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. 1981 Dec 1;91(3):695–705. doi: 10.1083/jcb.91.3.695

Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks

PMCID: PMC2112811  PMID: 6799521

Abstract

The ordered structure of the leading edge (lamellipodium) of cultured fibroblasts is readily revealed in cells extracted briefly in Triton X- 100-glutaraldehyde mixtures, fixed further in glutaraldehyde, and then negatively stained for electron microscopy. By this procedure, the leading edge regions show a highly organised, three-dimensional network of actin filaments together with variable numbers of radiating actin filament bundles or microspikes. The use of Phalloidin after glutaraldehyde fixation resulted in a marginal improvement in filament order. Processing of the cytoskeletons though the additional steps generally employed for conventional electron microscopy resulted in a marked deterioration or complete disruption of the order of the actin filament networks. In contrast, the actin filaments of the stress fiber bundles were essentially unaffected. Thus, postfixation in osmium tetroxide (1% for 7 min at room temperature) transformed the networks to a reticulum of kinked fibers, resembling those produced by the exposure of muscle F-actin to OsO4 in vitro (P. Maupin-Szamier and T. D. Pollard. 1978. J. Cell Biol. 77:837--852). While limited exposure to OsO4 (0.2+ for 20 min at 0 degrees C) obviated this destruction, dehydration in acetone or ethanol, with or without post-osmication, caused a further and unavoidable disordering and aggregation of the meshwork filaments. The meshwork regions of the leading edge then showed a striking resemblance to the networks hitherto described in critical point-dried preparations of cultured cells. I conclude that much of the "microtrabecular lattice" described by Wolosewick and Porter (1979. J. Cell Biol. 82:114--139) in the latter preparations constitutes actin meshworks and actin filament arrays, with their associated components, that have been distorted and aggregated by the preparative procedures employed.

