Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Aug 1;107(2):665–674. doi: 10.1083/jcb.107.2.665

Tension and compression in the cytoskeleton of PC-12 neurites. II: Quantitative measurements

PMCID: PMC2115196  PMID: 3417767

Abstract

We assessed the mechanical properties of PC-12 neurites by applying a force with calibrated glass needles and measured resulting changes in neurite length and deflection of the needle. We observed a linear relationship between force and length change that was not affected by multiple distensions and were thus able to determine neurite spring constants and initial, nondistended, rest tensions. 81 out of 82 neurites showed positive rest tensions ranging over three orders of magnitude with most values clustering around 30-40 mu dynes. Treatment with cytochalasin D significantly reduced neurite rest tensions to an average compression equal to 14% of the former tension and spring constants to an average of 17% of resting values. Treatment with nocodazole increased neurite rest tensions to an average of 282% of resting values but produced no change in spring constant. These observations suggest a particular type of complementary force interaction underlying axonal shape; the neurite actin network under tension and neurite microtubules under compression. Thermodynamics suggests that microtubule (MT) assembly may be regulated by changes in compressive load. We tested this effect by releasing neurite attachment to a polylysine-coated surface with polyaspartate, thus shifting external compressive support onto internal elements, and measuring the relative change in MT polymerization using quantitative Western blotting. Neurons grown on polylysine or collagen without further treatment had a 1:2 ratio of soluble to polymerized tubulin. When neurites grown on polylysine were treated with 1% polyaspartate for 15- 30 min, 80% of neurites retracted, shifting the soluble: polymerized tubulin ratio to 1:1. Polyaspartate treatment of cells grown on collagen, or grown on polylysine but treated with cytochalasin to reduce tension, caused neither retraction nor a change in the soluble:polymerized tubulin ratio. We suggest that the release of adhesion to the dish shifted the compressive load formerly borne by the dish onto Mts causing their partial depolymerization. Our observations are consistent with the possibility that alterations in MT compression during growth cone advance integrates MT assembly with the advance.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. Albrecht-Buehler G. Role of cortical tension in fibroblast shape and movement. Cell Motil Cytoskeleton. 1987;7(1):54–67. doi: 10.1002/cm.970070108. [DOI] [PubMed] [Google Scholar]
  2. Aletta J. M., Greene L. A. Sequential phosphorylation of chartin microtubule-associated proteins is regulated by the presence of microtubules. J Cell Biol. 1987 Jul;105(1):277–290. doi: 10.1083/jcb.105.1.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black M. M., Lasek R. J. Slow components of axonal transport: two cytoskeletal networks. J Cell Biol. 1980 Aug;86(2):616–623. doi: 10.1083/jcb.86.2.616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bray D. Axonal growth in response to experimentally applied mechanical tension. Dev Biol. 1984 Apr;102(2):379–389. doi: 10.1016/0012-1606(84)90202-1. [DOI] [PubMed] [Google Scholar]
  5. Bray D. Mechanical tension produced by nerve cells in tissue culture. J Cell Sci. 1979 Jun;37:391–410. doi: 10.1242/jcs.37.1.391. [DOI] [PubMed] [Google Scholar]
  6. Bray D., Thomas C., Shaw G. Growth cone formation in cultures of sensory neurons. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5226–5229. doi: 10.1073/pnas.75.10.5226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bray D., White J. G. Cortical flow in animal cells. Science. 1988 Feb 19;239(4842):883–888. doi: 10.1126/science.3277283. [DOI] [PubMed] [Google Scholar]
