Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 Jun 1;129(5):1275–1286. doi: 10.1083/jcb.129.5.1275

Actin filament organization in the fish keratocyte lamellipodium

PMCID: PMC2120461  PMID: 7775574

Abstract

From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation- release mechanism, which predicts the existence of short (less than 0.5 microns) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope. Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 degrees to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid- region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length. Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton, a steep gradient of phalloidin staining was observed from the front to the rear of the lamellipodium. With the protocol required to obtain the electron microscope images, namely Triton extraction followed by fixation, phalloidin staining was, significantly and preferentially reduced in the anterior part of the lamellipodium. This resulted in a lower gradient of filament density, consistent with that seen in the electron microscope, and indicated a loss of around 45% of the filamentous actin during Triton extraction. We conclude, first that the filament organization and length distribution does not support a nucleation release model, but is more consistent with a treadmilling-type mechanism of locomotion featuring actin filaments of graded length. Second, we suggest that two layers of filaments make up the lamellipodium; a lower, stabilized layer associated with the ventral membrane and an upper layer associated with the dorsal membrane that is composed of filaments of a shorter range of lengths than the lower layer and which is mainly lost in Triton.

Full Text

The Full Text of this article is available as a PDF (5.7 MB).

Selected References

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

  1. Abercrombie M., Heaysman J. E., Pegrum S. M. The locomotion of fibroblasts in culture. I. Movements of the leading edge. Exp Cell Res. 1970 Mar;59(3):393–398. doi: 10.1016/0014-4827(70)90646-4. [DOI] [PubMed] [Google Scholar]
  2. Adams R. J., Pollard T. D. Membrane-bound myosin-I provides new mechanisms in cell motility. Cell Motil Cytoskeleton. 1989;14(2):178–182. doi: 10.1002/cm.970140203. [DOI] [PubMed] [Google Scholar]
  3. Bereiter-Hahn J., Strohmeier R., Kunzenbacher I., Beck K., Vöth M. Locomotion of Xenopus epidermis cells in primary culture. J Cell Sci. 1981 Dec;52:289–311. doi: 10.1242/jcs.52.1.289. [DOI] [PubMed] [Google Scholar]
  4. Carlier M. F., Pantaloni D. Actin assembly in response to extracellular signals: role of capping proteins, thymosin beta 4 and profilin. Semin Cell Biol. 1994 Jun;5(3):183–191. doi: 10.1006/scel.1994.1023. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Cooper M. S., Schliwa M. Motility of cultured fish epidermal cells in the presence and absence of direct current electric fields. J Cell Biol. 1986 Apr;102(4):1384–1399. doi: 10.1083/jcb.102.4.1384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Edds K. T. Dynamic aspects of filopodial formation by reorganization of microfilaments. J Cell Biol. 1977 May;73(2):479–491. doi: 10.1083/jcb.73.2.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Fukui Y., Lynch T. J., Brzeska H., Korn E. D. Myosin I is located at the leading edges of locomoting Dictyostelium amoebae. Nature. 1989 Sep 28;341(6240):328–331. doi: 10.1038/341328a0. [DOI] [PubMed] [Google Scholar]
  10. Heath J. P., Holifield B. F. Cell locomotion: new research tests old ideas on membrane and cytoskeletal flow. Cell Motil Cytoskeleton. 1991;18(4):245–257. doi: 10.1002/cm.970180402. [DOI] [PubMed] [Google Scholar]
  11. Heath J., Holifield B. Cell locomotion. Actin alone in lamellipodia. Nature. 1991 Jul 11;352(6331):107–108. doi: 10.1038/352107a0. [DOI] [PubMed] [Google Scholar]
  12. Höglund A. S., Karlsson R., Arro E., Fredriksson B. A., Lindberg U. Visualization of the peripheral weave of microfilaments in glia cells. J Muscle Res Cell Motil. 1980 Jun;1(2):127–146. doi: 10.1007/BF00711795. [DOI] [PubMed] [Google Scholar]
