Advertisement
Journal Articles
Microtubule assembly is regulated by externally applied strain in cultured smooth muscle cells
A.J. Putnam, J.J. Cunningham, R.G. Dennis, J.J. Linderman, D.J. Mooney
Journal of Cell Science 1998 111: 3379-3387;

Summary

Mechanical forces clearly regulate the development and phenotype of a variety of tissues and cultured cells. However, it is not clear how mechanical information is transduced intracellularly to alter cellular function. Thermodynamic modeling predicts that mechanical forces influence microtubule assembly, and hence suggest microtubules as one potential cytoskeletal target for mechanical signals. In this study, the assembly of microtubules was analyzed in rat aortic smooth muscle cells cultured on silicon rubber substrates exposed to step increases in applied strain. Cytoskeletal and total cellular protein fractions were extracted from the cells following application of the external strain, and tubulin levels were quantified biochemically via a competitive ELISA and western blotting using bovine brain tubulin as a standard. In the first set of experiments, smooth muscle cells were subjected to a step-increase in strain and the distribution of tubulin between monomeric, polymeric, and total cellular pools was followed with time. Microtubule mass increased rapidly following application of the strain, with a statistically significant increase (P<0.05) in microtubule mass from 373+/−32 pg/cell (t=0) to 514+/−30 pg/cell (t=15 minutes). In parallel, the amount of soluble tubulin decreased approximately fivefold. The microtubule mass decreased after 1 hour to a value of 437+/−24 pg/cell. In the second set of experiments, smooth muscle cells were subjected to increasing doses of externally applied strain using a custom-built strain device. Monomeric, polymeric, and total tubulin fractions were extracted after 15 minutes of applied strain and quantified as for the earlier experiments. Microtubule mass increased with increasing strain while total cellular tubulin levels remained essentially constant at all strain levels. These findings are consistent with a thermodynamic model which predicts that microtubule assembly is promoted as a cell is stretched and compressional loads on the microtubules are presumably relieved. Furthermore, these data suggest microtubules are a potential target for translating changes in externally applied mechanical stimuli to alterations in cellular phenotype.

REFERENCES

    1. Barbee K. A.,
    2. Macarak E. J. and
    3. Thibault L. E.
    (1994). Strain measurements in cultured vascular smooth muscle cells subjected to mechanical deformation. Ann. Biomed. Eng 22, 14–22
    OpenUrlCrossRefPubMedWeb of Science
    1. Baudet C.,
    2. Naveilhan P.,
    3. Jehan F.,
    4. Brachet P. and
    5. Wion D.
    (1995). Expression of nerve growth factor gene is controlled by the microtubule network. J. Neurosci. Res 41, 462–470
    OpenUrlCrossRefPubMedWeb of Science
    1. Beloussov L. V.,
    2. Saveliev S. V.,
    3. Naumidi I. I. and
    4. Novoselov V. V.
    (1994). Mechanical stresses in embryonic tissues: patterns, morphogenetic role and involvement in regulatory feedback. Int. Rev. Cytol 150, 1–34
    OpenUrlPubMedWeb of Science
    1. Ben-Ze'ev A.,
    2. Farmer S. R. and
    3. Penman S.
    (1979). Mechanisms of regulating tubulin synthesis in cultured mammalian cells. Cell 17, 319–325
    OpenUrlCrossRefPubMedWeb of Science
    1. Birukov K. G.,
    2. Shirinsky V. P.,
    3. Stepanova O. V.,
    4. Tkachuk V. A.,
    5. Hahn A. W. A.,
    6. Resnik T. J. and
    7. Smirnov V. N.
    (1995). Stretch affects phenotype and proliferation of vascular smooth muscle cells. Mol. Cell. Biochem 144, 131–139
    OpenUrlCrossRefPubMedWeb of Science
    1. Buxbaum R. E. and
    2. Heidemann S. R.
    (1988). A thermodynamic model for force integration and microtubule assembly during axonal elongation. J. Theor. Biol 134, 379–390
    OpenUrlCrossRefPubMedWeb of Science
    1. Caron J. M.,
    2. Jones A. L. and
    3. Kirschner M. W.
    (1985). Autoregulation of tubulin synthesis in hepatocytes and fibroblasts. J. Cell Biol 101, 1763–1772
