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. 1994 Aug 2;126(4):955–966. doi: 10.1083/jcb.126.4.955

Dictyostelium discoideum mutants with conditional defects in phagocytosis

PMCID: PMC2120108  PMID: 7519624

Abstract

We have isolated and characterized Dictyostelium discoideum mutants with conditional defects in phagocytosis. Under suspension conditions, the mutants exhibited dramatic reductions in the uptake of bacteria and polystyrene latex beads. The initial binding of these ligands was unaffected, however, indicating that the defect was not in a plasma membrane receptor: Because of the phagocytosis defect, the mutants were unable to grow when cultured in suspensions of heat-killed bacteria. The mutants exhibited normal capacities for fluid phase endocytosis and grew as rapidly as parental (AX4) cells in axenic medium. Both the defects in phagocytosis and growth on bacteria were corrected when the mutant Dictyostelium cells were cultured on solid substrates. Reversion and genetic complementation analysis suggested that the mutant phenotypes were caused by single gene defects. While the precise site of action of the mutations was not established, the mutations are likely to affect an early signaling event because the binding of bacteria to mutant cells in suspension was unable to trigger the localized polymerization of actin filaments required for ingestion; other aspects of actin function appeared normal. This class of conditional phagocytosis mutant should prove to be useful for the expression cloning of the affected gene(s).

