Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Jul 1;107(1):201–209. doi: 10.1083/jcb.107.1.201

How actin binds and assembles onto plasma membranes from Dictyostelium discoideum

PMCID: PMC2115166  PMID: 3392099

Abstract

We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross- linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Bender N., Fasold H., Kenmoku A., Middelhoff G., Volk K. E. The selective blocking of the polymerization reaction of striated muscle actin leading to a derivative suitable for crystallization. Modification of Tyr-53 by 5-diazonium-(1H)tetrazole. Eur J Biochem. 1976 Apr 15;64(1):215–218. doi: 10.1111/j.1432-1033.1976.tb10290.x. [DOI] [PubMed] [Google Scholar]
  2. Bennett H., Condeelis J. Decoration with myosin subfragment-1 disrupts contacts between microfilaments and the cell membrane in isolated Dictyostelium cortices. J Cell Biol. 1984 Oct;99(4 Pt 1):1434–1440. doi: 10.1083/jcb.99.4.1434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bonder E. M., Fishkind D. J., Mooseker M. S. Direct measurement of critical concentrations and assembly rate constants at the two ends of an actin filament. Cell. 1983 Sep;34(2):491–501. doi: 10.1016/0092-8674(83)90382-3. [DOI] [PubMed] [Google Scholar]
  4. Burtnick L. D. Modification of actin with fluorescein isothiocyanate. Biochim Biophys Acta. 1984 Nov 23;791(1):57–62. doi: 10.1016/0167-4838(84)90281-4. [DOI] [PubMed] [Google Scholar]
  5. Carson M., Weber A., Zigmond S. H. An actin-nucleating activity in polymorphonuclear leukocytes is modulated by chemotactic peptides. J Cell Biol. 1986 Dec;103(6 Pt 2):2707–2714. doi: 10.1083/jcb.103.6.2707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen C. M., Foley S. F. Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane. J Cell Biol. 1980 Aug;86(2):694–698. doi: 10.1083/jcb.86.2.694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Coluccio L. M., Tilney L. G. Phalloidin enhances actin assembly by preventing monomer dissociation. J Cell Biol. 1984 Aug;99(2):529–535. doi: 10.1083/jcb.99.2.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooper J. A., Pollard T. D. Effect of capping protein on the kinetics of actin polymerization. Biochemistry. 1985 Jan 29;24(3):793–799. doi: 10.1021/bi00324a039. [DOI] [PubMed] [Google Scholar]
  9. Coué M., Korn E. D. Interaction of plasma gelsolin with G-actin and F-actin in the presence and absence of calcium ions. J Biol Chem. 1985 Dec 5;260(28):15033–15041. [PubMed] [Google Scholar]
  10. Dahlquist F. W. The meaning of Scatchard and Hill plots. Methods Enzymol. 1978;48:270–299. doi: 10.1016/s0076-6879(78)48015-2. [DOI] [PubMed] [Google Scholar]
  11. DeRosier D. J., Tilney L. G. The form and function of actin. A product of its unique design. Cell Muscle Motil. 1984;5:139–169. doi: 10.1007/978-1-4684-4592-3_3. [DOI] [PubMed] [Google Scholar]
  12. Edelman G. M. Surface modulation in cell recognition and cell growth. Science. 1976 Apr 16;192(4236):218–226. doi: 10.1126/science.769162. [DOI] [PubMed] [Google Scholar]
  13. Egelman E. H., Francis N., DeRosier D. J. F-actin is a helix with a random variable twist. Nature. 1982 Jul 8;298(5870):131–135. doi: 10.1038/298131a0. [DOI] [PubMed] [Google Scholar]
  14. Elzinga M., Collins J. H., Kuehl W. M., Adelstein R. S. Complete amino-acid sequence of actin of rabbit skeletal muscle. Proc Natl Acad Sci U S A. 1973 Sep;70(9):2687–2691. doi: 10.1073/pnas.70.9.2687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Elzinga M., Phelan J. J. F-actin is intermolecularly crosslinked by N,N'-p-phenylenedimaleimide through lysine-191 and cysteine-374. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6599–6602. doi: 10.1073/pnas.81.21.6599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fowler V. M., Luna E. J., Hargreaves W. R., Taylor D. L., Branton D. Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane. J Cell Biol. 1981 Feb;88(2):388–395. doi: 10.1083/jcb.88.2.388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fox J. E., Phillips D. R. Polymerization and organization of actin filaments within platelets. Semin Hematol. 1983 Oct;20(4):243–260. [PubMed] [Google Scholar]
