Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1984 Aug 1;99(2):536–549. doi: 10.1083/jcb.99.2.536

Analysis of the role of microfilaments and microtubules in acquisition of bipolarity and elongation of fibroblasts in hydrated collagen gels

PMCID: PMC2113263  PMID: 6146628

Abstract

Fibroblasts in situ reside within a collagenous stroma and are elongate and bipolar in shape. If isolated and grown on glass, they change from elongate to flat shape, lose filopodia, and acquire ruffles. This shape change can be reversed to resemble that in situ by suspending the cells in hydrated collagen gels. In this study of embryonic avian corneal fibroblasts grown in collagen gels, we describe for the first time the steps in the acquisition of the elongate shape and analyze the effect of cytoskeleton-disrupting drugs on filopodial activity, assumption of bipolarity, and cell elongation within extracellular matrix. We have previously shown by immunofluorescence that filopodia contain actin but not myosin and are free of organelles. The cell cortex is rich in actin and the cytosol, in myosin. By using antitubulin, we show in the present study that microtubules are aligned along the long axis of the bipolar cell body. The first step in assumption of the elongate shape is extension of filopodia by the round cells suspended in collagen, and this is not significantly affected by the drugs we used: taxol to stabilize microtubules; nocodazole to disassemble microtubules; and cytochalasin D to disrupt microfilaments. The second step, movement of filopodia to opposite ends of the cell, is disrupted by cytochalasin, but not by taxol or nocodazole. The third step, extension of pseudopodia and acquisition of bipolarity similarly requires intact actin, but not microtubules. If fibroblasts are allowed to become bipolar before drug treatment, moreover, they remain so in the presence of the drugs. To complete the fourth step, extensive elongation of the cell, both intact actin and microtubules are required. Retraction of the already elongated cell occurs on microtubule disruption, but retraction requires an intact actin cytoskeleton. We suggest that the cell interacts with surrounding collagen fibrils via its actin cytoskeleton to become bipolar in shape, and that microtubules interact with the actin cortex to bring about the final elongation of the fibroblast.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

