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. 1992 Dec 1;119(5):1219–1243. doi: 10.1083/jcb.119.5.1219

Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity

PMCID: PMC2289720  PMID: 1447299

Abstract

The organization and polarity of actin filaments in neuronal growth cones was studied with negative stain and freeze-etch EM using a permeabilization protocol that caused little detectable change in morphology when cultured nerve growth cones were observed by video- enhanced differential interference contrast microscopy. The lamellipodial actin cytoskeleton was composed of two distinct subpopulations: a population of 40-100-nm-wide filament bundles radiated from the leading edge, and a second population of branching short filaments filled the volume between the dorsal and ventral membrane surfaces. Together, the two populations formed the three- dimensional structural network seen within expanding lamellipodia. Interaction of the actin filaments with the ventral membrane surface occurred along the length of the filaments via membrane associated proteins. The long bundled filament population was primarily involved in these interactions. The filament tips of either population appeared to interact with the membrane only at the leading edge; this interaction was mediated by a globular Triton-insoluble material. Actin filament polarity was determined by decoration with myosin S1 or heavy meromyosin. Previous reports have suggested that the polarity of the actin filaments in motile cells is uniform, with the barbed ends toward the leading edge. We observed that the actin filament polarity within growth cone lamellipodia is not uniform; although the predominant orientation was with the barbed end toward the leading edge (47-56%), 22-25% of the filaments had the opposite orientation with their pointed ends toward the leading edge, and 19-31% ran parallel to the leading edge. The two actin filament populations display distinct polarity profiles: the longer filaments appear to be oriented predominantly with their barbed ends toward the leading edge, whereas the short filaments appear to be randomly oriented. The different length, organization and polarity of the two filament populations suggest that they differ in stability and function. The population of bundled long filaments, which appeared to be more ventrally located and in contact with membrane proteins, may be more stable than the population of short branched filaments. The location, organization, and polarity of the long bundled filaments suggest that they may be necessary for the expansion of lamellipodia and for the production of tension mediated by receptors to substrate adhesion molecules.