  18. GREENBURY C. L., MOORE D. H., NUNN L. A. THE REACTION WITH RED CELLS OF 7S RABBIT ANTIBODY, ITS SUB-UNITS AND THEIR RECOMBINANTS. Immunology. 1965 Apr;8:420–431. [PMC free article] [PubMed] [Google Scholar]
  19. Gilbert H. R., Frieden C. Preparation, purification and properties of a crosslinked trimer of G-actin. Biochem Biophys Res Commun. 1983 Mar 16;111(2):404–408. doi: 10.1016/0006-291x(83)90320-0. [DOI] [PubMed] [Google Scholar]
  20. Goodloe-Holland C. M., Luna E. J. A membrane cytoskeleton from Dictyostelium discoideum. III. Plasma membrane fragments bind predominantly to the sides of actin filaments. J Cell Biol. 1984 Jul;99(1 Pt 1):71–78. doi: 10.1083/jcb.99.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Greene L. E., Eisenberg E. The binding of heavy meromyosin to F-actin. J Biol Chem. 1980 Jan 25;255(2):549–554. [PubMed] [Google Scholar]
  22. Griffith L. M., Pollard T. D. Evidence for actin filament-microtubule interaction mediated by microtubule-associated proteins. J Cell Biol. 1978 Sep;78(3):958–965. doi: 10.1083/jcb.78.3.958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hegyi G., Premecz G., Sain B., Mühlrád A. Selective carbethoxylation of the histidine residues of actin by diethylpyrocarbonate. Eur J Biochem. 1974 May 2;44(1):7–12. doi: 10.1111/j.1432-1033.1974.tb03452.x. [DOI] [PubMed] [Google Scholar]
  24. Jacobson B. S. Actin binding to the cytoplasmic surface of the plasma membrane isolated from Dictyostelium discoideum. Biochem Biophys Res Commun. 1980 Dec 31;97(4):1493–1498. doi: 10.1016/s0006-291x(80)80034-9. [DOI] [PubMed] [Google Scholar]
  25. Kurth M. C., Wang L. L., Dingus J., Bryan J. Purification and characterization of a gelsolin-actin complex from human platelets. Evidence for Ca2+-insensitive functions. J Biol Chem. 1983 Sep 25;258(18):10895–10903. [PubMed] [Google Scholar]
  26. 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]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Laub F., Kaplan M., Gitler C. Actin polymerization accompanies Thy-1-capping on mouse thymocytes. FEBS Lett. 1981 Feb 9;124(1):35–38. doi: 10.1016/0014-5793(81)80048-8. [DOI] [PubMed] [Google Scholar]
  29. Lewis J. C. Cytoskeleton in platelet function. Cell Muscle Motil. 1984;5:341–377. doi: 10.1007/978-1-4684-4592-3_10. [DOI] [PubMed] [Google Scholar]
  30. Luna E. J., Fowler V. M., Swanson J., Branton D., Taylor D. L. A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity. J Cell Biol. 1981 Feb;88(2):396–409. doi: 10.1083/jcb.88.2.396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Luna E. J., Goodloe-Holland C. M., Ingalls H. M. A membrane cytoskeleton from Dictyostelium discoideum. II. Integral proteins mediate the binding of plasma membranes to F-actin affinity beads. J Cell Biol. 1984 Jul;99(1 Pt 1):58–70. doi: 10.1083/jcb.99.1.58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Melchior W. B., Jr, Fahrney D. Ethoxyformylation of proteins. Reaction of ethoxyformic anhydride with alpha-chymotrypsin, pepsin, and pancreatic ribonuclease at pH 4. Biochemistry. 1970 Jan 20;9(2):251–258. doi: 10.1021/bi00804a010. [DOI] [PubMed] [Google Scholar]
  33. Miles E. W. Modification of histidyl residues in proteins by diethylpyrocarbonate. Methods Enzymol. 1977;47:431–442. doi: 10.1016/0076-6879(77)47043-5. [DOI] [PubMed] [Google Scholar]
  34. Mockrin S. C., Korn E. D. Isolation and characterization of covalently cross-linked actin dimer. J Biol Chem. 1981 Aug 10;256(15):8228–8233. [PubMed] [Google Scholar]
  35. Mooseker M. S., Pollard T. D., Wharton K. A. Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border. J Cell Biol. 1982 Oct;95(1):223–233. doi: 10.1083/jcb.95.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mühlrad A., Corsi A., Granata A. L. Studies on the properties of chemically modified actin. I. Photooxidation, succinylation, nitration. Biochim Biophys Acta. 1968 Oct 1;162(3):435–443. doi: 10.1016/0005-2728(68)90129-1. [DOI] [PubMed] [Google Scholar]