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

  1. Albertini D. F., Anderson E. Microtubule and microfilament rearrangements during capping of concanavalin A receptors on cultured ovarian granulosa cells. J Cell Biol. 1977 Apr;73(1):111–127. doi: 10.1083/jcb.73.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Albrecht-Buehler G., Bushnell A. The orientation of centrioles in migrating 3T3 cells. Exp Cell Res. 1979 Apr;120(1):111–118. doi: 10.1016/0014-4827(79)90542-1. [DOI] [PubMed] [Google Scholar]
  3. Albrecht-Buehler G. Daughter 3T3 cells. Are they mirror images of each other? J Cell Biol. 1977 Mar;72(3):595–603. doi: 10.1083/jcb.72.3.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Albrecht-Buehler G. Filopodia of spreading 3T3 cells. Do they have a substrate-exploring function? J Cell Biol. 1976 May;69(2):275–286. doi: 10.1083/jcb.69.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Albrecht-Buehler G., Goldman R. D. Microspike-mediated particle transport towards the cell body during early spreading of 3T3 cells. Exp Cell Res. 1976 Feb;97(2):329–339. doi: 10.1016/0014-4827(76)90624-8. [DOI] [PubMed] [Google Scholar]
  6. Banzhaf W. C., Warren R. H., McClay D. R. Cortical reorganization following fertilization of sea urchin eggs: sensitivity to cytochalasin B. Dev Biol. 1980 Dec;80(2):506–515. doi: 10.1016/0012-1606(80)90424-8. [DOI] [PubMed] [Google Scholar]
  7. Bard J. B., Hay E. D. The behavior of fibroblasts from the developing avian cornea. Morphology and movement in situ and in vitro. J Cell Biol. 1975 Nov;67(2PT1):400–418. doi: 10.1083/jcb.67.2.400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bellows C. G., Melcher A. H., Aubin J. E. Contraction and organization of collagen gels by cells cultured from periodontal ligament, gingiva and bone suggest functional differences between cell types. J Cell Sci. 1981 Aug;50:299–314. doi: 10.1242/jcs.50.1.299. [DOI] [PubMed] [Google Scholar]
  9. Bellows C. G., Melcher A. H., Bhargava U., Aubin J. E. Fibroblasts contracting three-dimensional collagen gels exhibit ultrastructure consistent with either contraction or protein secretion. J Ultrastruct Res. 1982 Feb;78(2):178–192. doi: 10.1016/s0022-5320(82)80022-1. [DOI] [PubMed] [Google Scholar]
  10. Bernanke D. H., Markwald R. R. Migratory behavior of cardiac cushion tissue cells in a collagen-lattice culture system. Dev Biol. 1982 Jun;91(2):235–245. doi: 10.1016/0012-1606(82)90030-6. [DOI] [PubMed] [Google Scholar]
  11. Bourguignon L. Y., Singer S. J. Transmembrane interactions and the mechanism of capping of surface receptors by their specific ligands. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5031–5035. doi: 10.1073/pnas.74.11.5031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Byers H. R., Fujiwara K., Porter K. R. Visualization of microtubules of cells in situ by indirect immunofluorescence. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6657–6661. doi: 10.1073/pnas.77.11.6657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Condeelis J. Isolation of concanavalin A caps during various stages of formation and their association with actin and myosin. J Cell Biol. 1979 Mar;80(3):751–758. doi: 10.1083/jcb.80.3.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Conrad G. W., Hart G. W., Chen Y. Differences in vitro between fibroblast-like cells from cornea, heart, and skin of embryonic chicks. J Cell Sci. 1977 Aug;26:119–137. doi: 10.1242/jcs.26.1.119. [DOI] [PubMed] [Google Scholar]
  15. Davis E. M. Translocation of neural crest cells within a hydrated collagen lattice. J Embryol Exp Morphol. 1980 Feb;55:17–31. [PubMed] [Google Scholar]
  16. De Brabander M., Geuens G., Nuydens R., Willebrords R., De Mey J. Microtubule assembly in living cells after release from nocodazole block: the effects of metabolic inhibitors, taxol and PH. Cell Biol Int Rep. 1981 Sep;5(9):913–920. doi: 10.1016/0309-1651(81)90206-x. [DOI] [PubMed] [Google Scholar]
  17. De Brabander M., Geuens G., Nuydens R., Willebrords R., De Mey J. Taxol induces the assembly of free microtubules in living cells and blocks the organizing capacity of the centrosomes and kinetochores. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5608–5612. doi: 10.1073/pnas.78.9.5608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dodd N. J., Schor S. L., Rushton G. The effects of a collagenous extracellular matrix on fibroblast membrane organization. An ESR spin label study. Exp Cell Res. 1982 Oct;141(2):421–431. doi: 10.1016/0014-4827(82)90230-0. [DOI] [PubMed] [Google Scholar]
  19. Dvorak J. A., Stotler W. F. A controlled-environment culture system for high resolution light microscopy. Exp Cell Res. 1971 Sep;68(1):144–148. doi: 10.1016/0014-4827(71)90596-9. [DOI] [PubMed] [Google Scholar]
  20. Elsdale T., Bard J. Collagen substrata for studies on cell behavior. J Cell Biol. 1972 Sep;54(3):626–637. doi: 10.1083/jcb.54.3.626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fujiwara K., Pollard T. D. Simultaneous localization of myosin and tubulin in human tissue culture cells by double antibody staining. J Cell Biol. 1978 Apr;77(1):182–195. doi: 10.1083/jcb.77.1.182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gotlieb A. I., May L. M., Subrahmanyan L., Kalnins V. I. Distribution of microtubule organizing centers in migrating sheets of endothelial cells. J Cell Biol. 1981 Nov;91(2 Pt 1):589–594. doi: 10.1083/jcb.91.2.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Greenburg G., Hay E. D. Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells. J Cell Biol. 1982 Oct;95(1):333–339. doi: 10.1083/jcb.95.1.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Griffith L. M., Pollard T. D. Evidence for actin filament-microtubule interaction mediated by microtubule-associated proteins. J Cell Biol. 1978 Sep;78(3):958–965. doi: 10.1083/jcb.78.3.958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Grinnell F., Bennett M. H. Fibroblast adhesion on collagen substrata in the presence and absence of plasma fibronectin. J Cell Sci. 1981 Apr;48:19–34. doi: 10.1242/jcs.48.1.19. [DOI] [PubMed] [Google Scholar]
