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. 1996 May;7(5):803–823. doi: 10.1091/mbc.7.5.803

Re-expression of ABP-120 rescues cytoskeletal, motility, and phagocytosis defects of ABP-120- Dictyostelium mutants.

D Cox 1, D Wessels 1, D R Soll 1, J Hartwig 1, J Condeelis 1
PMCID: PMC275931  PMID: 8744952

Abstract

The actin binding protein ABP-120 has been proposed to cross-link actin filaments in nascent pseudopods, in a step required for normal pseudopod extension in motile Dictyostelium amoebae. To test this hypothesis, cell lines that lack ABP-120 were created independently either by chemical mutagenesis or homologous recombination. Different phenotypes were reported in these two studies. The chemical mutant shows only a subtle defect in actin cross-linking, while the homologous recombinant mutants show profound defects in actin cross-linking, cytoskeletal structure, pseudopod number and size, cell motility and chemotaxis and, as shown here, phagocytosis. To resolve the controversy as to what the ABP-120- phenotype is, ABP-120 was re-expressed in an ABP-120- cell line created by homologous recombination. Two independently "rescued" cell lines that express wild-type levels of ABP-120 were analyzed. In both rescued cell lines, actin incorporation into the cytoskeleton, pseudopod formation, cell morphology, instantaneous velocity, phagocytosis, and chemotaxis were restored to wild-type levels. There is no alteration in the expression levels of several related actin binding proteins in either the original ABP-120- cell line or in the rescued cell lines, leading to the conclusion that neither the aberrant phenotype observed in ABP-120- cells nor the normal phenotype reasserted in rescued cells can be attributed to alterations in the levels of other abundant and related actin binding proteins. Re-expression of ABP-120 in ABP-120- cells reestablishes normal structural and behavioral parameters, demonstrating that the severity and properties of the structural and behavioral defects of ABP-120- cell lines produced by homologous recombination are the direct result of the absence of ABP-120.