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Selected References

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

  1. André E., Brink M., Gerisch G., Isenberg G., Noegel A., Schleicher M., Segall J. E., Wallraff E. A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis. J Cell Biol. 1989 Mar;108(3):985–995. doi: 10.1083/jcb.108.3.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Axline S. G., Reaven E. P. Inhibition of phagocytosis and plasma membrane mobility of the cultivated macrophage by cytochalasin B. Role of subplasmalemmal microfilaments. J Cell Biol. 1974 Sep;62(3):647–659. doi: 10.1083/jcb.62.3.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beug H., Katz F. E., Gerisch G. Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium discoideum. J Cell Biol. 1973 Mar;56(3):647–658. doi: 10.1083/jcb.56.3.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bozzaro S., Roseman S. Adhesion of Dictyostelium discoideum cells to carbohydrates immobilized in polyacrylamide gels. I. Evidence for three sugar-specific cell surface receptors. J Biol Chem. 1983 Nov 25;258(22):13882–13889. [PubMed] [Google Scholar]
  5. Brink M., Gerisch G., Isenberg G., Noegel A. A., Segall J. E., Wallraff E., Schleicher M. A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor. J Cell Biol. 1990 Oct;111(4):1477–1489. doi: 10.1083/jcb.111.4.1477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chia C. P., Shariff A., Savage S. A., Luna E. J. The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly. J Cell Biol. 1993 Feb;120(4):909–922. doi: 10.1083/jcb.120.4.909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cox D., Condeelis J., Wessels D., Soll D., Kern H., Knecht D. A. Targeted disruption of the ABP-120 gene leads to cells with altered motility. J Cell Biol. 1992 Feb;116(4):943–955. doi: 10.1083/jcb.116.4.943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. De Lozanne A., Spudich J. A. Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science. 1987 May 29;236(4805):1086–1091. doi: 10.1126/science.3576222. [DOI] [PubMed] [Google Scholar]
  9. Di Virgilio F., Meyer B. C., Greenberg S., Silverstein S. C. Fc receptor-mediated phagocytosis occurs in macrophages at exceedingly low cytosolic Ca2+ levels. J Cell Biol. 1988 Mar;106(3):657–666. doi: 10.1083/jcb.106.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dynes J. L., Firtel R. A. Molecular complementation of a genetic marker in Dictyostelium using a genomic DNA library. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7966–7970. doi: 10.1073/pnas.86.20.7966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Falkow S., Isberg R. R., Portnoy D. A. The interaction of bacteria with mammalian cells. Annu Rev Cell Biol. 1992;8:333–363. doi: 10.1146/annurev.cb.08.110192.002001. [DOI] [PubMed] [Google Scholar]
  12. Francis C. L., Ryan T. A., Jones B. D., Smith S. J., Falkow S. Ruffles induced by Salmonella and other stimuli direct macropinocytosis of bacteria. Nature. 1993 Aug 12;364(6438):639–642. doi: 10.1038/364639a0. [DOI] [PubMed] [Google Scholar]
  13. Fukui Y., Lynch T. J., Brzeska H., Korn E. D. Myosin I is located at the leading edges of locomoting Dictyostelium amoebae. Nature. 1989 Sep 28;341(6240):328–331. doi: 10.1038/341328a0. [DOI] [PubMed] [Google Scholar]
  14. Fukui Y., Yumura S., Yumura T. K. Agar-overlay immunofluorescence: high-resolution studies of cytoskeletal components and their changes during chemotaxis. Methods Cell Biol. 1987;28:347–356. doi: 10.1016/s0091-679x(08)61655-6. [DOI] [PubMed] [Google Scholar]
  15. Gerisch G., Noegel A. A., Schleicher M. Genetic alteration of proteins in actin-based motility systems. Annu Rev Physiol. 1991;53:607–628. doi: 10.1146/annurev.ph.53.030191.003135. [DOI] [PubMed] [Google Scholar]
  16. Greenberg S., Burridge K., Silverstein S. C. Colocalization of F-actin and talin during Fc receptor-mediated phagocytosis in mouse macrophages. J Exp Med. 1990 Dec 1;172(6):1853–1856. doi: 10.1084/jem.172.6.1853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Greenberg S., Chang P., Silverstein S. C. Tyrosine phosphorylation is required for Fc receptor-mediated phagocytosis in mouse macrophages. J Exp Med. 1993 Feb 1;177(2):529–534. doi: 10.1084/jem.177.2.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Greenberg S., Chang P., Silverstein S. C. Tyrosine phosphorylation of the gamma subunit of Fc gamma receptors, p72syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages. J Biol Chem. 1994 Feb 4;269(5):3897–3902. [PubMed] [Google Scholar]
  19. Greenberg S., el Khoury J., di Virgilio F., Kaplan E. M., Silverstein S. C. Ca(2+)-independent F-actin assembly and disassembly during Fc receptor-mediated phagocytosis in mouse macrophages. J Cell Biol. 1991 May;113(4):757–767. doi: 10.1083/jcb.113.4.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gresham H. D., Graham I. L., Anderson D. C., Brown E. J. Leukocyte adhesion-deficient neutrophils fail to amplify phagocytic function in response to stimulation. Evidence for CD11b/CD18-dependent and -independent mechanisms of phagocytosis. J Clin Invest. 1991 Aug;88(2):588–597. doi: 10.1172/JCI115343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Griffin F. M., Jr, Griffin J. A., Leider J. E., Silverstein S. C. Studies on the mechanism of phagocytosis. I. Requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane. J Exp Med. 1975 Nov 1;142(5):1263–1282. doi: 10.1084/jem.142.5.1263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Griffin F. M., Jr, Griffin J. A., Silverstein S. C. Studies on the mechanism of phagocytosis. II. The interaction of macrophages with anti-immunoglobulin IgG-coated bone marrow-derived lymphocytes. J Exp Med. 1976 Sep 1;144(3):788–809. doi: 10.1084/jem.144.3.788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hishikawa T., Cheung J. Y., Yelamarty R. V., Knutson D. W. Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages. J Cell Biol. 1991 Oct;115(1):59–66. doi: 10.1083/jcb.115.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Howard P. K., Sefton B. M., Firtel R. A. Tyrosine phosphorylation of actin in Dictyostelium associated with cell-shape changes. Science. 1993 Jan 8;259(5092):241–244. doi: 10.1126/science.7678470. [DOI] [PubMed] [Google Scholar]