    OpenUrlAbstract/FREE Full Text
    1. Cassimeris L.,
    2. Pryer N. K. and
    3. Salmon E. D.
    (1988). Real-time observations of microtubule dynamic instability in living cells. J. Cell Biol 107, 2223–2231
    OpenUrlAbstract/FREE Full Text
    1. Cleveland D. W.,
    2. Lopata M. A.,
    3. Sherline P. and
    4. Kirschner M. W.
    (1981). Unpolymerized tubulin modulates the level of tubulin mRNAs. Cell 25, 537–546
    OpenUrlCrossRefPubMedWeb of Science
    1. Cole N. B. and
    2. Lippincott-Schwartz J.
    (1995). Organization of organelles and membrane traffic by microtubules. Curr. Opin. Cell Biol 7, 55–64
    OpenUrlCrossRefPubMedWeb of Science
    1. Danowski B. A.
    (1989). Fibroblast contractility and actin organization are stimulated by microtubule inhibitors. J. Cell Sci 93, 255–266
    OpenUrlAbstract/FREE Full Text
    1. Davies P. F. and
    2. Tripathi S. C.
    (1993). Mechanical stress mechanisms and the cell. An endothelial paradigm. Circ. Res 72, 239–245
    OpenUrlAbstract/FREE Full Text
    1. Davies P. F.,
    2. Robotewskyj A. and
    3. Griem M. L.
    (1994). Quantitative studies of endothelial cell adhesion: Directional remodeling of focal adhesion sites in response to flow forces. J. Clin. Invest 93, 2031–2038
    OpenUrlCrossRefPubMedWeb of Science
    1. Dennerll T. J.,
    2. Joshi H. C.,
    3. Steel V. L.,
    4. Buxbaum R. E. and
    5. Heidemann S. R.
    (1988). Tension and compression in the cytoskeleton of PC-12 neurites II: quantitative measurements. J. Cell Biol 107, 665–674
    OpenUrlAbstract/FREE Full Text
    1. Dogterom M. and
    2. Yurke B.
    (1997). Measurement of the force-velocity relation for growing microtubules. Science 278, 856–860
    OpenUrlAbstract/FREE Full Text
    1. Drubin D. G.,
    2. Feinstein S. C.,
    3. Shooter E. M. and
    4. Kirschner M. W.
    (1985). Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly promoting factors. J. Cell Biol 101, 1799–1807
    OpenUrlAbstract/FREE Full Text
    1. Duncan R. L. and
    2. Turner C. H.
    (1995). Mechanotransduction and the functional response of bone to mechanical strain. Calcif. Tissue Int 57, 344–358
    OpenUrlCrossRefPubMedWeb of Science
    1. Evangelisti R.,
    2. Becchetti E.,
    3. Baroni T.,
    4. Rossi L.,
    5. Arena N.,
    6. Valeno V.,
    7. Carinci P. and
    8. Locci P.
    (1995). Modulation of phenotypic expression of fibroblasts by alteration of the cytoskeleton. Cell. Biochem. Funct 13, 41–52
    OpenUrlCrossRefPubMedWeb of Science
    1. Gilbert J. A.,
    2. Weinhold P. S.,
    3. Banes A. J.,
    4. Link G. W. and
    5. Jones G. L.
    (1994). Strain profiles for circular cell culture plates containing flexible surfaces employed to mechanically deform cells in vitro. J. Biomechanics 27, 1169–1177
    OpenUrlCrossRefPubMedWeb of Science
    1. Heidemann S. R. and
    2. Buxbaum R. E.
    (1990). Tension as a regulator and integrator of axonal growth. Cell Motil. Cytoskel 17, 6–10
    OpenUrlCrossRefPubMedWeb of Science
    1. Ingber D. E.
    (1993). Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton. J. Cell Sci 104, 613–627