  37. Mühlrad A., Hegyi G., Horányi M. Studies on the properties of chemically modified actin. 3. Carbethoxylation. Biochim Biophys Acta. 1969 May;181(1):184–190. doi: 10.1016/0005-2795(69)90240-2. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Painter R. G., McIntosh A. T. The regional association of actin and myosin with sites of particle phagocytosis. J Supramol Struct. 1979;12(3):369–384. doi: 10.1002/jss.400120308. [DOI] [PubMed] [Google Scholar]
  40. Pfeiffer J. R., Seagrave J. C., Davis B. H., Deanin G. G., Oliver J. M. Membrane and cytoskeletal changes associated with IgE-mediated serotonin release from rat basophilic leukemia cells. J Cell Biol. 1985 Dec;101(6):2145–2155. doi: 10.1083/jcb.101.6.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pollard T. D., Cooper J. A. Actin and actin-binding proteins. A critical evaluation of mechanisms and functions. Annu Rev Biochem. 1986;55:987–1035. doi: 10.1146/annurev.bi.55.070186.005011. [DOI] [PubMed] [Google Scholar]
  42. Reynolds J. A. Interaction of divalent antibody with cell surface antigens. Biochemistry. 1979 Jan 23;18(2):264–269. doi: 10.1021/bi00569a004. [DOI] [PubMed] [Google Scholar]
  43. Schwartz M. A., Luna E. J. Binding and assembly of actin filaments by plasma membranes from Dictyostelium discoideum. J Cell Biol. 1986 Jun;102(6):2067–2075. doi: 10.1083/jcb.102.6.2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  45. Sutoh K. Actin-actin and actin-deoxyribonuclease I contact sites in the actin sequence. Biochemistry. 1984 Apr 24;23(9):1942–1946. doi: 10.1021/bi00304a009. [DOI] [PubMed] [Google Scholar]
  46. Svitkina T. M., Neyfakh A. A., Jr, Bershadsky A. D. Actin cytoskeleton of spread fibroblasts appears to assemble at the cell edges. J Cell Sci. 1986 Jun;82:235–248. doi: 10.1242/jcs.82.1.235. [DOI] [PubMed] [Google Scholar]
  47. Tilney L. G., Bonder E. M., DeRosier D. J. Actin filaments elongate from their membrane-associated ends. J Cell Biol. 1981 Aug;90(2):485–494. doi: 10.1083/jcb.90.2.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tilney L. G., Inoué S. Acrosomal reaction of Thyone sperm. II. The kinetics and possible mechanism of acrosomal process elongation. J Cell Biol. 1982 Jun;93(3):820–827. doi: 10.1083/jcb.93.3.820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Uyemura D. G., Brown S. S., Spudich J. A. Biochemical and structural characterization of actin from Dictyostelium discoideum. J Biol Chem. 1978 Dec 25;253(24):9088–9096. [PubMed] [Google Scholar]
  50. Wang Y. L. Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling. J Cell Biol. 1985 Aug;101(2):597–602. doi: 10.1083/jcb.101.2.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Weber A., Northrop J., Bishop M. F., Ferrone F. A., Mooseker M. S. Nucleation of actin polymerization by villin and elongation at subcritical monomer concentration. Biochemistry. 1987 May 5;26(9):2528–2536. doi: 10.1021/bi00383a019. [DOI] [PubMed] [Google Scholar]
  52. Wegner A., Isenberg G. 12-fold difference between the critical monomer concentrations of the two ends of actin filaments in physiological salt conditions. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4922–4925. doi: 10.1073/pnas.80.16.4922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wegner A. Treadmilling of actin at physiological salt concentrations. An analysis of the critical concentrations of actin filaments. J Mol Biol. 1982 Nov 15;161(4):607–615. doi: 10.1016/0022-2836(82)90411-9. [DOI] [PubMed] [Google Scholar]
  54. Wuestehube L. J., Luna E. J. F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes. J Cell Biol. 1987 Oct;105(4):1741–1751. doi: 10.1083/jcb.105.4.1741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yin H. L., Stossel T. P. Purification and structural properties of gelsolin, a Ca2+-activated regulatory protein of macrophages. J Biol Chem. 1980 Oct 10;255(19):9490–9493. [PubMed] [Google Scholar]

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

RESOURCES