  8. Cande W. Z., Snyder J., Smith D., Summers K., McIntosh J. R. A functional mitotic spindle prepared from mammalian cells in culture. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1559–1563. doi: 10.1073/pnas.71.4.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cooper J. A. Effects of cytochalasin and phalloidin on actin. J Cell Biol. 1987 Oct;105(4):1473–1478. doi: 10.1083/jcb.105.4.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Corvaja N., Di Luzio A., Biocca S., Cattaneo A., Calissano P. Morphological and ultrastructural changes in PC12 pheochromocytoma cells induced by a combined treatment with NGF and taxol. Exp Cell Res. 1982 Dec;142(2):385–395. doi: 10.1016/0014-4827(82)90380-9. [DOI] [PubMed] [Google Scholar]
  11. Daniels M. The role of microtubules in the growth and stabilization of nerve fibers. Ann N Y Acad Sci. 1975 Jun 30;253:535–544. doi: 10.1111/j.1749-6632.1975.tb19227.x. [DOI] [PubMed] [Google Scholar]
  12. De Brabander M., Geuens G., Van De Veire R., Thoneé F., Aerts F., Desplenter L., De Cree J., Borgers M. The effects of R U7934 (NSC 238159), a new antimicrotubular substance, on the ultrastructure of neoplastic cells in vivo. Eur J Cancer. 1977 Jun;13(6):511–528. doi: 10.1016/0014-2964(77)90113-x. [DOI] [PubMed] [Google Scholar]
  13. Drubin D. G., Feinstein S. C., Shooter E. M., Kirschner M. W. Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors. J Cell Biol. 1985 Nov;101(5 Pt 1):1799–1807. doi: 10.1083/jcb.101.5.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goldberg D. J., Burmeister D. W. Stages in axon formation: observations of growth of Aplysia axons in culture using video-enhanced contrast-differential interference contrast microscopy. J Cell Biol. 1986 Nov;103(5):1921–1931. doi: 10.1083/jcb.103.5.1921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. HIRAMOTO Y. MECHANICAL PROPERTIES OF SEA URCHIN EGGS. I. SURFACE FORCE AND ELASTIC MODULUS OF THE CELL MEMBRANE. Exp Cell Res. 1963 Oct;32:59–75. doi: 10.1016/0014-4827(63)90069-7. [DOI] [PubMed] [Google Scholar]
  16. Heidemann S. R., Joshi H. C., Schechter A., Fletcher J. R., Bothwell M. Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells. J Cell Biol. 1985 Mar;100(3):916–927. doi: 10.1083/jcb.100.3.916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hill T. L., Kirschner M. W. Bioenergetics and kinetics of microtubule and actin filament assembly-disassembly. Int Rev Cytol. 1982;78:1–125. [PubMed] [Google Scholar]
  18. Hirokawa N. Cross-linker system between neurofilaments, microtubules, and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method. J Cell Biol. 1982 Jul;94(1):129–142. doi: 10.1083/jcb.94.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jacobs J. R., Stevens J. K. Experimental modification of PC12 neurite shape with the microtubule-depolymerizing drug Nocodazole: a serial electron microscopic study of neurite shape control. J Cell Biol. 1986 Sep;103(3):907–915. doi: 10.1083/jcb.103.3.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Joshi H. C., Chu D., Buxbaum R. E., Heidemann S. R. Tension and compression in the cytoskeleton of PC 12 neurites. J Cell Biol. 1985 Sep;101(3):697–705. doi: 10.1083/jcb.101.3.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Landis S. C. Neuronal growth cones. Annu Rev Physiol. 1983;45:567–580. doi: 10.1146/annurev.ph.45.030183.003031. [DOI] [PubMed] [Google Scholar]
  24. Letourneau P. C., Shattuck T. A., Ressler A. H. "Pull" and "push" in neurite elongation: observations on the effects of different concentrations of cytochalasin B and taxol. Cell Motil Cytoskeleton. 1987;8(3):193–209. doi: 10.1002/cm.970080302. [DOI] [PubMed] [Google Scholar]
  25. McBeath E., Fujiwara K. Improved fixation for immunofluorescence microscopy using light-activated 1,3,5-triazido-2,4,6-trinitrobenzene (TTB). J Cell Biol. 1984 Dec;99(6):2061–2073. doi: 10.1083/jcb.99.6.2061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mizushima-Sugano J., Maeda T., Miki-Noumura T. Flexural rigidity of singlet microtubules estimated from statistical analysis of their contour lengths and end-to-end distances. Biochim Biophys Acta. 1983 Jan 25;755(2):257–262. doi: 10.1016/0304-4165(83)90212-x. [DOI] [PubMed] [Google Scholar]
