Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1980 May 1;85(2):346–360. doi: 10.1083/jcb.85.2.346

Cell differentiation and flagellar elongation in Naegleria gruberi. Dependence on transcription and translation

PMCID: PMC2110630  PMID: 6154711

Abstract

This paper presents evidence that the phenotypic transformation of Naegleria gruberi from amebae to flagellates that occurs when cells are placed in a nutrient-free aqueous environment is dependent on transcription and translation. RNA and protein are synthesized during the hour-long differentiation. Actinomycin D and daunomycin selectively inhibit RNA synthesis, and cycloheximide selectively inhibits protein synthesis, throughout the time required for differentiation. These inhibitors prevent differentiation if added soon after the cells are transferred to nonnutrient buffer but cease to block specific differentiation events at subsequent, reproducible times, the transition points. After each transition point, morphogenesis can occur in the presence of the inhibitor and in the virtual absence of transcription or translation. A map of the transition points indicates that RNA synthesis is required until halfway through the temporal process from initiation to flagellum assembly, and that protein synthesis is required until three-fourths of the way through. Even when flagellum outgrowth can occur in the presence of cycloheximide, the length of the flagella formed is determined by the extent of synthesis of an unknown "limiting precursor." The transition points for formation of flagella and for formation of the streamlined flagellate body shape are temporally separate. These results indicate that differentiation in Naegleria involves a redirection of cell metabolism to produce new RNA and protein molecules that are essential for morphogenesis.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Dingle A. D., Fulton C. Development of the flagellar apparatus of Naegleria. J Cell Biol. 1966 Oct;31(1):43–54. doi: 10.1083/jcb.31.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Farrell K. W. Flagellar regeneration in Chlamydomonas reinhardtii: evidence that cycloheximide pulses induce a delay in morphogenesis. J Cell Sci. 1976 May;20(3):639–654. doi: 10.1242/jcs.20.3.639. [DOI] [PubMed] [Google Scholar]
  3. Fico R. M., Chen T. K., Canellakis E. S. Bifunctional intercalators: relationship of antitumor activity of diacridines to the cell membrane. Science. 1977 Oct 7;198(4312):53–56. doi: 10.1126/science.897680. [DOI] [PubMed] [Google Scholar]
  4. Firtel R. A., Baxter L., Lodish H. F. Actinomycin D and the regulation of enzyme biosynthesis during development of Dictyostelium discoideum. J Mol Biol. 1973 Sep 15;79(2):315–327. doi: 10.1016/0022-2836(73)90008-9. [DOI] [PubMed] [Google Scholar]
  5. Fulton C. Axenic cultivation of Naegleria gruberi. Requirement for methionine. Exp Cell Res. 1974 Oct;88(2):365–370. doi: 10.1016/0014-4827(74)90253-5. [DOI] [PubMed] [Google Scholar]
  6. Fulton C. Cell differentiation in Naegleria gruberi. Annu Rev Microbiol. 1977;31:597–629. doi: 10.1146/annurev.mi.31.100177.003121. [DOI] [PubMed] [Google Scholar]
  7. Fulton C., Dingle A. D. Appearance of the flagellate phenotype in populations of Naegleria amebae. Dev Biol. 1967 Feb;15(2):165–191. doi: 10.1016/0012-1606(67)90012-7. [DOI] [PubMed] [Google Scholar]
  8. Fulton C., Dingle A. D. Basal bodies, but not centrioles, in Naegleria. J Cell Biol. 1971 Dec;51(3):826–836. doi: 10.1083/jcb.51.3.826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fulton C. Early events of cell differentiation in Naegleria gruberi. Synergistic control by electrolytes and a factor from yeast extract. Dev Biol. 1972 Aug;28(4):603–619. doi: 10.1016/0012-1606(72)90006-1. [DOI] [PubMed] [Google Scholar]
  10. Fulton C. Intracellular regulation of cell shape and motility in Naegleria. First insights and a working hypothesis. J Supramol Struct. 1977;6(1):13–43. doi: 10.1002/jss.400060103. [DOI] [PubMed] [Google Scholar]
  11. Gavin R. H., Frankel J. Macromolecular synthesis, differentiation and cell division in Tetrahymena pyriformis, mating type I variety 1. J Cell Physiol. 1969 Oct;74(2):123–134. doi: 10.1002/jcp.1040740204. [DOI] [PubMed] [Google Scholar]
  12. Goldstein E. S., Penman S. Regulation of protein synthesis in mammalian cells. V. Further studies on the effect of actinomycin D on translation control in HeLa cells. J Mol Biol. 1973 Oct 25;80(2):243–254. doi: 10.1016/0022-2836(73)90170-8. [DOI] [PubMed] [Google Scholar]
  13. Hartwell L. H., Hutchison H. T., Holland T. M., McLaughlin C. S. The effect of cycloheximide upon polyribosome stability in two yeast mutants defective respectively in the initiation of polypeptide chains and in messenger RNA synthesis. Mol Gen Genet. 1970;106(4):347–361. doi: 10.1007/BF00324052. [DOI] [PubMed] [Google Scholar]
  14. Honikel K. O., Santo R. E. A model for the in vitro inhibition of the DNa polymerase reaction with the base specific antibiotics chromomycin-A 3' actinomycin-C 3 and daunomycin. Biochim Biophys Acta. 1972 May 29;269(3):354–363. doi: 10.1016/0005-2787(72)90122-0. [DOI] [PubMed] [Google Scholar]