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Selected References

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  1. Begg D. A., Rodewald R., Rebhun L. I. The visualization of actin filament polarity in thin sections. Evidence for the uniform polarity of membrane-associated filaments. J Cell Biol. 1978 Dec;79(3):846–852. doi: 10.1083/jcb.79.3.846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bottenstein J. E., Sato G. H. Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci U S A. 1979 Jan;76(1):514–517. doi: 10.1073/pnas.76.1.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bridgman P. C., Carr C., Pedersen S. E., Cohen J. B. Visualization of the cytoplasmic surface of Torpedo postsynaptic membranes by freeze-etch and immunoelectron microscopy. J Cell Biol. 1987 Oct;105(4):1829–1846. doi: 10.1083/jcb.105.4.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bridgman P. C., Dailey M. E. The organization of myosin and actin in rapid frozen nerve growth cones. J Cell Biol. 1989 Jan;108(1):95–109. doi: 10.1083/jcb.108.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bridgman P. C., Nakajima Y. Distribution of filipin-sterol complexes on cultured muscle cells: cell-substratum contact areas associated with acetylcholine receptor clusters. J Cell Biol. 1983 Feb;96(2):363–372. doi: 10.1083/jcb.96.2.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bridgman P. C., Reese T. S. The structure of cytoplasm in directly frozen cultured cells. I. Filamentous meshworks and the cytoplasmic ground substance. J Cell Biol. 1984 Nov;99(5):1655–1668. doi: 10.1083/jcb.99.5.1655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burmeister D. W., Chen M., Bailey C. H., Goldberg D. J. The distribution and movement of organelles in maturing growth cones: correlated video-enhanced and electron microscopic studies. J Neurocytol. 1988 Dec;17(6):783–795. doi: 10.1007/BF01216706. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Dailey M. E., Bridgman P. C. Structure and organization of membrane organelles along distal microtubule segments in growth cones. J Neurosci Res. 1991 Sep;30(1):242–258. doi: 10.1002/jnr.490300125. [DOI] [PubMed] [Google Scholar]
  10. DeBiasio R. L., Wang L. L., Fisher G. W., Taylor D. L. The dynamic distribution of fluorescent analogues of actin and myosin in protrusions at the leading edge of migrating Swiss 3T3 fibroblasts. J Cell Biol. 1988 Dec;107(6 Pt 2):2631–2645. doi: 10.1083/jcb.107.6.2631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Forscher P., Smith S. J. Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone. J Cell Biol. 1988 Oct;107(4):1505–1516. doi: 10.1083/jcb.107.4.1505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. HUXLEY H. E. ELECTRON MICROSCOPE STUDIES ON THE STRUCTURE OF NATURAL AND SYNTHETIC PROTEIN FILAMENTS FROM STRIATED MUSCLE. J Mol Biol. 1963 Sep;7:281–308. doi: 10.1016/s0022-2836(63)80008-x. [DOI] [PubMed] [Google Scholar]
  13. Heuser J. E., Anderson R. G. Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J Cell Biol. 1989 Feb;108(2):389–400. doi: 10.1083/jcb.108.2.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Heuser J. E., Kirschner M. W. Filament organization revealed in platinum replicas of freeze-dried cytoskeletons. J Cell Biol. 1980 Jul;86(1):212–234. doi: 10.1083/jcb.86.1.212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Heuser J. E. Structure of the myosin crossbridge lattice in insect flight muscle. J Mol Biol. 1983 Sep 5;169(1):123–154. doi: 10.1016/s0022-2836(83)80178-8. [DOI] [PubMed] [Google Scholar]
  16. Hirokawa N., Tilney L. G., Fujiwara K., Heuser J. E. Organization of actin, myosin, and intermediate filaments in the brush border of intestinal epithelial cells. J Cell Biol. 1982 Aug;94(2):425–443. doi: 10.1083/jcb.94.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Johnson M. I., Argiro V. Techniques in the tissue culture of rat sympathetic neurons. Methods Enzymol. 1983;103:334–347. doi: 10.1016/s0076-6879(83)03022-0. [DOI] [PubMed] [Google Scholar]
  18. KIELLEY W. W., HARRINGTON W. F. A model for the myosin molecule. Biochim Biophys Acta. 1960 Jul 15;41:401–421. doi: 10.1016/0006-3002(60)90037-8. [DOI] [PubMed] [Google Scholar]
  19. Kucik D. F., Elson E. L., Wang Y. L. Actin tracks. Nature. 1991 Dec 5;354(6352):362–363. doi: 10.1038/354362b0. [DOI] [PubMed] [Google Scholar]
  20. Kuczmarski E. R., Rosenbaum J. L. Studies on the organization and localization of actin and myosin in neurons. J Cell Biol. 1979 Feb;80(2):356–371. doi: 10.1083/jcb.80.2.356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Letourneau P. C. Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos. J Cell Biol. 1983 Oct;97(4):963–973. doi: 10.1083/jcb.97.4.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Margossian S. S., Lowey S. Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol. 1982;85(Pt B):55–71. doi: 10.1016/0076-6879(82)85009-x. [DOI] [PubMed] [Google Scholar]
  23. Maupin P., Pollard T. D. Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation. J Cell Biol. 1983 Jan;96(1):51–62. doi: 10.1083/jcb.96.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mitchison T., Kirschner M. Cytoskeletal dynamics and nerve growth. Neuron. 1988 Nov;1(9):761–772. doi: 10.1016/0896-6273(88)90124-9. [DOI] [PubMed] [Google Scholar]
  25. Moore P. B., Huxley H. E., DeRosier D. J. Three-dimensional reconstruction of F-actin, thin filaments and decorated thin filaments. J Mol Biol. 1970 Jun 14;50(2):279–295. doi: 10.1016/0022-2836(70)90192-0. [DOI] [PubMed] [Google Scholar]
  26. Pollard T. D., Doberstein S. K., Zot H. G. Myosin-I. Annu Rev Physiol. 1991;53:653–681. doi: 10.1146/annurev.ph.53.030191.003253. [DOI] [PubMed] [Google Scholar]
  27. Rees R. P., Reese T. S. New structural features of freeze-substituted neuritic growth cones. Neuroscience. 1981;6(2):247–254. doi: 10.1016/0306-4522(81)90060-9. [DOI] [PubMed] [Google Scholar]
  28. Rinnerthaler G., Herzog M., Klappacher M., Kunka H., Small J. V. Leading edge movement and ultrastructure in mouse macrophages. J Struct Biol. 1991 Feb;106(1):1–16. doi: 10.1016/1047-8477(91)90058-5. [DOI] [PubMed] [Google Scholar]
  29. Ris H. The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections. J Cell Biol. 1985 May;100(5):1474–1487. doi: 10.1083/jcb.100.5.1474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ryder M. I., Weinreb R. N., Niederman R. The organization of actin filaments in human polymorphonuclear leukocytes. Anat Rec. 1984 May;209(1):7–20. doi: 10.1002/ar.1092090103. [DOI] [PubMed] [Google Scholar]
  31. Sellers J. R., Kachar B. Polarity and velocity of sliding filaments: control of direction by actin and of speed by myosin. Science. 1990 Jul 27;249(4967):406–408. doi: 10.1126/science.2377894. [DOI] [PubMed] [Google Scholar]
  32. Small J. V., Celis J. E. Filament arrangements in negatively stained cultured cells: the organization of actin. Cytobiologie. 1978 Feb;16(2):308–325. [PubMed] [Google Scholar]
  33. Small J. V., Isenberg G., Celis J. E. Polarity of actin at the leading edge of cultured cells. Nature. 1978 Apr 13;272(5654):638–639. doi: 10.1038/272638a0. [DOI] [PubMed] [Google Scholar]
  34. Small J. V. Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks. J Cell Biol. 1981 Dec;91(3 Pt 1):695–705. doi: 10.1083/jcb.91.3.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Small J. V., Rinnerthaler G., Hinssen H. Organization of actin meshworks in cultured cells: the leading edge. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):599–611. doi: 10.1101/sqb.1982.046.01.056. [DOI] [PubMed] [Google Scholar]
  36. Small J. V. The actin cytoskeleton. Electron Microsc Rev. 1988;1(1):155–174. doi: 10.1016/s0892-0354(98)90010-7. [DOI] [PubMed] [Google Scholar]
  37. Smith S. J. Neuronal cytomechanics: the actin-based motility of growth cones. Science. 1988 Nov 4;242(4879):708–715. doi: 10.1126/science.3055292. [DOI] [PubMed] [Google Scholar]
  38. Stossel T. P. Contribution of actin to the structure of the cytoplasmic matrix. J Cell Biol. 1984 Jul;99(1 Pt 2):15s–21s. doi: 10.1083/jcb.99.1.15s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Theriot J. A., Mitchison T. J. Actin microfilament dynamics in locomoting cells. Nature. 1991 Jul 11;352(6331):126–131. doi: 10.1038/352126a0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Wolosewick J. J., Porter K. R. Microtrabecular lattice of the cytoplasmic ground substance. Artifact or reality. J Cell Biol. 1979 Jul;82(1):114–139. doi: 10.1083/jcb.82.1.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yamada K. M., Spooner B. S., Wessells N. K. Ultrastructure and function of growth cones and axons of cultured nerve cells. J Cell Biol. 1971 Jun;49(3):614–635. doi: 10.1083/jcb.49.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]

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