  27. Morris J. R., Lasek R. J. Stable polymers of the axonal cytoskeleton: the axoplasmic ghost. J Cell Biol. 1982 Jan;92(1):192–198. doi: 10.1083/jcb.92.1.192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nicklas R. B. Measurements of the force produced by the mitotic spindle in anaphase. J Cell Biol. 1983 Aug;97(2):542–548. doi: 10.1083/jcb.97.2.542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pasternak C., Elson E. L. Lymphocyte mechanical response triggered by cross-linking surface receptors. J Cell Biol. 1985 Mar;100(3):860–872. doi: 10.1083/jcb.100.3.860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Petersen N. O., McConnaughey W. B., Elson E. L. Dependence of locally measured cellular deformability on position on the cell, temperature, and cytochalasin B. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5327–5331. doi: 10.1073/pnas.79.17.5327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rappaport R. Cell division: direct measurement of maximum tension exerted by furrow of echinoderm eggs. Science. 1967 Jun 2;156(3779):1241–1243. doi: 10.1126/science.156.3779.1241. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Schliwa M., Ezzell R. M., Euteneuer U. erythro-9-[3-(2-Hydroxynonyl)]adenine is an effective inhibitor of cell motility and actin assembly. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6044–6048. doi: 10.1073/pnas.81.19.6044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Schroeder T. E. The origin of cleavage forces in dividing eggs. A mechanism in two steps. Exp Cell Res. 1981 Jul;134(1):231–240. doi: 10.1016/0014-4827(81)90480-8. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Solomon F., Magendantz M., Salzman A. Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins. Cell. 1979 Oct;18(2):431–438. doi: 10.1016/0092-8674(79)90062-x. [DOI] [PubMed] [Google Scholar]
  38. Spero D. A., Roisen F. J. Neuro-2a neuroblastoma cells form neurites in the presence of taxol and cytochalasin D. Brain Res. 1985 Nov;355(1):155–159. doi: 10.1016/0165-3806(85)90016-1. [DOI] [PubMed] [Google Scholar]
  39. Tomasek J. J., Hay E. D. Analysis of the role of microfilaments and microtubules in acquisition of bipolarity and elongation of fibroblasts in hydrated collagen gels. J Cell Biol. 1984 Aug;99(2):536–549. doi: 10.1083/jcb.99.2.536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Trinkaus J. P. Further thoughts on directional cell movement during morphogenesis. J Neurosci Res. 1985;13(1-2):1–19. doi: 10.1002/jnr.490130102. [DOI] [PubMed] [Google Scholar]
  41. Vandenburgh H. H. Dynamic mechanical orientation of skeletal myofibers in vitro. Dev Biol. 1982 Oct;93(2):438–443. doi: 10.1016/0012-1606(82)90131-2. [DOI] [PubMed] [Google Scholar]
  42. Weingarten M. D., Suter M. M., Littman D. R., Kirschner M. W. Properties of the depolymerization products of microtubules from mammalian brain. Biochemistry. 1974 Dec 31;13(27):5529–5537. doi: 10.1021/bi00724a012. [DOI] [PubMed] [Google Scholar]
  43. Williams R. C., Jr, Detrich H. W., 3rd Separation of tubulin from microtubule-associated proteins on phosphocellulose. Accompanying alterations in concentrations of buffer components. Biochemistry. 1979 Jun 12;18(12):2499–2503. doi: 10.1021/bi00579a010. [DOI] [PubMed] [Google Scholar]
  44. Yamada K. M., Spooner B. S., Wessells N. K. Axon growth: roles of microfilaments and microtubules. Proc Natl Acad Sci U S A. 1970 Aug;66(4):1206–1212. doi: 10.1073/pnas.66.4.1206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yanagida T., Oosawa F. Polarized fluorescence from epsilon-ADP incorporated into F-actin in a myosin-free single fiber: conformation of F-actin and changes induced in it by heavy meromyosin. J Mol Biol. 1978 Dec 15;126(3):507–524. doi: 10.1016/0022-2836(78)90056-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES