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. 1994 Dec;5(12):1281–1288. doi: 10.1091/mbc.5.12.1281

Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover.

D J Mooney 1, L K Hansen 1, R Langer 1, J P Vacanti 1, D E Ingber 1
PMCID: PMC301157  PMID: 7696710

Abstract

Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and changing cell shape. This finding is consistent with a mechanism of MT regulation in which the ECM stabilizes MTs by both accepting transfer of mechanical loads and altering tubulin degradation in cells that continue to autoregulate tubulin synthesis.

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Selected References

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  1. Auffray C., Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem. 1980 Jun;107(2):303–314. doi: 10.1111/j.1432-1033.1980.tb06030.x. [DOI] [PubMed] [Google Scholar]
  2. Ben-Ze'ev A., Farmer S. R., Penman S. Mechanisms of regulating tubulin synthesis in cultured mammalian cells. Cell. 1979 Jun;17(2):319–325. doi: 10.1016/0092-8674(79)90157-0. [DOI] [PubMed] [Google Scholar]
  3. Bond J. F., Robinson G. S., Farmer S. R. Differential expression of two neural cell-specific beta-tubulin mRNAs during rat brain development. Mol Cell Biol. 1984 Jul;4(7):1313–1319. doi: 10.1128/mcb.4.7.1313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buxbaum R. E., Heidemann S. R. A thermodynamic model for force integration and microtubule assembly during axonal elongation. J Theor Biol. 1988 Oct 7;134(3):379–390. doi: 10.1016/s0022-5193(88)80068-7. [DOI] [PubMed] [Google Scholar]
  5. Caron J. M., Berlin R. D. Dynamic interactions between microtubules and artificial membranes. Biochemistry. 1987 Jun 16;26(12):3681–3688. doi: 10.1021/bi00386a063. [DOI] [PubMed] [Google Scholar]
  6. Caron J. M., Jones A. L., Kirschner M. W. Autoregulation of tubulin synthesis in hepatocytes and fibroblasts. J Cell Biol. 1985 Nov;101(5 Pt 1):1763–1772. doi: 10.1083/jcb.101.5.1763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Caron J. M., Jones A. L., Rall L. B., Kirschner M. W. Autoregulation of tubulin synthesis in enucleated cells. Nature. 1985 Oct 17;317(6038):648–651. doi: 10.1038/317648a0. [DOI] [PubMed] [Google Scholar]
  8. Cleveland D. W., Lopata M. A., Sherline P., Kirschner M. W. Unpolymerized tubulin modulates the level of tubulin mRNAs. Cell. 1981 Aug;25(2):537–546. doi: 10.1016/0092-8674(81)90072-6. [DOI] [PubMed] [Google Scholar]
  9. Cleveland D. W., Pittenger M. F., Feramisco J. R. Elevation of tubulin levels by microinjection suppresses new tubulin synthesis. Nature. 1983 Oct 20;305(5936):738–740. doi: 10.1038/305738a0. [DOI] [PubMed] [Google Scholar]
  10. Danowski B. A. Fibroblast contractility and actin organization are stimulated by microtubule inhibitors. J Cell Sci. 1989 Jun;93(Pt 2):255–266. doi: 10.1242/jcs.93.2.255. [DOI] [PubMed] [Google Scholar]
  11. Dennerll T. J., Joshi H. C., Steel V. L., Buxbaum R. E., Heidemann S. R. Tension and compression in the cytoskeleton of PC-12 neurites. II: Quantitative measurements. J Cell Biol. 1988 Aug;107(2):665–674. doi: 10.1083/jcb.107.2.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Erickson C. A., Trinkaus J. P. Microvilli and blebs as sources of reserve surface membrane during cell spreading. Exp Cell Res. 1976 May;99(2):375–384. doi: 10.1016/0014-4827(76)90595-4. [DOI] [PubMed] [Google Scholar]
  13. Heidemann S. R., Buxbaum R. E. Tension as a regulator and integrator of axonal growth. Cell Motil Cytoskeleton. 1990;17(1):6–10. doi: 10.1002/cm.970170103. [DOI] [PubMed] [Google Scholar]
  14. Hill T. L. Microfilament or microtubule assembly or disassembly against a force. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5613–5617. doi: 10.1073/pnas.78.9.5613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  16. Ingber D. E. Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton. J Cell Sci. 1993 Mar;104(Pt 3):613–627. doi: 10.1242/jcs.104.3.613. [DOI] [PubMed] [Google Scholar]
  17. Ingber D. E. Fibronectin controls capillary endothelial cell growth by modulating cell shape. Proc Natl Acad Sci U S A. 1990 May;87(9):3579–3583. doi: 10.1073/pnas.87.9.3579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ingber D. Integrins as mechanochemical transducers. Curr Opin Cell Biol. 1991 Oct;3(5):841–848. doi: 10.1016/0955-0674(91)90058-7. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Kirschner M., Mitchison T. Beyond self-assembly: from microtubules to morphogenesis. Cell. 1986 May 9;45(3):329–342. doi: 10.1016/0092-8674(86)90318-1. [DOI] [PubMed] [Google Scholar]
  21. Kolodney M. S., Wysolmerski R. B. Isometric contraction by fibroblasts and endothelial cells in tissue culture: a quantitative study. J Cell Biol. 1992 Apr;117(1):73–82. doi: 10.1083/jcb.117.1.73. [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. Lamoureux P., Steel V. L., Regal C., Adgate L., Buxbaum R. E., Heidemann S. R. Extracellular matrix allows PC12 neurite elongation in the absence of microtubules. J Cell Biol. 1990 Jan;110(1):71–79. doi: 10.1083/jcb.110.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mitchison T. J. Microtubule dynamics and kinetochore function in mitosis. Annu Rev Cell Biol. 1988;4:527–549. doi: 10.1146/annurev.cb.04.110188.002523. [DOI] [PubMed] [Google Scholar]
  25. Mitchison T., Kirschner M. Dynamic instability of microtubule growth. Nature. 1984 Nov 15;312(5991):237–242. doi: 10.1038/312237a0. [DOI] [PubMed] [Google Scholar]
  26. Mooney D., Hansen L., Vacanti J., Langer R., Farmer S., Ingber D. Switching from differentiation to growth in hepatocytes: control by extracellular matrix. J Cell Physiol. 1992 Jun;151(3):497–505. doi: 10.1002/jcp.1041510308. [DOI] [PubMed] [Google Scholar]
  27. Pipeleers D. G., Pipeleers-Marichal M. A., Kipnis D. M. Physiological regulation of total tubulin and polymerized tubulin in tissues. J Cell Biol. 1977 Aug;74(2):351–357. doi: 10.1083/jcb.74.2.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schwartz M. A. Transmembrane signalling by integrins. Trends Cell Biol. 1992 Oct;2(10):304–308. doi: 10.1016/0962-8924(92)90120-c. [DOI] [PubMed] [Google Scholar]
  29. Sims J. R., Karp S., Ingber D. E. Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape. J Cell Sci. 1992 Dec;103(Pt 4):1215–1222. doi: 10.1242/jcs.103.4.1215. [DOI] [PubMed] [Google Scholar]
  30. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Vale R. D. Microtubule-based motor proteins. Curr Opin Cell Biol. 1990 Feb;2(1):15–22. doi: 10.1016/s0955-0674(05)80025-0. [DOI] [PubMed] [Google Scholar]
  32. Wang N., Butler J. P., Ingber D. E. Mechanotransduction across the cell surface and through the cytoskeleton. Science. 1993 May 21;260(5111):1124–1127. doi: 10.1126/science.7684161. [DOI] [PubMed] [Google Scholar]

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