  26. Harris A. K., Stopak D., Wild P. Fibroblast traction as a mechanism for collagen morphogenesis. Nature. 1981 Mar 19;290(5803):249–251. doi: 10.1038/290249a0. [DOI] [PubMed] [Google Scholar]
  27. Hay E. D., Revel J. P. Fine structure of the developing avian cornea. Monogr Dev Biol. 1969;1:1–144. [PubMed] [Google Scholar]
  28. Herman B., Langevin M. A., Albertini D. F. The effects of taxol on the organization of the cytoskeleton in cultured ovarian granulosa cells. Eur J Cell Biol. 1983 Jul;31(1):34–45. [PubMed] [Google Scholar]
  29. Hsieh P., Chen L. B. Behavior of cells seeded in isolated fibronectin matrices. J Cell Biol. 1983 May;96(5):1208–1217. doi: 10.1083/jcb.96.5.1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kupfer A., Louvard D., Singer S. J. Polarization of the Golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental wound. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2603–2607. doi: 10.1073/pnas.79.8.2603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Revel J. P., Hoch P., Ho D. Adhesion of culture cells to their substratum. Exp Cell Res. 1974 Mar 15;84(1):207–218. doi: 10.1016/0014-4827(74)90398-x. [DOI] [PubMed] [Google Scholar]
  32. Rovensky Y. A., Slavnaya I. L. Spreading of fibroblast-like cells on grooved surfaces: a study by scanning electron microscopy. Exp Cell Res. 1974 Mar 15;84(1):199–206. doi: 10.1016/0014-4827(74)90397-8. [DOI] [PubMed] [Google Scholar]
  33. Sanders E. J., Prasad S. The culture of chick embryo mesoderm cells in hydrated collagen gels. J Exp Zool. 1983 Apr;226(1):81–92. doi: 10.1002/jez.1402260111. [DOI] [PubMed] [Google Scholar]
  34. Sattilaro R. F., Dentler W. L., LeCluyse E. L. Microtubule-associated proteins (MAPs) and the organization of actin filaments in vitro. J Cell Biol. 1981 Aug;90(2):467–473. doi: 10.1083/jcb.90.2.467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schiff P. B., Horwitz S. B. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1561–1565. doi: 10.1073/pnas.77.3.1561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Schor S. L., Allen T. D., Winn B. Lymphocyte migration into three-dimensional collagen matrices: a quantitative study. J Cell Biol. 1983 Apr;96(4):1089–1096. doi: 10.1083/jcb.96.4.1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schor S. L. Cell proliferation and migration on collagen substrata in vitro. J Cell Sci. 1980 Feb;41:159–175. doi: 10.1242/jcs.41.1.159. [DOI] [PubMed] [Google Scholar]
  39. Schor S. L., Schor A. M., Bazill G. W. The effects of fibronectin on the migration of human foreskin fibroblasts and Syrian hamster melanoma cells into three-dimensional gels of native collagen fibres. J Cell Sci. 1981 Apr;48:301–314. doi: 10.1242/jcs.48.1.301. [DOI] [PubMed] [Google Scholar]
  40. Solomon F. Detailed neurite morphologies of sister neurolbastoma cells are related. Cell. 1979 Jan;16(1):165–169. doi: 10.1016/0092-8674(79)90197-1. [DOI] [PubMed] [Google Scholar]
  41. Solomon F., Magendantz M. Cytochalasin separates microtubule disassembly from loss of asymmetric morphology. J Cell Biol. 1981 Apr;89(1):157–161. doi: 10.1083/jcb.89.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Solomon F. Neuroblastoma cells recapitulate their detailed neurite morphologies after reversible microtubule disassembly. Cell. 1980 Sep;21(2):333–338. doi: 10.1016/0092-8674(80)90469-9. [DOI] [PubMed] [Google Scholar]
  43. Sugrue S. P., Hay E. D. Response of basal epithelial cell surface and Cytoskeleton to solubilized extracellular matrix molecules. J Cell Biol. 1981 Oct;91(1):45–54. doi: 10.1083/jcb.91.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tomasek J. J., Hay E. D., Fujiwara K. Collagen modulates cell shape and cytoskeleton of embryonic corneal and fibroma fibroblasts: distribution of actin, alpha-actinin, and myosin. Dev Biol. 1982 Jul;92(1):107–122. doi: 10.1016/0012-1606(82)90155-5. [DOI] [PubMed] [Google Scholar]
  45. Vasiliev J. M., Gelfand I. M., Domnina L. V., Ivanova O. Y., Komm S. G., Olshevskaja L. V. Effect of colcemid on the locomotory behaviour of fibroblasts. J Embryol Exp Morphol. 1970 Nov;24(3):625–640. [PubMed] [Google Scholar]
  46. Weiss P., Garber B. Shape and Movement of Mesenchyme Cells as Functions of the Physical Structure of the Medium: Contributions to a Quantitative Morphology. Proc Natl Acad Sci U S A. 1952 Mar;38(3):264–280. doi: 10.1073/pnas.38.3.264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Willingham M. C., Yamada S. S., Bechtel P. J., Rutherford A. V., Pastan I. H. Ultrastructural immunocytochemical localization of myosin in cultured fibroblastic cells. J Histochem Cytochem. 1981 Nov;29(11):1289–1301. doi: 10.1177/29.11.7033361. [DOI] [PubMed] [Google Scholar]
  48. Willingham M. C., Yamada S. S., Davies P. J., Rutherford A. V., Gallo M. G., Pastan I. Intracellular localization of actin in cultured fibroblasts by electron microscopic immunocytochemistry. J Histochem Cytochem. 1981 Jan;29(1):17–37. doi: 10.1177/29.1.7009728. [DOI] [PubMed] [Google Scholar]
  49. Witkowski J. A., Brighton W. D. Stages of spreading of human diploid cells on glass surfaces. Exp Cell Res. 1971 Oct;68(2):372–380. doi: 10.1016/0014-4827(71)90162-5. [DOI] [PubMed] [Google Scholar]

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

RESOURCES