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J Cell Biol. 1982 June 1; 93(3): 615–631.
PMCID: PMC2112125

Synthesis, transport, and utilization of specific flagellar proteins during flagellar regeneration in Chlamydomonas

Abstract

We labeled gametes of Chlamydomonas with 10-min pulses of 35SO4(-2) before and at various times after deflagellation, and isolated whole cells and flagella immediately after the pulse. The labeled proteins were separated by one- or two-dimensional gel electrophoresis, and the amount of isotope incorporated into specific proteins was determined. Individual proteins were identified with particular structures by correlating missing axonemal polypeptides with ultrastructural defects in paralyzed mutants, or by polypeptide analysis of flagellar fractions. Synthesis of most flagellar proteins appeared to be coordinately induced after flagellar amputation. The rate of synthesis for most quantified proteins increased at least 4- to 10-fold after deflagellation. The kinetics of synthesis of proteins contained together within a structure (e.g., the radial spoke proteins [RSP] ) were frequently similar; however, the kinetics of synthesis of proteins contained in different structures (e.g., RSP vs. alpha- and beta- tubulins) were different. Most newly synthesized flagellar proteins were rapidly transported into the flagellum with kinetics reflecting the rate of growth of the organelle; exceptions included a central tubule complex protein (CT1) and an actinlike component, both of which appeared to be supplied almost entirely from pre-existing, unlabeled pools. Isotope dilution experiments showed that, for most quantified axonemal proteins, a minimum of 35-40% of the polypeptide chains used in assembling a new axoneme was synthesized during regeneration; these proteins appeared to have predeflagellation pools of approximately the same size relative to their stoichiometries in the axoneme. In contrast, CT1 and the actinlike protein had comparatively large pools.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abreu SL, Brinster RL. Synthesis of tubulin and actin during the preimplantation development of the mouse. Exp Cell Res. 1978 Jun;114(1):135–141. [PubMed]
  • Bergman K, Goodenough UW, Goodenough DA, Jawitz J, Martin H. Gametic differentiation in Chlamydomonas reinhardtii. II. Flagellar membranes and the agglutination reaction. J Cell Biol. 1975 Dec;67(3):606–622. [PMC free article] [PubMed]
  • Bonner WM, Laskey RA. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Brown DL, Rogers KA. Hydrostatic pressure-induced internalization of flagellar axonemes, disassembly, and reutilization during flagellar regeneration in Polytomella. Exp Cell Res. 1978 Dec;117(2):313–324. [PubMed]
  • Collis PS, Weeks DP. Selective inhibition of tubulin synthesis by amiprophos methyl during flagellar regeneration in Chlamydomonas reinhardi. Science. 1978 Oct 27;202(4366):440–442. [PubMed]
  • Dentler WL, Rosenbaum JL. Flagellar elongation and shortening in Chlamydomonas. III. structures attached to the tips of flagellar microtubules and their relationship to the directionality of flagellar microtubule assembly. J Cell Biol. 1977 Sep;74(3):747–759. [PMC free article] [PubMed]
  • Dentler WL, Pratt MM, Stephens RE. Microtubule-membrane interactions in cilia. II. Photochemical cross-linking of bridge structures and the identification of a membrane-associated dynein-like ATPase. J Cell Biol. 1980 Feb;84(2):381–403. [PMC free article] [PubMed]
  • Fulton C, Kowit JD. Programmed synthesis of flagellar tubulin during cell differentiation in Naegleria. Ann N Y Acad Sci. 1975 Jun 30;253:318–332. [PubMed]
  • Gitelman SE, Witman GB. Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella. J Cell Biol. 1980 Dec;87(3 Pt 1):764–770. [PMC free article] [PubMed]
  • Guttman SD, Gorovsky MA. Cilia regeneration in starved tetrahymena: an inducible system for studying gene expression and organelle biogenesis. Cell. 1979 Jun;17(2):307–317. [PubMed]
  • Guttman SD, Glover CV, Allis CD, Gorovsky MA. Heat shock, deciliation and release from anoxia induce the synthesis of the same set of polypeptides in starved T. pyriformis. Cell. 1980 Nov;22(1 Pt 1):299–307. [PubMed]
  • Howell SH, Posakony JW, Hill KR. The cell cycle program of polypeptide labeling in Chlamydomonas reinhardtii. J Cell Biol. 1977 Feb;72(2):223–241. [PMC free article] [PubMed]
