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. 1987 Nov 1;105(5):2279–2292. doi: 10.1083/jcb.105.5.2279

Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii

PMCID: PMC2114871  PMID: 2824527

Abstract

When Chlamydomonas reinhardtii gametes of opposite mating type are mixed together, they adhere by a flagella-mediated agglutination that triggers three rapid mating responses: flagellar tip activation, cell wall loss, and mating structure activation accompanied by actin polymerization. Here we show that a transient 10-fold elevation of intracellular cAMP levels is also triggered by sexual agglutination. We further show that gametes of a single mating type can be induced to undergo all three mating responses when presented with exogenous dibutyryl-cAMP (db-cAMP). These events are also induced by cyclic nucleotide phosphodiesterase inhibitors, which elevate endogenous cAMP levels and act synergistically with db-cAMP. Non-agglutinating mutants of opposite mating type will fuse efficiently in the presence of db- cAMP. No activation of mating events is induced by calcium plus ionophores, 8-bromo-cGMP, dibutyryl-cGMP, nigericin at alkaline pH, phorbol esters, or forskolin. H-8, an inhibitor of cyclic nucleotide- dependent protein kinase, inhibits mating events in agglutinating cells and antagonizes the effects of cAMP on non-agglutinating cells. Adenylate cyclase activity was detected in both the gamete cell body and flagella, with the highest specific activity displayed in flagellar membrane fractions. The flagellar membrane adenylate cyclase is preferentially stimulated by Mn++, unresponsive to NaF, GTP, GTP gamma S, AlF4-, and forskolin, and is inhibited by trifluoperazine. Cyclic nucleotide phosphodiesterase activity is also present in flagella. Our observations indicate that cAMP is a sufficient initial signal for all of the known mating reaction events in C. reinhardtii, and suggest that the flagellar cyclase and/or phosphodiesterase may be important loci of control for the agglutination-stimulated production of this signal.

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Selected References

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  1. Adair W. S. Characterization of Chlamydomonas sexual agglutinins. J Cell Sci Suppl. 1985;2:233–260. doi: 10.1242/jcs.1985.supplement_2.13. [DOI] [PubMed] [Google Scholar]
  2. Adair W. S., Hwang C., Goodenough U. W. Identification and visualization of the sexual agglutinin from the mating-type plus flagellar membrane of Chlamydomonas. Cell. 1983 May;33(1):183–193. doi: 10.1016/0092-8674(83)90347-1. [DOI] [PubMed] [Google Scholar]
  3. Aurbach G. D. Polypeptide and amine hormone regulation of adenylate cyclase. Annu Rev Physiol. 1982;44:653–666. doi: 10.1146/annurev.ph.44.030182.003253. [DOI] [PubMed] [Google Scholar]
  4. Beavo J. A., Rogers N. L., Crofford O. B., Baird C. E., Hardman J. G., Sutherland E. W., Newman E. V. Effects of phosphodiesterase inhibitors on cyclic AMP levels and on lipolysis. Ann N Y Acad Sci. 1971 Dec 30;185:129–136. doi: 10.1111/j.1749-6632.1971.tb45243.x. [DOI] [PubMed] [Google Scholar]
  5. Begg D. A., Rebhun L. I. pH regulates the polymerization of actin in the sea urchin egg cortex. J Cell Biol. 1979 Oct;83(1):241–248. doi: 10.1083/jcb.83.1.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bentley J. K., Garbers D. L., Domino S. E., Noland T. D., Van Dop C. Spermatozoa contain a guanine nucleotide-binding protein ADP-ribosylated by pertussis toxin. Biochem Biophys Res Commun. 1986 Jul 31;138(2):728–734. doi: 10.1016/s0006-291x(86)80557-5. [DOI] [PubMed] [Google Scholar]
  7. Bergman K., Goodenough U. W., Goodenough D. A., 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. doi: 10.1083/jcb.67.3.606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bloodgood R. A., Leffler E. M., Bojczuk A. T. Reversible inhibition of Chlamydomonas flagellar surface motility. J Cell Biol. 1979 Sep;82(3):664–674. doi: 10.1083/jcb.82.3.664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bloodgood R. A., Levin E. N. Transient increase in calcium efflux accompanies fertilization in Chlamydomonas. J Cell Biol. 1983 Aug;97(2):397–404. doi: 10.1083/jcb.97.2.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bloodgood R. A. Motility occurring in association with the surface of the Chlamydomonas flagellum. J Cell Biol. 1977 Dec;75(3):983–989. doi: 10.1083/jcb.75.3.983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bradford M. M. 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. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  12. Brooker G. Newer developments in the determination of cyclic AMP and other cyclic nucleotides, adenylate cyclase, and phosphodiesterase. Methods Biochem Anal. 1974;22:95–121. doi: 10.1002/9780470110423.ch2. [DOI] [PubMed] [Google Scholar]
  13. Cambier J. C., Justement L. B., Newell M. K., Chen Z. Z., Harris L. K., Sandoval V. M., Klemsz M. J., Ransom J. T. Transmembrane signals and intracellular "second messengers" in the regulation of quiescent B-lymphocyte activation. Immunol Rev. 1987 Feb;95:37–57. doi: 10.1111/j.1600-065x.1987.tb00499.x. [DOI] [PubMed] [Google Scholar]
  14. Chasin M., Harris D. N. Inhibitory and activators of cyclic nucleotide phosphodiesterase. Adv Cyclic Nucleotide Res. 1976;7:225–264. [PubMed] [Google Scholar]
  15. Clark R. B. Desensitization of hormonal stimuli coupled to regulation of cyclic AMP levels. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1986;20:151–209. [PubMed] [Google Scholar]
  16. Connolly T. M., Lawing W. J., Jr, Majerus P. W. Protein kinase C phosphorylates human platelet inositol trisphosphate 5'-phosphomonoesterase, increasing the phosphatase activity. Cell. 1986 Sep 12;46(6):951–958. doi: 10.1016/0092-8674(86)90077-2. [DOI] [PubMed] [Google Scholar]
  17. Cooper J. B., Adair W. S., Mecham R. P., Heuser J. E. Chlamydomonas agglutinin is a hydroxyproline-rich glycoprotein. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5898–5901. doi: 10.1073/pnas.80.19.5898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Detmers P. A., Condeelis J. Trifluoperazine and W-7 inhibit mating in Chlamydomonas at an early stage of gametic interaction. Exp Cell Res. 1986 Apr;163(2):317–326. doi: 10.1016/0014-4827(86)90063-7. [DOI] [PubMed] [Google Scholar]
  19. Detmers P. A., Goodenough U. W., Condeelis J. Elongation of the fertilization tubule in Chlamydomonas: new observations on the core microfilaments and the effect of transient intracellular signals on their structural integrity. J Cell Biol. 1983 Aug;97(2):522–532. doi: 10.1083/jcb.97.2.522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fischer U., Amrhein N. Cyclic nucleotide phosphodiesterase of Chlamydomonas reinhardtii. Biochim Biophys Acta. 1974 Apr 25;341(2):412–420. doi: 10.1016/0005-2744(74)90234-4. [DOI] [PubMed] [Google Scholar]
  21. Frazier W., Glaser L. Surface components and cell recognition. Annu Rev Biochem. 1979;48:491–523. doi: 10.1146/annurev.bi.48.070179.002423. [DOI] [PubMed] [Google Scholar]
  22. Garbers D. L., Kopf G. S. The regulation of spermatozoa by calcium cyclic nucleotides. Adv Cyclic Nucleotide Res. 1980;13:251–306. [PubMed] [Google Scholar]
  23. Garbers D. L., Tubb D. J., Kopf G. S. Regulation of sea urchin sperm cyclic AMP-dependent protein kinases by an egg associated factor. Biol Reprod. 1980 Apr;22(3):526–532. doi: 10.1093/biolreprod/22.3.526. [DOI] [PubMed] [Google Scholar]
  24. Gilman A. G. G proteins and dual control of adenylate cyclase. Cell. 1984 Mar;36(3):577–579. doi: 10.1016/0092-8674(84)90336-2. [DOI] [PubMed] [Google Scholar]
  25. Goodenough U. W., Adair W. S., Collin-Osdoby P., Heuser J. E. Structure of the Chlamydomonas agglutinin and related flagellar surface proteins in vitro and in situ. J Cell Biol. 1985 Sep;101(3):924–941. doi: 10.1083/jcb.101.3.924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Goodenough U. W., Detmers P. A., Hwang C. Activation for cell fusion in Chlamydomonas: analysis of wild-type gametes and nonfusing mutants. J Cell Biol. 1982 Feb;92(2):378–386. doi: 10.1083/jcb.92.2.378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Goodenough U. W., Jurivich D. Tipping and mating-structure activation induced in Chlamydomonas gametes by flagellar membrane antisera. J Cell Biol. 1978 Dec;79(3):680–693. doi: 10.1083/jcb.79.3.680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Goodenough U. W., StClair H. S. BALD-2: a mutation affecting the formation of doublet and triplet sets of microtubules in Chlamydomonas reinhardtii. J Cell Biol. 1975 Sep;66(3):480–491. doi: 10.1083/jcb.66.3.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Goodenough U. W., Weiss R. L. Gametic differentiation in Chlamydomonas reinhardtii. III. Cell wall lysis and microfilament-associated mating structure activation in wild-type and mutant strains. J Cell Biol. 1975 Dec;67(3):623–637. doi: 10.1083/jcb.67.3.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Heuser J. Three-dimensional visualization of coated vesicle formation in fibroblasts. J Cell Biol. 1980 Mar;84(3):560–583. doi: 10.1083/jcb.84.3.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hidaka H., Inagaki M., Kawamoto S., Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 1984 Oct 9;23(21):5036–5041. doi: 10.1021/bi00316a032. [DOI] [PubMed] [Google Scholar]
  32. Hildebrandt J. D., Codina J., Tash J. S., Kirchick H. J., Lipschultz L., Sekura R. D., Birnbaumer L. The membrane-bound spermatozoal adenylyl cyclase system does not share coupling characteristics with somatic cell adenylyl cyclases. Endocrinology. 1985 Apr;116(4):1357–1366. doi: 10.1210/endo-116-4-1357. [DOI] [PubMed] [Google Scholar]
  33. Hoffman J. L., Goodenough U. W. Experimental dissection of flagellar surface motility in Chlamydomonas. J Cell Biol. 1980 Aug;86(2):656–665. doi: 10.1083/jcb.86.2.656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Hwang C. J., Monk B. C., Goodenough U. W. Linkage of Mutations Affecting minus Flagellar Membrane Agglutinability to the mt Mating-Type Locus of Chlamydomonas. Genetics. 1981 Sep;99(1):41–47. doi: 10.1093/genetics/99.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Johnson U. G., Porter K. R. Fine structure of cell division in Chlamydomonas reinhardi. Basal bodies and microtubules. J Cell Biol. 1968 Aug;38(2):403–425. doi: 10.1083/jcb.38.2.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kamiya R., Witman G. B. Submicromolar levels of calcium control the balance of beating between the two flagella in demembranated models of Chlamydomonas. J Cell Biol. 1984 Jan;98(1):97–107. doi: 10.1083/jcb.98.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kaska D. D., Piscopo I. C., Gibor A. Intracellular calcium redistribution during mating in Chlamydomonas reinhardii. Exp Cell Res. 1985 Oct;160(2):371–379. doi: 10.1016/0014-4827(85)90183-1. [DOI] [PubMed] [Google Scholar]
  38. Kopf G. S., Vacquier V. D. Characterization of a calmodulin-stimulated adenylate cyclase from abalone spermatozoa. J Biol Chem. 1984 Jun 25;259(12):7590–7596. [PubMed] [Google Scholar]
  39. Lefebvre P. A., Silflow C. D., Wieben E. D., Rosenbaum J. L. Increased levels of mRNAs for tubulin and other flagellar proteins after amputation or shortening of Chlamydomonas flagella. Cell. 1980 Jun;20(2):469–477. doi: 10.1016/0092-8674(80)90633-9. [DOI] [PubMed] [Google Scholar]
  40. Leichtling B. H., Coffman D. S., Yaeger E. S., Rickenberg H. V., al-Jumaliy W., Haley B. E. Occurrence of the adenylate cyclase "G protein" in membranes of Dictyostelium discoideum. Biochem Biophys Res Commun. 1981 Oct 30;102(4):1187–1195. doi: 10.1016/s0006-291x(81)80137-4. [DOI] [PubMed] [Google Scholar]
