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. 1985 Jun 1;100(6):1884–1893. doi: 10.1083/jcb.100.6.1884

Ligand-induced changes in the location of actin, myosin, 95K (alpha- actinin), and 120K protein in amebae of Dictyostelium discoideum

PMCID: PMC2113601  PMID: 3889011

Abstract

In this study we investigated concanavalin A (Con A) induced changes in the locations of actin, myosin, 120K, and 95K (alpha-actinin) to determine the extent to which actin and myosin are reorganized during capping and the roles that 120K and 95K might play in this reorganization. We observed the location of each protein by indirect immunofluorescence using affinity purified antibodies. Four morphological states were distinguished in vegetative Dictyostelium amebae: ameboid cells before Con A binding, patched cells, capped cells, and ameboid cells with caps. The location of each protein was distinct in ameboid cells both before and after capping Actin and 120K were found in the cell cortex usually associated with surface projections, and myosin and 95K were diffusely distributed. Myosin was excluded from surface projections in ameboid cells. During patching, all four proteins were localized below Con A patches. During capping, actin, myosin, and 95K protein moved with the Con A patches into the cap whereas 120K protein was excluded from the cap. During the late stages of cap formation actin and myosin were progressively lost from the cap, and 120K became concentrated in new actin-filled projections that formed away from the cap. However, 95K remained tightly associated with the cap. Poisoning cells with sodium azide inhibited capping but not patching of ligand. In azide-poisoned cells, myosin and 95K did not co-patch with Con A, whereas copatching of 120K and actin with Con A occurred as usual. Our results support the hypothesis that capping is an actomyosin-mediated motile event that involves a sliding interaction between actin filaments, which are anchored through the membrane to ligand patches, and myosin in the cortex. They are also consistent with a role for 120K in the formation of surface projections by promoting growth and/or cross-linking of actin filaments within projections, and with a role for 95K in regulating actomyosin-mediated contractility, earlier proposals based on the in vitro properties of these two proteins (Condeelis, J., M. Vahey, J. M. Carboni, J. DeMey, S. Ogihara, 1984, J. Cell Biol., 99:119s-126s).

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Selected References

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  1. Allwood G., Asherson G. L., Davey M. J., Goodford P. J. The early uptake of radioactive calcium by human lymphocytes treated with phytohaemagglutinin. Immunology. 1971 Sep;21(3):509–516. [PMC free article] [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. Braun J., Sha'afi R. I., Unanue E. R. Crosslinking by ligands to surface immunoglobulin triggers mobilization of intracellular 45Ca2+ in B lymphocytes. J Cell Biol. 1979 Sep;82(3):755–766. doi: 10.1083/jcb.82.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brier J., Fechheimer M., Swanson J., Taylor D. L. Abundance, relative gelation activity, and distribution of the 95,000-dalton actin-binding protein from Dictyostelium discoideum. J Cell Biol. 1983 Jul;97(1):178–185. doi: 10.1083/jcb.97.1.178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Condeelis J. Isolation of concanavalin A caps during various stages of formation and their association with actin and myosin. J Cell Biol. 1979 Mar;80(3):751–758. doi: 10.1083/jcb.80.3.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Condeelis J., Vahey M. A calcium- and pH-regulated protein from Dictyostelium discoideum that cross-links actin filaments. J Cell Biol. 1982 Aug;94(2):466–471. doi: 10.1083/jcb.94.2.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Detmers P. A., Goodenough U. W., Condeelis J. Elongation of the fertilization tubule in Chlamydomonas: new observations on the core microfilaments and the effect of transient intracellular signals on their structural integrity. J Cell Biol. 1983 Aug;97(2):522–532. doi: 10.1083/jcb.97.2.522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eckert B. S., Lazarides E. Localization of actin in Dictyostelium amebas by immunofluorescence. J Cell Biol. 1978 Jun;77(3):714–721. doi: 10.1083/jcb.77.3.714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Feramisco J. R., Blose S. H. Distribution of fluorescently labeled alpha-actinin in living and fixed fibroblasts. J Cell Biol. 1980 Aug;86(2):608–615. doi: 10.1083/jcb.86.2.608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Futrelle R. P., Traut J., McKee W. G. Cell behavior in Dictyostelium discoideum: preaggregation response to localized cyclic AMP pulses. J Cell Biol. 1982 Mar;92(3):807–821. doi: 10.1083/jcb.92.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Geiger B. Membrane-cytoskeleton interaction. Biochim Biophys Acta. 1983 Aug 11;737(3-4):305–341. doi: 10.1016/0304-4157(83)90005-9. [DOI] [PubMed] [Google Scholar]
  13. Geiger B., Singer S. J. The participation of alpha-actinin in the capping of cell membrane components. Cell. 1979 Jan;16(1):213–222. doi: 10.1016/0092-8674(79)90202-2. [DOI] [PubMed] [Google Scholar]
  14. Giffard R. G., Spudich J. A., Spudich A. Ca2+-sensitive isolation of a cortical actin matrix from Dictyostelium amoebae. J Muscle Res Cell Motil. 1983 Feb;4(1):115–131. doi: 10.1007/BF00711962. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Gordon D. J., Eisenberg E., Korn E. D. Characterization of cytoplasmic actin isolated from Acanthamoeba castellanii by a new method. J Biol Chem. 1976 Aug 10;251(15):4778–4786. [PubMed] [Google Scholar]
  17. Kuczmarski E. R., Spudich J. A. Regulation of myosin self-assembly: phosphorylation of Dictyostelium heavy chain inhibits formation of thick filaments. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7292–7296. doi: 10.1073/pnas.77.12.7292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Loomis W. F., Jr Sensitivity of Dictyostelium discoideum to nucleic acid analogues. Exp Cell Res. 1971 Feb;64(2):484–486. doi: 10.1016/0014-4827(71)90107-8. [DOI] [PubMed] [Google Scholar]
  20. Loor F., Forni L., Pernis B. The dynamic state of the lymphocyte membrane. Factors affecting the distribution and turnover of surface immunoglobulins. Eur J Immunol. 1972 Jun;2(3):203–212. doi: 10.1002/eji.1830020304. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Matsudaira P. T., Burgess D. R. Identification and organization of the components in the isolated microvillus cytoskeleton. J Cell Biol. 1979 Dec;83(3):667–673. doi: 10.1083/jcb.83.3.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mockrin S. C., Spudich J. A. Calcium control of actin-activated myosin adenosine triphosphatase from Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2321–2325. doi: 10.1073/pnas.73.7.2321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mooseker M. S., Tilney L. G. Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells. J Cell Biol. 1975 Dec;67(3):725–743. doi: 10.1083/jcb.67.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rubino S., Fighetti M., Unger E., Cappuccinelli P. Location of actin, myosin, and microtubular structures during directed locomotion of Dictyostelium amebae. J Cell Biol. 1984 Feb;98(2):382–390. doi: 10.1083/jcb.98.2.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Salisbury J. L., Condeelis J. S., Maihle N. J., Satir P. Calmodulin localization during capping and receptor-mediated endocytosis. Nature. 1981 Nov 12;294(5837):163–166. doi: 10.1038/294163a0. [DOI] [PubMed] [Google Scholar]
  27. Schreiner G. F., Fujiwara K., Pollard T. D., Unanue E. R. Redistribution of myosin accompanying capping of surface Ig. J Exp Med. 1977 May 1;145(5):1393–1398. doi: 10.1084/jem.145.5.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Singer S. J., Ash J. F., Bourguignon L. Y., Heggeness M. H., Louvard D. Transmembrane interactions and the mechanisms of transport of proteins across membranes. J Supramol Struct. 1978;9(3):373–389. doi: 10.1002/jss.400090308. [DOI] [PubMed] [Google Scholar]
  29. Spudich J. A. Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. II. Purification, properties, and membrane association of actin from amoebae of Dictyostelium discoideum. J Biol Chem. 1974 Sep 25;249(18):6013–6020. [PubMed] [Google Scholar]
  30. Tilney L. G. Polymerization of actin. V. A new organelle, the actomere, that initates the assembly of actin filaments in Thyone sperm. J Cell Biol. 1978 May;77(2):551–564. [PMC free article] [PubMed] [Google Scholar]
  31. 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. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vicker M. G., Schill W., Drescher K. Chemoattraction and chemotaxis in Dictyostelium discoideum: myxamoeba cannot read spatial gradients of cyclic adenosine monophosphate. J Cell Biol. 1984 Jun;98(6):2204–2214. doi: 10.1083/jcb.98.6.2204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wilson D. F., Chance B. Reversal of azide inhibition by uncouplers. Biochem Biophys Res Commun. 1966 Jun 13;23(5):751–756. doi: 10.1016/0006-291x(66)90465-7. [DOI] [PubMed] [Google Scholar]

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