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. 1992 Feb 1;116(3):737–744. doi: 10.1083/jcb.116.3.737

Inositol phospholipid metabolism may trigger flagellar excision in Chlamydomonas reinhardtii

PMCID: PMC2289324  PMID: 1309818

Abstract

Chlamydomonas reinhardtii cells shed their flagella in response to environmental stress. Under favorable conditions, flagella are quickly regrown. To learn more about the signals that trigger flagellar excision and regrowth we have investigated inositol phospholipid metabolites, molecules implicated in signal transduction in several other systems. After deflagellation by low pH or mastoparan, a potent activator of G proteins, there was a rapid increase in levels of inositol 1,4,5-trisphosphate measured by use of receptor-binding assays and HPLC. This increase was concomitant with a decrease in levels of phosphatidylinositol 4,5-bisphosphate and was followed by an increase in phosphatidic acid, results consistent with activation of phospholipase C and diacylglycerol kinase. Additional experiments suggest that this activated phospholipase C is not important for flagellar regrowth but plays a role in informing the excision apparatus of the environmental stress. Addition of neomycin (an inhibitor of phospholipase C) before exposure of cells to low pH or mastoparan prevented the increase in inositol 1,4,5-trisphosphate and also prevented deflagellation. Addition of neomycin after deflagellation blocked increases in inositol 1,4,5-trisphosphate that normally followed deflagellation, but did not block flagellar assembly. Furthermore, a flagellar excision-defective mutant, fa-1, did not shed its flagella in response to low pH or mastoparan, yet both of these agents activated phospholipase C in these cells. The results suggest that activation of phospholipase C, possibly via a G protein, is a proximal step in the signal transduction pathway inducing deflagellation in Chlamydomonas.