  41. Martin N. C., Goodenough U. W. Gametic differentiation in Chlamydomonas reinhardtii. I. Production of gametes and their fine structure. J Cell Biol. 1975 Dec;67(3):587–605. doi: 10.1083/jcb.67.3.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Mesland D. A., Hoffman J. L., Caligor E., Goodenough U. W. Flagellar tip activation stimulated by membrane adhesions in Chlamydomonas gametes. J Cell Biol. 1980 Mar;84(3):599–617. doi: 10.1083/jcb.84.3.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Minami S. A., Goodenough U. W. Novel glycopolypeptide synthesis induced by gametic cell fusion in Chlamydomonas reinhardtii. J Cell Biol. 1978 Apr;77(1):165–181. doi: 10.1083/jcb.77.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Nakamura T., Gold G. H. A cyclic nucleotide-gated conductance in olfactory receptor cilia. 1987 Jan 29-Feb 4Nature. 325(6103):442–444. doi: 10.1038/325442a0. [DOI] [PubMed] [Google Scholar]
  45. Omann G. M., Allen R. A., Bokoch G. M., Painter R. G., Traynor A. E., Sklar L. A. Signal transduction and cytoskeletal activation in the neutrophil. Physiol Rev. 1987 Jan;67(1):285–322. doi: 10.1152/physrev.1987.67.1.285. [DOI] [PubMed] [Google Scholar]
  46. Pace U., Hanski E., Salomon Y., Lancet D. Odorant-sensitive adenylate cyclase may mediate olfactory reception. Nature. 1985 Jul 18;316(6025):255–258. doi: 10.1038/316255a0. [DOI] [PubMed] [Google Scholar]
  47. Rubin R. W., Filner P. Adenosine 3',5'-cyclic monophosphate in Chlamydomonas reinhardtii. Influence on flagellar function and regeneration. J Cell Biol. 1973 Mar;56(3):628–635. doi: 10.1083/jcb.56.3.628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Salomon Y. Adenylate cyclase assay. Adv Cyclic Nucleotide Res. 1979;10:35–55. [PubMed] [Google Scholar]
  49. Schlegel R., Pardee A. B. Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells. Science. 1986 Jun 6;232(4755):1264–1266. doi: 10.1126/science.2422760. [DOI] [PubMed] [Google Scholar]
  50. Schramm M., Selinger Z. Message transmission: receptor controlled adenylate cyclase system. Science. 1984 Sep 21;225(4668):1350–1356. doi: 10.1126/science.6147897. [DOI] [PubMed] [Google Scholar]
  51. Simon L. N., Shuman D. A., Robins R. K. The chemistry and biological properties of nucleotides related to nucleoside 3',5'-cyclic phosphates. Adv Cyclic Nucleotide Res. 1973;3:225–353. [PubMed] [Google Scholar]
  52. Snell W. J., Buchanan M., Clausell A. Lidocaine reversibly inhibits fertilization in Chlamydomonas: a possible role for calcium in sexual signalling. J Cell Biol. 1982 Sep;94(3):607–612. doi: 10.1083/jcb.94.3.607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Snell W. J. Study of the release of cell wall degrading enzymes during adhesion of Chlamydomonas gametes. Exp Cell Res. 1982 Mar;138(1):109–119. doi: 10.1016/0014-4827(82)90096-9. [DOI] [PubMed] [Google Scholar]
  54. Steiner A. L., Pagliara A. S., Chase L. R., Kipnis D. M. Radioimmunoassay for cyclic nucleotides. II. Adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in mammalian tissues and body fluids. J Biol Chem. 1972 Feb 25;247(4):1114–1120. [PubMed] [Google Scholar]
  55. Sternweis P. C., Gilman A. G. Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4888–4891. doi: 10.1073/pnas.79.16.4888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Thompson W. J., Terasaki W. L., Epstein P. M., Strada S. J. Assay of cyclic nucleotide phosphodiesterase and resolution of multiple molecular forms of the enzyme. Adv Cyclic Nucleotide Res. 1979;10:69–92. [PubMed] [Google Scholar]
  57. Tilney L. G., Kiehart D. P., Sardet C., Tilney M. Polymerization of actin. IV. Role of Ca++ and H+ in the assembly of actin and in membrane fusion in the acrosomal reaction of echinoderm sperm. J Cell Biol. 1978 May;77(2):536–550. doi: 10.1083/jcb.77.2.536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Torruella M., Flawiá M. M., Eisenschlos C., Molina y Vedia L., Rubinstein C. P., Torres H. N. Trypanosoma cruzi adenylate cyclase activity. Purification and characterization. Biochem J. 1986 Feb 15;234(1):145–150. doi: 10.1042/bj2340145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Trimmer J. S., Vacquier V. D. Activation of sea urchin gametes. Annu Rev Cell Biol. 1986;2:1–26. doi: 10.1146/annurev.cb.02.110186.000245. [DOI] [PubMed] [Google Scholar]
  60. Weiss R. L., Goodenough D. A., Goodenough U. W. Membrane differentiations at sites specialized for cell fusion. J Cell Biol. 1977 Jan;72(1):144–160. doi: 10.1083/jcb.72.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Werth D. K., Niedel J. E., Pastan I. Vinculin, a cytoskeletal substrate of protein kinase C. J Biol Chem. 1983 Oct 10;258(19):11423–11426. [PubMed] [Google Scholar]
  62. 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]

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