Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 Mar 1;128(5):819–835. doi: 10.1083/jcb.128.5.819

Genetic deletion of ABP-120 alters the three-dimensional organization of actin filaments in Dictyostelium pseudopods

PMCID: PMC2120398  PMID: 7876307

Abstract

This study extends the observations on the defects in pseudopod formation of ABP-120+ and ABP-120- cells by a detailed morphological and biochemical analysis of the actin based cytoskeleton. Both ABP-120+ and ABP-120- cells polymerize the same amount of F-actin in response to stimulation with cAMP. However, unlike ABP-120+ cells, ABP-120- cells do not incorporate actin into the Triton X-100-insoluble cytoskeleton at 30-50 s, the time when ABP-120 is incorporated into the cytoskeleton and when pseudopods are extended after cAMP stimulation in wild-type cells. By confocal and electron microscopy, pseudopods extended by ABP- 120- cells are not as large or thick as those produced by ABP-120+ cells and in the electron microscope, an altered filament network is found in pseudopods of ABP-120- cells when compared to pseudopods of ABP-120+ cells. The actin filaments found in areas of pseudopods in ABP- 120+ cells either before or after stimulation were long, straight, and arranged into space filling orthogonal networks. Protrusions of ABP-120- cells are less three-dimensional, denser, and filled with multiple foci of aggregated filaments consistent with collapse of the filament network due to the absence of ABP-120-mediated cross-linking activity. The different organization of actin filaments may account for the diminished size of protrusions observed in living and fixed ABP-120- cells compared to ABP-120+ cells and is consistent with the role of ABP- 120 in regulating pseudopod extension through its cross-linking of actin filaments.

Full Text

The Full Text of this article is available as a PDF (11.2 MB).

Selected References

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

  1. Bresnick A. R., Janmey P. A., Condeelis J. Evidence that a 27-residue sequence is the actin-binding site of ABP-120. J Biol Chem. 1991 Jul 15;266(20):12989–12993. [PubMed] [Google Scholar]
  2. Bresnick A. R., Warren V., Condeelis J. Identification of a short sequence essential for actin binding by Dictyostelium ABP-120. J Biol Chem. 1990 Jun 5;265(16):9236–9240. [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Condeelis J. Are all pseudopods created equal? Cell Motil Cytoskeleton. 1992;22(1):1–6. doi: 10.1002/cm.970220102. [DOI] [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., Hall A., Bresnick A., Warren V., Hock R., Bennett H., Ogihara S. Actin polymerization and pseudopod extension during amoeboid chemotaxis. Cell Motil Cytoskeleton. 1988;10(1-2):77–90. doi: 10.1002/cm.970100113. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Condeelis J., Ogihara S., Bennett H., Carboni J., Hall A. Ultrastructural localization of cytoskeletal proteins in Dictyostelium amoebae. Methods Cell Biol. 1987;28:191–207. doi: 10.1016/s0091-679x(08)61645-3. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Cooper J. A. The role of actin polymerization in cell motility. Annu Rev Physiol. 1991;53:585–605. doi: 10.1146/annurev.ph.53.030191.003101. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Egelhoff T. T., Spudich J. A. Molecular genetics of cell migration: Dictyostelium as a model system. Trends Genet. 1991 May;7(5):161–166. doi: 10.1016/0168-9525(91)90380-9. [DOI] [PubMed] [Google Scholar]
  16. Fukui Y. Toward a new concept of cell motility: cytoskeletal dynamics in amoeboid movement and cell division. Int Rev Cytol. 1993;144:85–127. doi: 10.1016/s0074-7696(08)61514-4. [DOI] [PubMed] [Google Scholar]
  17. Gorlin J. B., Yamin R., Egan S., Stewart M., Stossel T. P., Kwiatkowski D. J., Hartwig J. H. Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring. J Cell Biol. 1990 Sep;111(3):1089–1105. doi: 10.1083/jcb.111.3.1089. [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. Hall A. L., Warren V., Dharmawardhane S., Condeelis J. Identification of actin nucleation activity and polymerization inhibitor in ameboid cells: their regulation by chemotactic stimulation. J Cell Biol. 1989 Nov;109(5):2207–2213. doi: 10.1083/jcb.109.5.2207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Hartwig J. H., Yin H. L. The organization and regulation of the macrophage actin skeleton. Cell Motil Cytoskeleton. 1988;10(1-2):117–125. doi: 10.1002/cm.970100116. [DOI] [PubMed] [Google Scholar]
  23. Hock R. S., Condeelis J. S. Isolation of a 240-kilodalton actin-binding protein from Dictyostelium discoideum. J Biol Chem. 1987 Jan 5;262(1):394–400. [PubMed] [Google Scholar]
  24. Lee J., Ishihara A., Theriot J. A., Jacobson K. Principles of locomotion for simple-shaped cells. Nature. 1993 Mar 11;362(6416):167–171. doi: 10.1038/362167a0. [DOI] [PubMed] [Google Scholar]
  25. Lewis A. K., Bridgman P. C. Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity. J Cell Biol. 1992 Dec;119(5):1219–1243. doi: 10.1083/jcb.119.5.1219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Luna E. J., Hitt A. L. Cytoskeleton--plasma membrane interactions. Science. 1992 Nov 6;258(5084):955–964. doi: 10.1126/science.1439807. [DOI] [PubMed] [Google Scholar]
  27. Niederman R., Amrein P. C., Hartwig J. Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein. J Cell Biol. 1983 May;96(5):1400–1413. doi: 10.1083/jcb.96.5.1400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Noegel A. A., Rapp S., Lottspeich F., Schleicher M., Stewart M. The Dictyostelium gelation factor shares a putative actin binding site with alpha-actinins and dystrophin and also has a rod domain containing six 100-residue motifs that appear to have a cross-beta conformation. J Cell Biol. 1989 Aug;109(2):607–618. doi: 10.1083/jcb.109.2.607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Ohta Y., Stossel T. P., Hartwig J. H. Ligand-sensitive binding of actin-binding protein to immunoglobulin G Fc receptor I (Fc gamma RI). Cell. 1991 Oct 18;67(2):275–282. doi: 10.1016/0092-8674(91)90179-3. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Oster G. F. On the crawling of cells. J Embryol Exp Morphol. 1984 Nov;83 (Suppl):329–364. [PubMed] [Google Scholar]
  33. Podolski J. L., Steck T. L. Length distribution of F-actin in Dictyostelium discoideum. J Biol Chem. 1990 Jan 25;265(3):1312–1318. [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Wessels D., Soll D. R., Knecht D., Loomis W. F., De Lozanne A., Spudich J. Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain. Dev Biol. 1988 Jul;128(1):164–177. doi: 10.1016/0012-1606(88)90279-5. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Zigmond S. H. Recent quantitative studies of actin filament turnover during cell locomotion. Cell Motil Cytoskeleton. 1993;25(4):309–316. doi: 10.1002/cm.970250402. [DOI] [PubMed] [Google Scholar]

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

RESOURCES