  25. Joiner K. A., Fuhrman S. A., Miettinen H. M., Kasper L. H., Mellman I. Toxoplasma gondii: fusion competence of parasitophorous vacuoles in Fc receptor-transfected fibroblasts. Science. 1990 Aug 10;249(4969):641–646. doi: 10.1126/science.2200126. [DOI] [PubMed] [Google Scholar]
  26. Jung G., Hammer J. A., 3rd Generation and characterization of Dictyostelium cells deficient in a myosin I heavy chain isoform. J Cell Biol. 1990 Jun;110(6):1955–1964. doi: 10.1083/jcb.110.6.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kayman S. C., Clarke M. Relationship between axenic growth of Dictyostelium discoideum strains and their track morphology on substrates coated with gold particles. J Cell Biol. 1983 Oct;97(4):1001–1010. doi: 10.1083/jcb.97.4.1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kayman S. C., Reichel M., Clarke M. Motility mutants of Dictyostelium discoideum. J Cell Biol. 1982 Jun;93(3):705–711. doi: 10.1083/jcb.93.3.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Knecht D. A., Loomis W. F. Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science. 1987 May 29;236(4805):1081–1086. doi: 10.1126/science.3576221. [DOI] [PubMed] [Google Scholar]
  30. Lennartz M. R., Cole F. S., Shepherd V. L., Wileman T. E., Stahl P. D. Isolation and characterization of a mannose-specific endocytosis receptor from human placenta. J Biol Chem. 1987 Jul 25;262(21):9942–9944. [PubMed] [Google Scholar]
  31. Lennartz M. R., Wileman T. E., Stahl P. D. Isolation and characterization of a mannose-specific endocytosis receptor from rabbit alveolar macrophages. Biochem J. 1987 Aug 1;245(3):705–711. doi: 10.1042/bj2450705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lew D. P., Andersson T., Hed J., Di Virgilio F., Pozzan T., Stendahl O. Ca2+-dependent and Ca2+-independent phagocytosis in human neutrophils. Nature. 1985 Jun 6;315(6019):509–511. doi: 10.1038/315509a0. [DOI] [PubMed] [Google Scholar]
  33. Loomis W. F. Genetic tools for Dictyostelium discoideum. Methods Cell Biol. 1987;28:31–65. doi: 10.1016/s0091-679x(08)61636-2. [DOI] [PubMed] [Google Scholar]
  34. Malawista S. E., Gee J. B., Bensch K. G. Cytochalasin B reversibly inhibits phagocytosis: functional, metabolic, and ultrastructural effects in human blood leukocytes and rabbit alveolar macrophages. Yale J Biol Med. 1971 Dec;44(3):286–300. [PMC free article] [PubMed] [Google Scholar]
  35. McNeil P. L., Swanson J. A., Wright S. D., Silverstein S. C., Taylor D. L. Fc-receptor-mediated phagocytosis occurs in macrophages without an increase in average [Ca++]i. J Cell Biol. 1986 May;102(5):1586–1592. doi: 10.1083/jcb.102.5.1586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rabinowitz S., Horstmann H., Gordon S., Griffiths G. Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages. J Cell Biol. 1992 Jan;116(1):95–112. doi: 10.1083/jcb.116.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schweiger A., Mihalache O., Ecke M., Gerisch G. Stage-specific tyrosine phosphorylation of actin in Dictyostelium discoideum cells. J Cell Sci. 1992 Jul;102(Pt 3):601–609. doi: 10.1242/jcs.102.3.601. [DOI] [PubMed] [Google Scholar]
  38. Schweinle J. E., Ezekowitz R. A., Tenner A. J., Kuhlman M., Joiner K. A. Human mannose-binding protein activates the alternative complement pathway and enhances serum bactericidal activity on a mannose-rich isolate of Salmonella. J Clin Invest. 1989 Dec;84(6):1821–1829. doi: 10.1172/JCI114367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shariff A., Luna E. J. Dictyostelium discoideum plasma membranes contain an actin-nucleating activity that requires ponticulin, an integral membrane glycoprotein. J Cell Biol. 1990 Mar;110(3):681–692. doi: 10.1083/jcb.110.3.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Silverstein S. C., Steinman R. M., Cohn Z. A. Endocytosis. Annu Rev Biochem. 1977;46:669–722. doi: 10.1146/annurev.bi.46.070177.003321. [DOI] [PubMed] [Google Scholar]
  41. Stossel T. P. From signal to pseudopod. How cells control cytoplasmic actin assembly. J Biol Chem. 1989 Nov 5;264(31):18261–18264. [PubMed] [Google Scholar]
  42. Sussman M. Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. Methods Cell Biol. 1987;28:9–29. doi: 10.1016/s0091-679x(08)61635-0. [DOI] [PubMed] [Google Scholar]
  43. Turner R., North M. J., Harwood J. M. Putrescine uptake by the cellular slime mould dictyostelium discoideum. Biochem J. 1979 Apr 15;180(1):119–127. doi: 10.1042/bj1800119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ukkonen P., Lewis V., Marsh M., Helenius A., Mellman I. Transport of macrophage Fc receptors and Fc receptor-bound ligands to lysosomes. J Exp Med. 1986 Apr 1;163(4):952–971. doi: 10.1084/jem.163.4.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Valtonen M. V., Plosila M., Valtonen V. V., Mäkelä P. H. Effect of the quality of the lipopolysaccharide on mouse virulence of Salmonella enteritidis. Infect Immun. 1975 Oct;12(4):828–832. doi: 10.1128/iai.12.4.828-832.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vogel G., Thilo L., Schwarz H., Steinhart R. Mechanism of phagocytosis in Dictyostelium discoideum: phagocytosis is mediated by different recognition sites as disclosed by mutants with altered phagocytotic properties. J Cell Biol. 1980 Aug;86(2):456–465. doi: 10.1083/jcb.86.2.456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wallraff E., Schleicher M., Modersitzki M., Rieger D., Isenberg G., Gerisch G. Selection of Dictyostelium mutants defective in cytoskeletal proteins: use of an antibody that binds to the ends of alpha-actinin rods. EMBO J. 1986 Jan;5(1):61–67. doi: 10.1002/j.1460-2075.1986.tb04178.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Westphal O., Jann K., Himmelspach K. Chemistry and immunochemistry of bacterial lipopolysaccharides as cell wall antigens and endotoxins. Prog Allergy. 1983;33:9–39. [PubMed] [Google Scholar]
  49. Witke W., Schleicher M., Noegel A. A. Redundancy in the microfilament system: abnormal development of Dictyostelium cells lacking two F-actin cross-linking proteins. Cell. 1992 Jan 10;68(1):53–62. doi: 10.1016/0092-8674(92)90205-q. [DOI] [PubMed] [Google Scholar]
  50. Wuestehube L. J., Chia C. P., Luna E. J. Indirect immunofluorescence localization of ponticulin in motile cells. Cell Motil Cytoskeleton. 1989;13(4):245–263. doi: 10.1002/cm.970130404. [DOI] [PubMed] [Google Scholar]

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