  • Huang B, Piperno G, Luck DJ. Paralyzed flagella mutants of Chlamydomonas reinhardtii. Defective for axonemal doublet microtubule arms. J Biol Chem. 1979 Apr 25;254(8):3091–3099. [PubMed]
  • Jarvik JW, Rosenbaum JL. Oversized flagellar membrane protein in paralyzed mutants of Chlamydomonas reinhardrii. J Cell Biol. 1980 May;85(2):258–272. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Laemmli UK, Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. [PubMed]
  • Laskey RA, Mills AD. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. [PubMed]
  • Lefebvre PA, Nordstrom SA, Moulder JE, Rosenbaum JL. Flagellar elongation and shortening in Chlamydomonas. IV. Effects of flagellar detachment, regeneration, and resorption on the induction of flagellar protein synthesis. J Cell Biol. 1978 Jul;78(1):8–27. [PMC free article] [PubMed]
  • Lefebvre PA, Silflow CD, Wieben ED, Rosenbaum JL. Increased levels of mRNAs for tubulin and other flagellar proteins after amputation or shortening of Chlamydomonas flagella. Cell. 1980 Jun;20(2):469–477. [PubMed]
  • Levy EM. Flagellar elongation: an example of controlled growth. J Theor Biol. 1974 Jan;43(1):133–149. [PubMed]
  • Lewis M, Helmsing PJ, Ashburner M. Parallel changes in puffing activity and patterns of protein synthesis in salivary glands of Drosophila. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3604–3608. [PubMed]
  • Lindquist S. Varying patterns of protein synthesis in Drosophila during heat shock: implications for regulation. Dev Biol. 1980 Jun 15;77(2):463–479. [PubMed]
  • Minami SA, Collis PS, Young EE, Weeks DP. Tubulin induction in C. reinhardii: requirement for tubulin mRNA synthesis. Cell. 1981 Apr;24(1):89–95. [PubMed]
  • Mirault ME, Goldschmidt-Clermont M, Moran L, Arrigo AP, Tissières A. The effect of heat shock on gene expression in Drosophila melanogaster. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):819–827. [PubMed]
  • O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed]
  • O'Farrell PZ, Goodman HM, O'Farrell PH. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. [PubMed]
  • Piperno G, Huang B, Luck DJ. Two-dimensional analysis of flagellar proteins from wild-type and paralyzed mutants of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1600–1604. [PubMed]
  • Piperno G, Luck DJ. An actin-like protein is a component of axonemes from Chlamydomonas flagella. J Biol Chem. 1979 Apr 10;254(7):2187–2190. [PubMed]
  • Piperno G, Luck DJ. Axonemal adenosine triphosphatases from flagella of Chlamydomonas reinhardtii. Purification of two dyneins. J Biol Chem. 1979 Apr 25;254(8):3084–3090. [PubMed]
  • Rosenbaum JL, Moulder JE, Ringo DL. Flagellar elongation and shortening in Chlamydomonas. The use of cycloheximide and colchicine to study the synthesis and assembly of flagellar proteins. J Cell Biol. 1969 May;41(2):600–619. [PMC free article] [PubMed]
  • SAGER R, GRANICK S. Nutritional studies with Chlamydomonas reinhardi. Ann N Y Acad Sci. 1953 Oct 14;56(5):831–838. [PubMed]
  • Silflow CD, Rosenbaum JL. Multiple alpha- and beta-tubulin genes in Chlamydomonas and regulation of tubulin mRNA levels after deflagellation. Cell. 1981 Apr;24(1):81–88. [PubMed]
  • Stephens RE. Thermal fractionation of outer fiber doublet microtubules into A- and B-subfiber components. A- and B-tubulin. J Mol Biol. 1970 Feb 14;47(3):353–363. [PubMed]
  • Stephens RE. Differential protein synthesis and utilization during cilia formation in sea urchin embryos. Dev Biol. 1977 Dec;61(2):311–329. [PubMed]
  • Valenzuela P, Quiroga M, Zaldivar J, Rutter WJ, Kirschner MW, Cleveland DW. Nucleotide and corresponding amino acid sequences encoded by alpha and beta tubulin mRNAs. Nature. 1981 Feb 19;289(5799):650–655. [PubMed]
  • Weeks DP, Collis PS. Induction of microtubule protein synthesis in Chlamydomonas reinhardi during flagellar regeneration. Cell. 1976 Sep;9(1):15–27. [PubMed]
  • Witman GB, Carlson K, Berliner J, Rosenbaum JL. Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes. J Cell Biol. 1972 Sep;54(3):507–539. [PMC free article] [PubMed]
  • Witman GB. The site of in vivo assembly of flagellar microtubules. Ann N Y Acad Sci. 1975 Jun 30;253:178–191. [PubMed]
  • Witman GB, 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. [PMC free article] [PubMed]

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