-myosin-ATP interaction. Annu Rev Biochem. 1980;49:921–956. doi: 10.1146/annurev.bi.49.070180.004421. [DOI] [PubMed] [Google Scholar]
  2. Amos W. B. Reversible mechanochemical cycle in the contraction of Vorticella. Nature. 1971 Jan 8;229(5280):127–128. doi: 10.1038/229127a0. [DOI] [PubMed] [Google Scholar]
  3. Amos W. B., Routledge L. M., Yew F. F. Calcium-binding proteins in a vorticellid contractile organelle. J Cell Sci. 1975 Oct;19(1):203–213. doi: 10.1242/jcs.19.1.203. [DOI] [PubMed] [Google Scholar]
  4. Anderson R. G. Biochemical and cytochemical evidence for ATPase activity in basal bodies isolated from oviduct. J Cell Biol. 1977 Aug;74(2):547–560. doi: 10.1083/jcb.74.2.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beckerle M. C., Porter K. R. Analysis of the role of microtubules and actin in erythrophore intracellular motility. J Cell Biol. 1983 Feb;96(2):354–362. doi: 10.1083/jcb.96.2.354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cachon J., Cachon M. Movement by non-actin filament mechanisms. Biosystems. 1981;14(3-4):313–326. doi: 10.1016/0303-2647(81)90038-1. [DOI] [PubMed] [Google Scholar]
  7. Cachon J., Cachon M., Tilney L. G., Tilney M. S. Movement generated by interactions between the dense material at the ends of microtubles and non-actin-containing microfilaments in Sticholonche zanclea. J Cell Biol. 1977 Feb;72(2):314–338. doi: 10.1083/jcb.72.2.314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheung W. Y. Calmodulin plays a pivotal role in cellular regulation. Science. 1980 Jan 4;207(4426):19–27. doi: 10.1126/science.6243188. [DOI] [PubMed] [Google Scholar]
  9. Dabrowska R., Sherry J. M., Aromatorio D. K., Hartshorne D. J. Modulator protein as a component of the myosin light chain kinase from chicken gizzard. Biochemistry. 1978 Jan 24;17(2):253–258. doi: 10.1021/bi00595a010. [DOI] [PubMed] [Google Scholar]
  10. De Mey J., Moeremans M., Geuens G., Nuydens R., De Brabander M. High resolution light and electron microscopic localization of tubulin with the IGS (immuno gold staining) method. Cell Biol Int Rep. 1981 Sep;5(9):889–899. doi: 10.1016/0309-1651(81)90204-6. [DOI] [PubMed] [Google Scholar]
  11. Ebashi S., Endo M. Calcium ion and muscle contraction. Prog Biophys Mol Biol. 1968;18:123–183. doi: 10.1016/0079-6107(68)90023-0. [DOI] [PubMed] [Google Scholar]
  12. Gitelman S. E., Witman G. B. Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella. J Cell Biol. 1980 Dec;87(3 Pt 1):764–770. doi: 10.1083/jcb.87.3.764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Head J. F., Perry S. V. The interaction of the calcium-binding protein (troponin C) with bivalent cations and the inhibitory protein (troponin I). Biochem J. 1974 Feb;137(2):145–154. doi: 10.1042/bj1370145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Klee C. B., Crouch T. H., Richman P. G. Calmodulin. Annu Rev Biochem. 1980;49:489–515. doi: 10.1146/annurev.bi.49.070180.002421. [DOI] [PubMed] [Google Scholar]
  15. Kretsinger R. H. Structure and evolution of calcium-modulated proteins. CRC Crit Rev Biochem. 1980;8(2):119–174. doi: 10.3109/10409238009105467. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Larson D. E., Dingle A. D. Isolation, ultrastructure, and protein composition of the flagellar rootlet of Naegleria gruberi. J Cell Biol. 1981 Jun;89(3):424–432. doi: 10.1083/jcb.89.3.424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maihle N. J., Dedman J. R., Means A. R., Chafouleas J. G., Satir B. H. Presence and indirect immunofluorescent localization of calmodulin in Paramecium tetraurelia. J Cell Biol. 1981 Jun;89(3):695–699. doi: 10.1083/jcb.89.3.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Means A. R., Chafouleas J. G. Regulation by and of calmodulin in mammalian cells. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):903–908. doi: 10.1101/sqb.1982.046.01.084. [DOI] [PubMed] [Google Scholar]