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Selected References

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

  1. Bray D., Vasiliev J. Cell motility. Networks from mutants. Nature. 1989 Mar 16;338(6212):203–204. doi: 10.1038/338203a0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Carboni J. M., Condeelis J. S. Ligand-induced changes in the location of actin, myosin, 95K (alpha-actinin), and 120K protein in amebae of Dictyostelium discoideum. J Cell Biol. 1985 Jun;100(6):1884–1893. doi: 10.1083/jcb.100.6.1884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cohen C. J., Bacon R., Clarke M., Joiner K., Mellman I. Dictyostelium discoideum mutants with conditional defects in phagocytosis. J Cell Biol. 1994 Aug;126(4):955–966. doi: 10.1083/jcb.126.4.955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cohen S. M., Knecht D., Lodish H. F., Loomis W. F. DNA sequences required for expression of a Dictyostelium actin gene. EMBO J. 1986 Dec 1;5(12):3361–3366. doi: 10.1002/j.1460-2075.1986.tb04651.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Condeelis J., Bresnick A., Demma M., Dharmawardhane S., Eddy R., Hall A. L., Sauterer R., Warren V. Mechanisms of amoeboid chemotaxis: an evaluation of the cortical expansion model. Dev Genet. 1990;11(5-6):333–340. doi: 10.1002/dvg.1020110504. [DOI] [PubMed] [Google Scholar]
  7. Condeelis J. Life at the leading edge: the formation of cell protrusions. Annu Rev Cell Biol. 1993;9:411–444. doi: 10.1146/annurev.cb.09.110193.002211. [DOI] [PubMed] [Google Scholar]
  8. Condeelis J. Understanding the cortex of crawling cells: insights from Dictyostelium. Trends Cell Biol. 1993 Nov;3(11):371–376. doi: 10.1016/0962-8924(93)90085-f. [DOI] [PubMed] [Google Scholar]
  9. Condeelis J., Vahey M., Carboni J. M., DeMey J., Ogihara S. Properties of the 120,000- and 95,000-dalton actin-binding proteins from Dictyostelium discoideum and their possible functions in assembling the cytoplasmic matrix. J Cell Biol. 1984 Jul;99(1 Pt 2):119s–126s. doi: 10.1083/jcb.99.1.119s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Cox D., Ridsdale J. A., Condeelis J., Hartwig J. Genetic deletion of ABP-120 alters the three-dimensional organization of actin filaments in Dictyostelium pseudopods. J Cell Biol. 1995 Mar;128(5):819–835. doi: 10.1083/jcb.128.5.819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dharmawardhane S., Warren V., Hall A. L., Condeelis J. Changes in the association of actin-binding proteins with the actin cytoskeleton during chemotactic stimulation of Dictyostelium discoideum. Cell Motil Cytoskeleton. 1989;13(1):57–63. doi: 10.1002/cm.970130107. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Franke J., Kessin R. A defined minimal medium for axenic strains of Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1977 May;74(5):2157–2161. doi: 10.1073/pnas.74.5.2157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Gerisch G., Segall J. E., Wallraff E. Isolation and behavioral analysis of mutants defective in cytoskeletal proteins. Cell Motil Cytoskeleton. 1989;14(1):75–79. doi: 10.1002/cm.970140115. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Hall A. L., Schlein A., Condeelis J. Relationship of pseudopod extension to chemotactic hormone-induced actin polymerization in amoeboid cells. J Cell Biochem. 1988 Jul;37(3):285–299. doi: 10.1002/jcb.240370304. [DOI] [PubMed] [Google Scholar]
  19. Hartwig J. H., Kwiatkowski D. J. Actin-binding proteins. Curr Opin Cell Biol. 1991 Feb;3(1):87–97. doi: 10.1016/0955-0674(91)90170-4. [DOI] [PubMed] [Google Scholar]
  20. Hartwig J. H., Shevlin P. The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages. J Cell Biol. 1986 Sep;103(3):1007–1020. doi: 10.1083/jcb.103.3.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Harvath L. Regulation of neutrophil chemotaxis: correlations with actin polymerization. Cancer Invest. 1990;8(6):651–654. doi: 10.3109/07357909009018937. [DOI] [PubMed] [Google Scholar]
  22. Howard T. H., Oresajo C. O. A method for quantifying F-actin in chemotactic peptide activated neutrophils: study of the effect of tBOC peptide. Cell Motil. 1985;5(6):545–557. doi: 10.1002/cm.970050609. [DOI] [PubMed] [Google Scholar]
  23. Knecht D. A., Jung J., Matthews L. Quantification of transformation efficiency using a new method for clonal growth and selection of axenic Dictyostelium cells. Dev Genet. 1990;11(5-6):403–409. doi: 10.1002/dvg.1020110513. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Kraft B., Steinbrech D., Yang M., Soll D. R. High-frequency switching in Dictyostelium. Dev Biol. 1988 Nov;130(1):198–208. doi: 10.1016/0012-1606(88)90426-5. [DOI] [PubMed] [Google Scholar]
  26. Matsudaira P. Modular organization of actin crosslinking proteins. Trends Biochem Sci. 1991 Mar;16(3):87–92. doi: 10.1016/0968-0004(91)90039-x. [DOI] [PubMed] [Google Scholar]
  27. Ogihara S., Carboni J., Condeelis J. Electron microscopic localization of myosin II and ABP-120 in the cortical actin matrix of Dictyostelium amoebae using IgG-gold conjugates. Dev Genet. 1988;9(4-5):505–520. doi: 10.1002/dvg.1020090427. [DOI] [PubMed] [Google Scholar]
  28. Reaven E. P., Axline S. G. Subplasmalemmal microfilaments and microtubules in resting and phagocytizing cultivated macrophages. J Cell Biol. 1973 Oct;59(1):12–27. doi: 10.1083/jcb.59.1.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sheetz M. P., Wayne D. B., Pearlman A. L. Extension of filopodia by motor-dependent actin assembly. Cell Motil Cytoskeleton. 1992;22(3):160–169. doi: 10.1002/cm.970220303. [DOI] [PubMed] [Google Scholar]
  30. Shelden E., Knecht D. A. Mutants lacking myosin II cannot resist forces generated during multicellular morphogenesis. J Cell Sci. 1995 Mar;108(Pt 3):1105–1115. doi: 10.1242/jcs.108.3.1105. [DOI] [PubMed] [Google Scholar]
  31. Shutt D. C., Wessels D., Wagenknecht K., Chandrasekhar A., Hitt A. L., Luna E. J., Soll D. R. Ponticulin plays a role in the positional stabilization of pseudopods. J Cell Biol. 1995 Dec;131(6 Pt 1):1495–1506. doi: 10.1083/jcb.131.6.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Soll D. R. "DMS," a computer-assisted system for quantitating motility, the dynamics of cytoplasmic flow, and pseudopod formation: its application to Dictyostelium chemotaxis. Cell Motil Cytoskeleton. 1988;10(1-2):91–106. doi: 10.1002/cm.970100114. [DOI] [PubMed] [Google Scholar]
  33. Soll D. R. Methods for manipulating and investigating developmental timing in Dictyostelium discoideum. Methods Cell Biol. 1987;28:413–431. doi: 10.1016/s0091-679x(08)61660-x. [DOI] [PubMed] [Google Scholar]
  34. Soll D. R. The use of computers in understanding how animal cells crawl. Int Rev Cytol. 1995;163:43–104. [PubMed] [Google Scholar]
  35. Soll D. R., Voss E., Varnum-Finney B., Wessels D. "Dynamic Morphology System": a method for quantitating changes in shape, pseudopod formation, and motion in normal and mutant amoebae of Dictyostelium discoideum. J Cell Biochem. 1988 Jun;37(2):177–192. doi: 10.1002/jcb.240370205. [DOI] [PubMed] [Google Scholar]
  36. Stendahl O. I., Hartwig J. H., Brotschi E. A., Stossel T. P. Distribution of actin-binding protein and myosin in macrophages during spreading and phagocytosis. J Cell Biol. 1980 Feb;84(2):215–224. doi: 10.1083/jcb.84.2.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stephens C. G., Snyderman R. Cyclic nucleotides regulate the morphologic alterations required for chemotaxis in monocytes. J Immunol. 1982 Mar;128(3):1192–1197. [PubMed] [Google Scholar]
  38. Sylwester A., Wessels D., Anderson S. A., Warren R. Q., Shutt D. C., Kennedy R. C., Soll D. R. HIV-induced syncytia of a T cell line form single giant pseudopods and are motile. J Cell Sci. 1993 Nov;106(Pt 3):941–953. doi: 10.1242/jcs.106.3.941. [DOI] [PubMed] [Google Scholar]
  39. Titus M. A., Wessels D., Spudich J. A., Soll D. The unconventional myosin encoded by the myoA gene plays a role in Dictyostelium motility. Mol Biol Cell. 1993 Feb;4(2):233–246. doi: 10.1091/mbc.4.2.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Varnum-Finney B. J., Voss E., Soll D. R. Frequency and orientation of pseudopod formation of Dictyostelium discoideum amebae chemotaxing in a spatial gradient: further evidence for a temporal mechanism. Cell Motil Cytoskeleton. 1987;8(1):18–26. doi: 10.1002/cm.970080104. [DOI] [PubMed] [Google Scholar]
  41. Varnum B., Soll D. R. Effects of cAMP on single cell motility in Dictyostelium. J Cell Biol. 1984 Sep;99(3):1151–1155. doi: 10.1083/jcb.99.3.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Wessels D., Murray J., Jung G., Hammer J. A., 3rd, Soll D. R. Myosin IB null mutants of Dictyostelium exhibit abnormalities in motility. Cell Motil Cytoskeleton. 1991;20(4):301–315. doi: 10.1002/cm.970200406. [DOI] [PubMed] [Google Scholar]
  44. Wessels D., Schroeder N. A., Voss E., Hall A. L., Condeelis J., Soll D. R. cAMP-mediated inhibition of intracellular particle movement and actin reorganization in Dictyostelium. J Cell Biol. 1989 Dec;109(6 Pt 1):2841–2851. doi: 10.1083/jcb.109.6.2841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wessels D., Titus M., Soll D. R. A Dictyostelium myosin I plays a crucial role in regulating the frequency of pseudopods formed on the substratum. Cell Motil Cytoskeleton. 1996;33(1):64–79. doi: 10.1002/(SICI)1097-0169(1996)33:1<64::AID-CM7>3.0.CO;2-I. [DOI] [PubMed] [Google Scholar]
  46. Witke W., Nellen W., Noegel A. Homologous recombination in the Dictyostelium alpha-actinin gene leads to an altered mRNA and lack of the protein. EMBO J. 1987 Dec 20;6(13):4143–4148. doi: 10.1002/j.1460-2075.1987.tb02760.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Wolosewick J. J., Condeelis J. Fine structure of gels prepared from an actin-binding protein and actin: comparison to cytoplasmic extracts and cortical cytoplasm in amoeboid cells of cortical cytoplasm in amoeboid cells of Dictyostelium discoideum. J Cell Biochem. 1986;30(3):227–243. doi: 10.1002/jcb.240300305. [DOI] [PubMed] [Google Scholar]
  49. Zigmond S. H., Sullivan S. J. Sensory adaptation of leukocytes to chemotactic peptides. J Cell Biol. 1979 Aug;82(2):517–527. doi: 10.1083/jcb.82.2.517. [DOI] [PMC free article] [PubMed] [Google Scholar]

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