Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1984 Nov 1;99(5):1655–1668. doi: 10.1083/jcb.99.5.1655

The structure of cytoplasm in directly frozen cultured cells. I. Filamentous meshworks and the cytoplasmic ground substance

PMCID: PMC2113346  PMID: 6436253

Abstract

Cultured fibroblasts or epithelial cells derived from Xenopus laevis embryos were directly frozen, freeze-substituted by an improved method, and then either critical-point-dried and viewed as whole mounts, or embedded and thin sectioned. In thin regions of these cells, where ice crystal artifacts are absent, the cytoplasm consisted of a dense, highly interconnected meshwork of filaments, embedded in a finely granular ground substance. The meshwork in directly frozen, intact cells was compared with that in cells that were lysed (physically, with detergents, or with filipin), or fixed with glutaraldehyde before freezing. Although filaments tended to be less numerous in lysed cells, their overall organization was the same as that in intact cells. However, fixation with glutaraldehyde before freezing distorted the meshwork to variable degrees depending on the osmolarity of the fixation buffer, and also obscured the granular ground substance which is obvious in directly frozen cells. With optimal preparative methods, the cytoplasm of these directly frozen cells is shown to consist of a cytoskeleton composed of discrete interwoven filaments interconnected by numerous finer filaments and a readily extractable granular matrix which presumably represents aggregations of cytoplasmic proteins.

Full Text

The Full Text of this article is available as a PDF (2.6 MB).

Selected References

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

  1. Barlow D. I., Sleigh M. A. Freeze substitution for preservation of ciliated surfaces for scanning electron microscopy. J Microsc. 1979 Jan;115(1):81–95. doi: 10.1111/j.1365-2818.1979.tb00154.x. [DOI] [PubMed] [Google Scholar]
  2. Bridgman P. C., Nakajima S., Greenberg A. S., Nakajima Y. Freeze-fracture and electrophysiological studies of newly developed acetylcholine receptors in Xenopus embryonic muscle cells. J Cell Biol. 1984 Jun;98(6):2160–2173. doi: 10.1083/jcb.98.6.2160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buckley I. K., Porter K. R. Electron microscopy of critical point dried whole cultured cells. J Microsc. 1975 Jul;104(2):107–120. doi: 10.1111/j.1365-2818.1975.tb04010.x. [DOI] [PubMed] [Google Scholar]
  4. Handley D. A., Alexander J. T., Chien S. The design and use of a simple device for rapid quench-freezing of biological samples. J Microsc. 1981 Mar;121(Pt 3):273–282. doi: 10.1111/j.1365-2818.1981.tb01224.x. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Heuser J. E., Reese T. S., Dennis M. J., Jan Y., Jan L., Evans L. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol. 1979 May;81(2):275–300. doi: 10.1083/jcb.81.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hirokawa N., Cheney R. E., Willard M. Location of a protein of the fodrin-spectrin-TW260/240 family in the mouse intestinal brush border. Cell. 1983 Mar;32(3):953–965. doi: 10.1016/0092-8674(83)90080-6. [DOI] [PubMed] [Google Scholar]
  8. Hirokawa N., Heuser J. E. Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells. J Cell Biol. 1981 Nov;91(2 Pt 1):399–409. doi: 10.1083/jcb.91.2.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jehl B., Bauer R., Dörge A., Rick R. The use of propane/isopentane mixtures for rapid freezing of biological specimens. J Microsc. 1981 Sep;123(Pt 3):307–309. doi: 10.1111/j.1365-2818.1981.tb02475.x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. Norman A. W., Spielvogel A. M., Wong R. G. Polyene antibiotic - sterol interaction. Adv Lipid Res. 1976;14:127–170. [PubMed] [Google Scholar]
  15. Peng H. B., Nakajima Y. Membrane particle aggregates in innervated and noninnervated cultures of Xenopus embryonic muscle cells. Proc Natl Acad Sci U S A. 1978 Jan;75(1):500–504. doi: 10.1073/pnas.75.1.500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Porter K. R., Anderson K. L. The structure of the cytoplasmic matrix preserved by freeze-drying and freeze-substitution. Eur J Cell Biol. 1982 Nov;29(1):83–96. [PubMed] [Google Scholar]
  17. Porter K. R., Tucker J. B. The ground substance of the living cell. Sci Am. 1981 Mar;244(3):56–67. doi: 10.1038/scientificamerican0381-56. [DOI] [PubMed] [Google Scholar]
  18. Pryzwansky K. B., Schliwa M., Porter K. R. Comparison of the three-dimensional organization of unextracted and Triton-extracted human neutrophilic polymorphonuclear leukocytes. Eur J Cell Biol. 1983 Mar;30(1):112–125. [PubMed] [Google Scholar]
  19. Robinson J. M., Karnovsky M. J. Evaluation of the polyene antibiotic filipin as a cytochemical probe for membrane cholesterol. J Histochem Cytochem. 1980 Feb;28(2):161–168. doi: 10.1177/28.2.6766487. [DOI] [PubMed] [Google Scholar]
  20. Schliwa M. Action of cytochalasin D on cytoskeletal networks. J Cell Biol. 1982 Jan;92(1):79–91. doi: 10.1083/jcb.92.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schliwa M., van Blerkom J. Structural interaction of cytoskeletal components. J Cell Biol. 1981 Jul;90(1):222–235. doi: 10.1083/jcb.90.1.222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schnapp B. J., Reese T. S. Cytoplasmic structure in rapid-frozen axons. J Cell Biol. 1982 Sep;94(3):667–669. doi: 10.1083/jcb.94.3.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Silva M. T., Guerra F. C., Magalhães M. M. The fixative action of uranyl acetate in electron microscopy. Experientia. 1968 Oct 15;24(10):1074–1074. doi: 10.1007/BF02138757. [DOI] [PubMed] [Google Scholar]
  24. Silva M. T., Santos Mota J. M., Melo J. V., Guerra F. C. Uranyl salts as fixatives for electron microscopy. Study of the membrane ultrastructure and phospholipid loss in bacilli. Biochim Biophys Acta. 1971 Jun 1;233(3):513–520. doi: 10.1016/0005-2736(71)90151-9. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Tao-Cheng J. H., Hirosawa K., Nakajima Y. Ultrastructure of the crayfish stretch receptor in relation to its function. J Comp Neurol. 1981 Jul 20;200(1):1–21. doi: 10.1002/cne.902000102. [DOI] [PubMed] [Google Scholar]
  28. VANHARREVELD A., CROWELL J., MALHOTRA S. K. A STUDY OF EXTRACELLULAR SPACE IN CENTRAL NERVOUS TISSUE BY FREEZE-SUBSTITUTION. J Cell Biol. 1965 Apr;25:117–137. doi: 10.1083/jcb.25.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. de Kruijff B., Gerritsen W. J., Oerlemans A., Demel R. A., van Deenen L. L. Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. I. Specificity of the membrane permeability changes induced by the polyene antibiotics. Biochim Biophys Acta. 1974 Feb 26;339(1):30–43. doi: 10.1016/0005-2736(74)90330-7. [DOI] [PubMed] [Google Scholar]

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

RESOURCES