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Selected References

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  1. Auger K. R., Carpenter C. L., Cantley L. C., Varticovski L. Phosphatidylinositol 3-kinase and its novel product, phosphatidylinositol 3-phosphate, are present in Saccharomyces cerevisiae. J Biol Chem. 1989 Dec 5;264(34):20181–20184. [PubMed] [Google Scholar]
  2. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  3. Baker E. J., Schloss J. A., Rosenbaum J. L. Rapid changes in tubulin RNA synthesis and stability induced by deflagellation in Chlamydomonas. J Cell Biol. 1984 Dec;99(6):2074–2081. doi: 10.1083/jcb.99.6.2074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Batty I. R., Nahorski S. R., Irvine R. F. Rapid formation of inositol 1,3,4,5-tetrakisphosphate following muscarinic receptor stimulation of rat cerebral cortical slices. Biochem J. 1985 Nov 15;232(1):211–215. doi: 10.1042/bj2320211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bazenet C. E., Brockman J. L., Lewis D., Chan C., Anderson R. A. Erythroid membrane-bound protein kinase binds to a membrane component and is regulated by phosphatidylinositol 4,5-bisphosphate. J Biol Chem. 1990 May 5;265(13):7369–7376. [PubMed] [Google Scholar]
  6. Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. doi: 10.1038/341197a0. [DOI] [PubMed] [Google Scholar]
  7. Bloomquist B. T., Shortridge R. D., Schneuwly S., Perdew M., Montell C., Steller H., Rubin G., Pak W. L. Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction. Cell. 1988 Aug 26;54(5):723–733. doi: 10.1016/s0092-8674(88)80017-5. [DOI] [PubMed] [Google Scholar]
  8. Bocckino S. B., Wilson P., Exton J. H. An enzymatic assay for picomole levels of phosphatidate. Anal Biochem. 1989 Jul;180(1):24–27. doi: 10.1016/0003-2697(89)90083-3. [DOI] [PubMed] [Google Scholar]
  9. Breer H., Boekhoff I., Tareilus E. Rapid kinetics of second messenger formation in olfactory transduction. Nature. 1990 May 3;345(6270):65–68. doi: 10.1038/345065a0. [DOI] [PubMed] [Google Scholar]
  10. Carney D. H., Scott D. L., Gordon E. A., LaBelle E. F. Phosphoinositides in mitogenesis: neomycin inhibits thrombin-stimulated phosphoinositide turnover and initiation of cell proliferation. Cell. 1985 Sep;42(2):479–488. doi: 10.1016/0092-8674(85)90105-9. [DOI] [PubMed] [Google Scholar]
  11. Challiss R. A., Chilvers E. R., Willcocks A. L., Nahorski S. R. Heterogeneity of [3H]inositol 1,4,5-trisphosphate binding sites in adrenal-cortical membranes. Characterization and validation of a radioreceptor assay. Biochem J. 1990 Jan 15;265(2):421–427. doi: 10.1042/bj2650421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cheshire J. L., Keller L. R. Uncoupling of Chlamydomonas flagellar gene expression and outgrowth from flagellar excision by manipulation of Ca2+. J Cell Biol. 1991 Dec;115(6):1651–1659. doi: 10.1083/jcb.115.6.1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Coté G. G., Depass A. L., Quarmby L. M., Tate B. F., Morse M. J., Satter R. L., Crain R. C. Separation and Characterization of Inositol Phospholipids from the Pulvini of Samanea saman. Plant Physiol. 1989 Aug;90(4):1422–1428. doi: 10.1104/pp.90.4.1422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Einspahr K. J., Peeler T. C., Thompson G. A., Jr Rapid changes in polyphosphoinositide metabolism associated with the response of Dunaliella salina to hypoosmotic shock. J Biol Chem. 1988 Apr 25;263(12):5775–5779. [PubMed] [Google Scholar]
  15. Gabev E., Kasianowicz J., Abbott T., McLaughlin S. Binding of neomycin to phosphatidylinositol 4,5-bisphosphate (PIP2). Biochim Biophys Acta. 1989 Feb 13;979(1):105–112. doi: 10.1016/0005-2736(89)90529-4. [DOI] [PubMed] [Google Scholar]
  16. Ginsburg G., Kimmel A. R. Inositol trisphosphate and diacylglycerol can differentially modulate gene expression in Dictyostelium. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9332–9336. doi: 10.1073/pnas.86.23.9332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Hannun Y. A., Bell R. M. Functions of sphingolipids and sphingolipid breakdown products in cellular regulation. Science. 1989 Jan 27;243(4890):500–507. doi: 10.1126/science.2643164. [DOI] [PubMed] [Google Scholar]
  19. Hannun Y. A., Bell R. M. Rat brain protein kinase C. Kinetic analysis of substrate dependence, allosteric regulation, and autophosphorylation. J Biol Chem. 1990 Feb 15;265(5):2962–2972. [PubMed] [Google Scholar]
  20. Hata Y., Ogata E., Kojima I. Platelet-derived growth factor stimulates synthesis of 1,2-diacylglycerol from monoacylglycerol in Balb/c 3T3 cells. Biochem J. 1989 Sep 15;262(3):947–952. doi: 10.1042/bj2620947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Higashijima T., Burnier J., Ross E. M. Regulation of Gi and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines. Mechanism and structural determinants of activity. J Biol Chem. 1990 Aug 25;265(24):14176–14186. [PubMed] [Google Scholar]
  22. Irvine R. F., Letcher A. J., Lander D. J., Drøbak B. K., Dawson A. P., Musgrave A. Phosphatidylinositol(4,5)bisphosphate and Phosphatidylinositol(4)phosphate in Plant Tissues. Plant Physiol. 1989 Mar;89(3):888–892. doi: 10.1104/pp.89.3.888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kanoh H., Yamada K., Sakane F. Diacylglycerol kinase: a key modulator of signal transduction? Trends Biochem Sci. 1990 Feb;15(2):47–50. doi: 10.1016/0968-0004(90)90172-8. [DOI] [PubMed] [Google Scholar]