  13. Karlsson R., Lassing I., Höglund A. S., Lindberg U. The organization of microfilaments in spreading platelets: a comparison with fibroblasts and glial cells. J Cell Physiol. 1984 Oct;121(1):96–113. doi: 10.1002/jcp.1041210113. [DOI] [PubMed] [Google Scholar]
  14. Kolega J. Effects of mechanical tension on protrusive activity and microfilament and intermediate filament organization in an epidermal epithelium moving in culture. J Cell Biol. 1986 Apr;102(4):1400–1411. doi: 10.1083/jcb.102.4.1400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kucik D. F., Elson E. L., Sheetz M. P. Cell migration does not produce membrane flow. J Cell Biol. 1990 Oct;111(4):1617–1622. doi: 10.1083/jcb.111.4.1617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lee J., Ishihara A., Jacobson K. How do cells move along surfaces? Trends Cell Biol. 1993 Nov;3(11):366–370. doi: 10.1016/0962-8924(93)90084-e. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. Okabe S., Hirokawa N. Incorporation and turnover of biotin-labeled actin microinjected into fibroblastic cells: an immunoelectron microscopic study. J Cell Biol. 1989 Oct;109(4 Pt 1):1581–1595. doi: 10.1083/jcb.109.4.1581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Okamoto Y., Sekine T. A streamlined method of subfragment one preparation from myosin. J Biochem. 1985 Oct;98(4):1143–1145. doi: 10.1093/oxfordjournals.jbchem.a135365. [DOI] [PubMed] [Google Scholar]
  23. Oster G. F., Perelson A. S. The physics of cell motility. J Cell Sci Suppl. 1987;8:35–54. doi: 10.1242/jcs.1987.supplement_8.3. [DOI] [PubMed] [Google Scholar]
  24. Peskin C. S., Odell G. M., Oster G. F. Cellular motions and thermal fluctuations: the Brownian ratchet. Biophys J. 1993 Jul;65(1):316–324. doi: 10.1016/S0006-3495(93)81035-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Small J. V., Herzog M., Häner M., Abei U. Visualization of actin filaments in keratocyte lamellipodia: negative staining compared with freeze-drying. J Struct Biol. 1994 Sep-Oct;113(2):135–141. doi: 10.1006/jsbi.1994.1043. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Small J. V. Lamellipodia architecture: actin filament turnover and the lateral flow of actin filaments during motility. Semin Cell Biol. 1994 Jun;5(3):157–163. doi: 10.1006/scel.1994.1020. [DOI] [PubMed] [Google Scholar]
  30. Small J. V. Microfilament-based motility in non-muscle cells. Curr Opin Cell Biol. 1989 Feb;1(1):75–79. doi: 10.1016/s0955-0674(89)80040-7. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Small J. V., Rohlfs A., Herzog M. Actin and cell movement. Symp Soc Exp Biol. 1993;47:57–71. [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. 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]
  37. Stossel T. P. On the crawling of animal cells. Science. 1993 May 21;260(5111):1086–1094. doi: 10.1126/science.8493552. [DOI] [PubMed] [Google Scholar]
  38. Symons M. H., Mitchison T. J. Control of actin polymerization in live and permeabilized fibroblasts. J Cell Biol. 1991 Aug;114(3):503–513. doi: 10.1083/jcb.114.3.503. [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. Theriot J. A., Mitchison T. J. Comparison of actin and cell surface dynamics in motile fibroblasts. J Cell Biol. 1992 Oct;119(2):367–377. doi: 10.1083/jcb.119.2.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Theriot J. A., Mitchison T. J. The nucleation-release model of actin filament dynamics in cell motility. Trends Cell Biol. 1992 Aug;2(8):219–222. doi: 10.1016/0962-8924(92)90298-2. [DOI] [PubMed] [Google Scholar]
  42. Theriot J. A. Regulation of the actin cytoskeleton in living cells. Semin Cell Biol. 1994 Jun;5(3):193–199. doi: 10.1006/scel.1994.1024. [DOI] [PubMed] [Google Scholar]
  43. Wagner M. C., Barylko B., Albanesi J. P. Tissue distribution and subcellular localization of mammalian myosin I. J Cell Biol. 1992 Oct;119(1):163–170. doi: 10.1083/jcb.119.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Yin H. L., Hartwig J. H. The structure of the macrophage actin skeleton. J Cell Sci Suppl. 1988;9:169–184. doi: 10.1242/jcs.1988.supplement_9.9. [DOI] [PubMed] [Google Scholar]
  46. 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