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Selected References

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  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. 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]
  3. Bereiter-Hahn J., Fox C. H., Thorell B. Quantitative reflection contrast microscopy of living cells. J Cell Biol. 1979 Sep;82(3):767–779. doi: 10.1083/jcb.82.3.767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blikstad I., Markey F., Carlsson L., Persson T., Lindberg U. Selective assay of monomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease I. Cell. 1978 Nov;15(3):935–943. doi: 10.1016/0092-8674(78)90277-5. [DOI] [PubMed] [Google Scholar]
  5. Bray D., Thomas C. Unpolymerized actin in fibroblasts and brain. J Mol Biol. 1976 Aug 25;105(4):527–544. doi: 10.1016/0022-2836(76)90233-3. [DOI] [PubMed] [Google Scholar]
  6. Brown S., Levinson W., Spudich J. A. Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction. J Supramol Struct. 1976;5(2):119–130. doi: 10.1002/jss.400050203. [DOI] [PubMed] [Google Scholar]
  7. Buckley I. K., Porter K. R. Cytoplasmic fibrils in living cultured cells. A light and electron microscope study. Protoplasma. 1967;64(4):349–380. doi: 10.1007/BF01666538. [DOI] [PubMed] [Google Scholar]
  8. Buckley I. K. Three dimensional fine structure of cultured cells: possible implications for subcellular motility. Tissue Cell. 1975;7(1):51–72. doi: 10.1016/s0040-8166(75)80007-3. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Gicquaud C., Gruda J., Pollender J. M. La phalloïdine protège la F-actine contre les effets destructeurs de l'acide osmique et du permanganate. Eur J Cell Biol. 1980 Feb;20(3):234–239. [PubMed] [Google Scholar]
  11. Goldman R. D., Lazarides E., Pollack R., Weber K. The distribution of actin in non-muscle cells. The use of actin antibody in the localization of actin within the microfilament bundles of mouse 3T3 cells. Exp Cell Res. 1975 Feb;90(2):333–344. doi: 10.1016/0014-4827(75)90323-7. [DOI] [PubMed] [Google Scholar]
  12. Haemmerli G., Felix H., Sträuli P. Motility of L 5222 rat leukemia cells in the flattened state. Evidence against emperipolesis. Virchows Arch B Cell Pathol. 1977 Jun 24;24(2):165–178. doi: 10.1007/BF02889277. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Huxley H. E. The mechanism of muscular contraction. Science. 1969 Jun 20;164(3886):1356–1365. doi: 10.1126/science.164.3886.1356. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Ingram V. M. A side view of moving fibroblasts. Nature. 1969 May 17;222(5194):641–644. doi: 10.1038/222641a0. [DOI] [PubMed] [Google Scholar]
  17. Lazarides E. Two general classes of cytoplasmic actin filaments in tissue culture cells: the role of tropomyosin. J Supramol Struct. 1976;5(4):531(383)–563(415). doi: 10.1002/jss.400050410. [DOI] [PubMed] [Google Scholar]
  18. Lazarides E., Weber K. Actin antibody: the specific visualization of actin filaments in non-muscle cells. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2268–2272. doi: 10.1073/pnas.71.6.2268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Maupin-Szamier P., Pollard T. D. Actin filament destruction by osmium tetroxide. J Cell Biol. 1978 Jun;77(3):837–852. doi: 10.1083/jcb.77.3.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mikawa T. 'Freezing' of Ca-regulated conformation of reconstituted thin filament of skeletal muscle by glutaraldehyde. Nature. 1979 Mar 29;278(5703):473–474. doi: 10.1038/278473a0. [DOI] [PubMed] [Google Scholar]
  21. Osborn M., Webster R. E., Weber K. Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell. J Cell Biol. 1978 Jun;77(3):R27–R34. doi: 10.1083/jcb.77.3.r27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Otto J. J., Kane R. E., Bryan J. Formation of filopodia in coelomocytes: localization of fascin, a 58,000 dalton actin cross-linking protein. Cell. 1979 Jun;17(2):285–293. doi: 10.1016/0092-8674(79)90154-5. [DOI] [PubMed] [Google Scholar]
  23. Reedy M. K., Holmes K. C., Tregear R. T. Induced changes in orientation of the cross-bridges of glycerinated insect flight muscle. Nature. 1965 Sep 18;207(5003):1276–1280. doi: 10.1038/2071276a0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Small J. V., Squire J. M. Structural basis of contraction in vertebrate smooth muscle. J Mol Biol. 1972 Jun 14;67(1):117–149. doi: 10.1016/0022-2836(72)90390-7. [DOI] [PubMed] [Google Scholar]
  27. Spooner B. S., Yamada K. M., Wessells N. K. Microfilaments and cell locomotion. J Cell Biol. 1971 Jun;49(3):595–613. doi: 10.1083/jcb.49.3.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tilney L. G. Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments. J Cell Biol. 1975 Feb;64(2):289–310. doi: 10.1083/jcb.64.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Valentine R. C., Shapiro B. M., Stadtman E. R. Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli. Biochemistry. 1968 Jun;7(6):2143–2152. doi: 10.1021/bi00846a017. [DOI] [PubMed] [Google Scholar]
  30. Wieland T. Modification of actins by phallotoxins. Naturwissenschaften. 1977 Jun;64(6):303–309. doi: 10.1007/BF00446784. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Wolosewick J. J., Porter K. R. Stereo high-voltage electron microscopy of whole cells of the human diploid line, WI-38. Am J Anat. 1976 Nov;147(3):303–323. doi: 10.1002/aja.1001470305. [DOI] [PubMed] [Google Scholar]
  33. Wray J., Vibert P., Cohen C. Actin filaments in muscle: pattern of myosin and tropomyosin/troponin attachments. J Mol Biol. 1978 Sep 25;124(3):501–521. doi: 10.1016/0022-2836(78)90184-5. [DOI] [PubMed] [Google Scholar]
  34. Wulf E., Deboben A., Bautz F. A., Faulstich H., Wieland T. Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4498–4502. doi: 10.1073/pnas.76.9.4498. [DOI] [PMC free article] [PubMed] [Google Scholar]

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