  15. Kay J. E., Korner A. Effect of cycloheximide on protein and ribonucleic acid synthesis in cultured human lymphocytes. Biochem J. 1966 Sep;100(3):815–822. doi: 10.1042/bj1000815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kowit J. D., Fulton C. Programmed synthesis of tubulin for the flagella that develop during cell differentiation in Naegleria gruberi. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2877–2881. doi: 10.1073/pnas.71.7.2877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. Lai E. Y., Walsh C., Wardell D., Fulton C. Programmed appearance of translatable flagellar tubulin mRNA during cell differentiation in Naegleria. Cell. 1979 Aug;17(4):867–878. doi: 10.1016/0092-8674(79)90327-1. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. McMahon D. Cycloheximide is not a specific inhibitor of protein synthesis in vivo. Plant Physiol. 1975 May;55(5):815–821. doi: 10.1104/pp.55.5.815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Munro H. N. The determination of nucleic acids. Methods Biochem Anal. 1966;14:113–176. doi: 10.1002/9780470110324.ch5. [DOI] [PubMed] [Google Scholar]
  22. PAUL J., STRUTHERS M. G. Actinomycin D-resistant RNA synthesis in animal cells. Biochem Biophys Res Commun. 1963 Apr 23;11:135–139. doi: 10.1016/0006-291x(63)90080-9. [DOI] [PubMed] [Google Scholar]
  23. Palmiter R. D., Schimke R. T. Regulation of protein synthesis in chick oviduct. 3. Mechanism of ovalbumin "superinduction" by actinomycin D. J Biol Chem. 1973 Mar 10;248(5):1502–1512. [PubMed] [Google Scholar]
  24. Preston T. M., O'Dell D. S. Studies on the amoeboid and flagellate stages of Naegleria. Ann Soc Belg Med Trop. 1974;54(4-5):279–286. [PubMed] [Google Scholar]
  25. Preston T. M., O'Dell D. S. Synergistic effect of polymixin B with other antibiotics on the transformation of Naegleria gruberi. Exp Cell Res. 1971 Oct;68(2):465–466. doi: 10.1016/0014-4827(71)90177-7. [DOI] [PubMed] [Google Scholar]
  26. Rhoads R. E., McKnight G. S., Schimke R. T. Quantitative measurement of ovalbumin messenger ribonucleic acid activity. Localization in polysomes, induction by estrogen, and effect of actinomycin D. J Biol Chem. 1973 Mar 25;248(6):2031–2039. [PubMed] [Google Scholar]
  27. 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]
  28. Sauer H. W., Babcock K. L., Rusch H. P. Sporulation in Physarum polycephalum: a model system for studies on differentiation. Exp Cell Res. 1969 Oct;57(2):319–327. doi: 10.1016/0014-4827(69)90156-6. [DOI] [PubMed] [Google Scholar]
  29. Schimke R. T., Doyle D. Control of enzyme levels in animal tissues. Annu Rev Biochem. 1970;39:929–976. doi: 10.1146/annurev.bi.39.070170.004433. [DOI] [PubMed] [Google Scholar]
  30. Schwartz R. J. Control of glutamine synthetase synthesis in the embryonic chick neural retina. A caution in the use of actinomycin D. J Biol Chem. 1973 Sep 25;248(18):6426–6435. [PubMed] [Google Scholar]
  31. Singer R. H., Penman S. Messenger RNA in HeLa cells: kinetics of formation and decay. J Mol Biol. 1973 Aug 5;78(2):321–334. doi: 10.1016/0022-2836(73)90119-8. [DOI] [PubMed] [Google Scholar]
  32. Soll D. R., Sonneborn D. R. Zoospore germination in Blastocladiella emersonii: cell differentiation without protein synthesis? Proc Natl Acad Sci U S A. 1971 Feb;68(2):459–463. doi: 10.1073/pnas.68.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Steinberg R. A., Levinson B. B., Tomkins G. M. "Superinduction" of tyrosine aminotransferase by actinomycin D: a reevaluation. Cell. 1975 May;5(1):29–35. doi: 10.1016/0092-8674(75)90088-4. [DOI] [PubMed] [Google Scholar]
  34. Sussman M., Loomis W. F., Jr, Ashworth J. M., Sussman R. R. The effect of actinomycin D on cellular slime mold morphogenesis. Biochem Biophys Res Commun. 1967 Feb 8;26(3):353–359. doi: 10.1016/0006-291x(67)90131-3. [DOI] [PubMed] [Google Scholar]
  35. Wade J., Satir P. The effect of mercaptoethanol on flagellar morphogenesis in the amoeboflagellate Naegleria gruberi (Schardinger). Exp Cell Res. 1968 Apr;50(1):81–92. doi: 10.1016/0014-4827(68)90396-0. [DOI] [PubMed] [Google Scholar]
  36. Walsh C., Fulton C. Transcription during cell differentiation in Naegleria gruberi. Preferential synthesis of messenger RNA. Biochim Biophys Acta. 1973 Jun 8;312(1):52–71. doi: 10.1016/0005-2787(73)90052-x. [DOI] [PubMed] [Google Scholar]
  37. Ward D. C., Reich E., Goldberg I. H. Base specificity in the interaction of polynucleotides with antibiotic drugs. Science. 1965 Sep 10;149(3689):1259–1263. doi: 10.1126/science.149.3689.1259. [DOI] [PubMed] [Google Scholar]
  38. Weeks D. P., Collis P. S. Induction of microtubule protein synthesis in Chlamydomonas reinhardi during flagellar regeneration. Cell. 1976 Sep;9(1):15–27. doi: 10.1016/0092-8674(76)90048-9. [DOI] [PubMed] [Google Scholar]
  39. Yuyama S. The effects of selected chemical agents on the amoeba-flagellate transformation in Naegleria gruberi. J Protozool. 1971 May;18(2):337–343. doi: 10.1111/j.1550-7408.1971.tb03328.x. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES