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J Cell Biol. Jun 1, 1990; 110(6): 1955–1964.
PMCID: PMC2116136

Generation and characterization of Dictyostelium cells deficient in a myosin I heavy chain isoform

Abstract

Motile activities such as chemotaxis and phagocytosis, which occur in Dictyostelium cells lacking myosin II, may be dependent upon myosin I. To begin to explore this possibility, we have engineered a disruption of the Dictyostelium myosin I heavy chain (DMIHC) gene described recently (Jung, G., C. L. Saxe III, A. R. Kimmel, and J. A. Hammer III. 1989. Proc. Natl. Acad. Sci. USA. 86:6186-6190). The double-crossover, gene disruption event that occurred resulted in replacement of the middle approximate one-third of the gene with the neomycin resistance marker. The resulting cells are devoid of both the 3.6-kb DMIHC gene transcript and the 124-kD DMIHC polypeptide. DMIHC- cells are capable of chemotactic streaming and aggregation, but these processes are delayed. Furthermore, the rate of phagocytosis by DMIHC- cells is reduced, as assessed by growth rate on lawns of heat-killed bacteria and on the initial rate of uptake of FITC-labeled bacteria. Therefore, this Dictyostelium myosin I isoform appears to play a role in supporting chemotaxis and phagocytosis, but it is clearly not required for these processes to occur. Using a portion of the DMIHC gene as a probe, we have cloned three additional Dictyostelium small myosin heavy chain genes. Comparison of these four genes with three genes described recently by Titus et al. (Titus, M. A., H. M. Warrick, and J. A. Spudich. 1989. Cell Reg. 1:55-63) indicates that there are at least five small myosin heavy chain genes in Dictyostelium. The probability that there is considerable overlap of function between these small myosin isoforms indicates that multiple gene disruptions within a single cell may be necessary to generate a more striking myosin I- phenotype.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adams RJ, Pollard TD. Propulsion of organelles isolated from Acanthamoeba along actin filaments by myosin-I. Nature. 1986 Aug 21;322(6081):754–756. [PubMed]
  • Adams RJ, Pollard TD. Membrane-bound myosin-I provides new mechanisms in cell motility. Cell Motil Cytoskeleton. 1989;14(2):178–182. [PubMed]
  • Albanesi JP, Fujisaki H, Hammer JA, 3rd, Korn ED, Jones R, Sheetz MP. Monomeric Acanthamoeba myosins I support movement in vitro. J Biol Chem. 1985 Jul 25;260(15):8649–8652. [PubMed]
  • Chia CP, Luna EJ. Phagocytosis in Dictyostelium discoideum is inhibited by antibodies directed primarily against common carbohydrate epitopes of a major cell-surface plasma membrane glycoprotein. Exp Cell Res. 1989 Mar;181(1):11–26. [PubMed]
  • Côté GP, Albanesi JP, Ueno T, Hammer JA, 3rd, Korn ED. Purification from Dictyostelium discoideum of a low-molecular-weight myosin that resembles myosin I from Acanthamoeba castellanii. J Biol Chem. 1985 Apr 25;260(8):4543–4546. [PubMed]
  • DeLozanne A, Lewis M, Spudich JA, Leinwand LA. Cloning and characterization of a nonmuscle myosin heavy chain cDNA. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6807–6810. [PMC free article] [PubMed]
  • Earnshaw WC, Sullivan KF, Machlin PS, Cooke CA, Kaiser DA, Pollard TD, Rothfield NF, Cleveland DW. Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen. J Cell Biol. 1987 Apr;104(4):817–829. [PMC free article] [PubMed]
  • Egelhoff TT, Brown SS, Manstein DJ, Spudich JA. Hygromycin resistance as a selectable marker in Dictyostelium discoideum. Mol Cell Biol. 1989 May;9(5):1965–1968. [PMC free article] [PubMed]
  • Fujisaki H, Albanesi JP, Korn ED. Experimental evidence for the contractile activities of Acanthamoeba myosins IA and IB. J Biol Chem. 1985 Sep 15;260(20):11183–11189. [PubMed]
  • Fukui Y, Lynch TJ, Brzeska H, Korn ED. Myosin I is located at the leading edges of locomoting Dictyostelium amoebae. Nature. 1989 Sep 28;341(6240):328–331. [PubMed]
  • Hammer JA, 3rd, Korn ED, Paterson BM. Isolation of a non-muscle myosin heavy chain gene from Acanthamoeba. J Biol Chem. 1986 Feb 5;261(4):1949–1956. [PubMed]
  • Hammer JA, 3rd, Bowers B, Paterson BM, Korn ED. Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail. J Cell Biol. 1987 Aug;105(2):913–925. [PMC free article] [PubMed]
  • Jung G, Korn ED, Hammer JA., 3rd The heavy chain of Acanthamoeba myosin IB is a fusion of myosin-like and non-myosin-like sequences. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6720–6724. [PMC free article] [PubMed]
  • Jung G, Saxe CL, 3rd, Kimmel AR, Hammer JA., 3rd Dictyostelium discoideum contains a gene encoding a myosin I heavy chain. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6186–6190. [PMC free article] [PubMed]
  • Jung G, Schmidt CJ, Hammer JA., 3rd Myosin I heavy-chain genes of Acanthamoeba castellanii: cloning of a second gene and evidence for the existence of a third isoform. Gene. 1989 Oct 30;82(2):269–280. [PubMed]
  • Knecht DA, Loomis WF. Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science. 1987 May 29;236(4805):1081–1086. [PubMed]
  • Korn ED, Hammer JA., 3rd Myosins of nonmuscle cells. Annu Rev Biophys Biophys Chem. 1988;17:23–45. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Loomis WF. Genetic tools for Dictyostelium discoideum. Methods Cell Biol. 1987;28:31–65. [PubMed]
  • Manstein DJ, Titus MA, De Lozanne A, Spudich JA. Gene replacement in Dictyostelium: generation of myosin null mutants. EMBO J. 1989 Mar;8(3):923–932. [PMC free article] [PubMed]
  • Nellen W, Saur U. Cell-cycle dependent transformation competence in Dictyostelium discoideum. Biochem Biophys Res Commun. 1988 Jul 15;154(1):54–59. [PubMed]
  • Nellen W, Datta S, Reymond C, Sivertsen A, Mann S, Crowley T, Firtel RA. Molecular biology in Dictyostelium: tools and applications. Methods Cell Biol. 1987;28:67–100. [PubMed]
  • Rothstein RJ. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PMC free article] [PubMed]
  • Titus MA, Warrick HM, Spudich JA. Multiple actin-based motor genes in Dictyostelium. Cell Regul. 1989 Nov;1(1):55–63. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Wessels D, Soll DR, Knecht D, Loomis WF, De Lozanne A, Spudich J. Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain. Dev Biol. 1988 Jul;128(1):164–177. [PubMed]

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