  24. Keller L. R., Schloss J. A., Silflow C. D., Rosenbaum J. L. Transcription of alpha- and beta-tubulin genes in vitro in isolated Chlamydomonas reinhardi nuclei. J Cell Biol. 1984 Mar;98(3):1138–1143. doi: 10.1083/jcb.98.3.1138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kikkawa U., Kishimoto A., Nishizuka Y. The protein kinase C family: heterogeneity and its implications. Annu Rev Biochem. 1989;58:31–44. doi: 10.1146/annurev.bi.58.070189.000335. [DOI] [PubMed] [Google Scholar]
  26. Lefebvre P. A., Nordstrom S. A., Moulder J. E., Rosenbaum J. L. 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. doi: 10.1083/jcb.78.1.8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. MORRISON W. R. A FAST, SIMPLE AND RELIABLE METHOD FOR THE MICRODETERMINATION OF PHOSPHORUS IN BIOLOGICAL MATERIALS. Anal Biochem. 1964 Feb;7:218–224. doi: 10.1016/0003-2697(64)90231-3. [DOI] [PubMed] [Google Scholar]
  28. Matozaki T., Williams J. A. Multiple sources of 1,2-diacylglycerol in isolated rat pancreatic acini stimulated by cholecystokinin. Involvement of phosphatidylinositol bisphosphate and phosphatidylcholine hydrolysis. J Biol Chem. 1989 Sep 5;264(25):14729–14734. [PubMed] [Google Scholar]
  29. Minami S. A., Collis P. S., Young E. E., Weeks D. P. Tubulin induction in C. reinhardii: requirement for tubulin mRNA synthesis. Cell. 1981 Apr;24(1):89–95. doi: 10.1016/0092-8674(81)90504-3. [DOI] [PubMed] [Google Scholar]
  30. Moolenaar W. H., Kruijer W., Tilly B. C., Verlaan I., Bierman A. J., de Laat S. W. Growth factor-like action of phosphatidic acid. Nature. 1986 Sep 11;323(6084):171–173. doi: 10.1038/323171a0. [DOI] [PubMed] [Google Scholar]
  31. Morse M. J., Crain R. C., Coté G. G., Satter R. L. Light-Stimulated Inositol Phospholipid Turnover in Samanea saman Pulvini : Increased Levels of Diacylglycerol. Plant Physiol. 1989 Mar;89(3):724–727. doi: 10.1104/pp.89.3.724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pike M. C., Bruck M. E., Arndt C., Lee C. S. Chemoattractants stimulate phosphatidylinositol-4-phosphate kinase in human polymorphonuclear leukocytes. J Biol Chem. 1990 Feb 5;265(4):1866–1873. [PubMed] [Google Scholar]
  33. Preiss J., Loomis C. R., Bishop W. R., Stein R., Niedel J. E., Bell R. M. Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J Biol Chem. 1986 Jul 5;261(19):8597–8600. [PubMed] [Google Scholar]
  34. Rosenbaum J. L., Moulder J. E., Ringo D. L. 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. doi: 10.1083/jcb.41.2.600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Schaeffer E., Smith D., Mardon G., Quinn W., Zuker C. Isolation and characterization of two new drosophila protein kinase C genes, including one specifically expressed in photoreceptor cells. Cell. 1989 May 5;57(3):403–412. doi: 10.1016/0092-8674(89)90915-x. [DOI] [PubMed] [Google Scholar]
  37. Schuring F., Brederoo J., Musgrave A., van den Ende H. Increase in calcium triggers mating structure activation in Chlamydomonas eugametos. FEMS Microbiol Lett. 1990 Sep 15;59(3):237–240. doi: 10.1016/0378-1097(90)90226-g. [DOI] [PubMed] [Google Scholar]
  38. Slivka S. R., Insel P. A. Phorbol ester and neomycin dissociate bradykinin receptor-mediated arachidonic acid release and polyphosphoinositide hydrolysis in Madin-Darby canine kidney cells. Evidence that bradykinin mediates noninterdependent activation of phospholipases A2 and C. J Biol Chem. 1988 Oct 15;263(29):14640–14647. [PubMed] [Google Scholar]
  39. Smith J. B., Dwyer S. D., Smith L. Lowering extracellular pH evokes inositol polyphosphate formation and calcium mobilization. J Biol Chem. 1989 May 25;264(15):8723–8728. [PubMed] [Google Scholar]
  40. Smrcka A. V., Hepler J. R., Brown K. O., Sternweis P. C. Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq. Science. 1991 Feb 15;251(4995):804–807. doi: 10.1126/science.1846707. [DOI] [PubMed] [Google Scholar]
  41. Sylvia V., Curtin G., Norman J., Stec J., Busbee D. Activation of a low specific activity form of DNA polymerase alpha by inositol-1,4-bisphosphate. Cell. 1988 Aug 26;54(5):651–658. doi: 10.1016/s0092-8674(88)80009-6. [DOI] [PubMed] [Google Scholar]
  42. Tysnes O. B., Steen V. M., Holmsen H. Neomycin inhibits platelet functions and inositol phospholipid metabolism upon stimulation with thrombin, but not with ionomycin or 12-O-tetradecanoyl-phorbol 13-acetate. Eur J Biochem. 1988 Oct 15;177(1):219–223. doi: 10.1111/j.1432-1033.1988.tb14365.x. [DOI] [PubMed] [Google Scholar]
  43. VITALE J. J., HEGSTED D. M., NAKAMURA M., CONNORS P. The effect of thyroxine on magnesium requirement. J Biol Chem. 1957 Jun;226(2):597–601. [PubMed] [Google Scholar]
  44. Van Haastert P. J., De Vries M. J., Penning L. C., Roovers E., Van der Kaay J., Erneux C., Van Lookeren Campagne M. M. Chemoattractant and guanosine 5'-[gamma-thio]triphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3H]inositol by electroporation. Biochem J. 1989 Mar 1;258(2):577–586. doi: 10.1042/bj2580577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Van Haastert P. J. Determination of inositol 1,4,5-trisphosphate levels in Dictyostelium by isotope dilution assay. Anal Biochem. 1989 Feb 15;177(1):115–119. doi: 10.1016/0003-2697(89)90024-9. [DOI] [PubMed] [Google Scholar]
  46. Whitman M., Downes C. P., Keeler M., Keller T., Cantley L. Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature. 1988 Apr 14;332(6165):644–646. doi: 10.1038/332644a0. [DOI] [PubMed] [Google Scholar]
  47. Wood S. F., Szuts E. Z., Fein A. Inositol trisphosphate production in squid photoreceptors. Activation by light, aluminum fluoride, and guanine nucleotides. J Biol Chem. 1989 Aug 5;264(22):12970–12976. [PubMed] [Google Scholar]

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