  20. Melkonian M. Ultrastructural aspects of basal body associated fibrous structures in green algae: a critical review. Biosystems. 1980;12(1-2):85–104. doi: 10.1016/0303-2647(80)90040-4. [DOI] [PubMed] [Google Scholar]
  21. Naito Y., Kaneko H. Reactivated triton-extracted models o paramecium: modification of ciliary movement by calcium ions. Science. 1972 May 5;176(4034):523–524. doi: 10.1126/science.176.4034.523. [DOI] [PubMed] [Google Scholar]
  22. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  23. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  24. Plancke Y. D., Lazarides E. Evidence for a phosphorylated form of calmodulin in chicken brain and muscle. Mol Cell Biol. 1983 Aug;3(8):1412–1420. doi: 10.1128/mcb.3.8.1412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. SJOSTRAND F. S. The ultrastructure of the innersegments of the retinal rods of the guinea pig eye as revealed by electron microscopy. J Cell Physiol. 1953 Aug;42(1):45–70. doi: 10.1002/jcp.1030420104. [DOI] [PubMed] [Google Scholar]
  26. Salisbury J. L. Calcium-sequestering vesicles and contractile flagellar roots. J Cell Sci. 1982 Dec;58:433–443. doi: 10.1242/jcs.58.1.433. [DOI] [PubMed] [Google Scholar]
  27. Salisbury J. L., Floyd G. L. Calcium-induced contraction of the rhizoplast of a quadriflagellate green alga. Science. 1978 Dec 1;202(4371):975–977. doi: 10.1126/science.202.4371.975. [DOI] [PubMed] [Google Scholar]
  28. Sleigh M. Contractility of the roots of flagella and cilia. Nature. 1979 Jan 25;277(5694):263–264. doi: 10.1038/277263a0. [DOI] [PubMed] [Google Scholar]
  29. Stephens R. E. The basal apparatus. Mass isolation from the molluscan ciliated gill epithelium and a preliminary characterization of striated rootlets. J Cell Biol. 1975 Feb;64(2):408–420. doi: 10.1083/jcb.64.2.408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tucker R. W., Pardee A. B., Fujiwara K. Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells. Cell. 1979 Jul;17(3):527–535. doi: 10.1016/0092-8674(79)90261-7. [DOI] [PubMed] [Google Scholar]
  32. Waisman D. M., Singh T. J., Wang J. H. The modulator-dependent protein kinase. A multifunctional protein kinase activatable by the Ca2+-dependent modulator protein of the cyclic nucleotide system. J Biol Chem. 1978 May 25;253(10):3387–3390. [PubMed] [Google Scholar]
  33. Welsh M. J., Dedman J. R., Brinkley B. R., Means A. R. Calcium-dependent regulator protein: localization in mitotic apparatus of eukaryotic cells. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1867–1871. doi: 10.1073/pnas.75.4.1867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wolniak S. M., Hepler P. K., Jackson W. T. Ionic changes in the mitotic apparatus at the metaphase/anaphase transition. J Cell Biol. 1983 Mar;96(3):598–605. doi: 10.1083/jcb.96.3.598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yagi K., Yazawa M., Kakiuchi S., Ohshima M., Uenishi K. Identification of an activator protein for myosin light chain kinase as the Ca2+-dependent modulator protein. J Biol Chem. 1978 Mar 10;253(5):1338–1340. [PubMed] [Google Scholar]

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