    OpenUrlFREE Full Text
    1. Ingber D. E.,
    2. Dike L.,
    3. Hansen L.,
    4. Karp S.,
    5. Liley H.,
    6. Maniotis A.,
    7. McNamee H.,
    8. Mooney D.,
    9. Plopper G.,
    10. Sims J. and
    11. Wang N.
    (1994). Cellular tensegrity: exploring how mechanical changes in the cytoskeleton regulate cell growth, migration and tissue pattern during morphogenesis. Int. Rev. Cytol 150, 173–224
    OpenUrlCrossRefPubMedWeb of Science
    1. Joshi H. C.,
    2. Chu D.,
    3. Buxbaum R. E. and
    4. Heidemann S. R.
    (1985). Tension and compression in the cytoskeleton of PC12 neurites. J. Cell Biol 101, 697–705
    OpenUrlAbstract/FREE Full Text
    1. Juliano R. L. and
    2. Haskill S.
    (1993). Signal transduction from the extracellular matrix. J. Cell Biol 120, 577–585
    OpenUrlFREE Full Text
    1. Kolodney M. S. and
    2. Elson E. L.
    (1995). Contraction due to microtubule disruption is associated with increased phosphorylation of myosin regulatory light chain. Proc. Nat. Acad. Sci. USA 92, 10252–10256
    OpenUrlAbstract/FREE Full Text
    1. Laemmli U. K.
    (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
    OpenUrlCrossRefPubMedWeb of Science
    1. Lee A. A.,
    2. Delhaas T.,
    3. Waldman L. K.,
    4. MacKenna D. A.,
    5. Villarreal F. J. and
    6. McCulloch A. D.
    (1996). An equibiaxial strain system for cultured cells. Am J. Physiol 271, 1400–.
    OpenUrl
    1. Leiber D.,
    2. Jasper J. R.,
    3. Alousi A. A.,
    4. Martin J.,
    5. Bernstein D. and
    6. Insel P. A.
    (1993). Alteration in Gs-mediated signal transduction in S49 lymphoma cells treated with inhibitors of microtubules. J. Biol. Chem 268, 3833–3837
    OpenUrlAbstract/FREE Full Text
    1. Middleton C. A.,
    2. Brown A. F.,
    3. Brown R. M. and
    4. Roberts D. J. H.
    (1988). The shape of cultured epithelial cells does not depend on the integrity of their microtubules. J. Cell Sci 91, 337–345
    OpenUrlAbstract/FREE Full Text
    1. Mooney D. J.,
    2. Hansen L. K.,
    3. Langer R.,
    4. Vacanti J. P. and
    5. Ingber D. E.
    (1994). Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover. Mol. Biol. Cell 5, 1281–1288
    OpenUrlAbstract/FREE Full Text
    1. Mooney D. J.,
    2. Langer R. and
    3. Ingber D. E.
    (1995). Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix. J. Cell Sci 108, 2311–2320
    OpenUrlAbstract/FREE Full Text
    1. Rosania G. R. and
    2. Swanson J. A.
    (1996). Microtubules can modulate pseudopod activity from a distance inside macrophages. Cell Motil. Cytoskel 34, 230–245
    OpenUrlCrossRefPubMedWeb of Science
    1. Rosette C. and
    2. Karin M.
    (1995). Cytoskeletal control of gene expression: depolymerization of microtubules activates NF-B. J. Cell Biol 128, 1111–1119
    OpenUrlAbstract/FREE Full Text
    1. Rothman A.,
    2. Kulik T. J.,
    3. Taubman M. B.,
    4. Berk B. C.,
    5. Smith C. W. J. and
    6. Nadal-Ginard B.
    (1992). Development and characterization of a cloned rat pulmonary arterial smooth muscle cell line that maintains differentiated properties through multiple subcultures. Circulation 86, 1977–1986
    OpenUrlAbstract/FREE Full Text
    1. Ruoslahti E.
    (1991). Integrins. J. Clin. Invest 87, 1–5
    OpenUrlCrossRefPubMedWeb of Science
    1. Schiff P. B. and
    2. Horwitz S. B.
    (1980). Taxol stabilizes microtubules in mouse fibroblast cells. Proc. Nat. Acad. Sci. USA 77, 1561–1565
    OpenUrlAbstract/FREE Full Text
    1. Schmidt C.,
    2. Pommerenke H.,
    3. Durr F.,
    4. Nebe B. and
    5. Rychly J.
    (1988). Mechanical stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskeletally anchored proteins. J. Biol. Chem 273, 5081–5085
    OpenUrl
    1. Shinohara-Gotoh Y.,
    2. Nishida E.,
    3. Hoshi M. and
    4. Sakai H.
    (1991). Activation of microtubule-associated protein kinase by microtubule disruption in quiescent rat 3Y1 cells. Exp. Cell Res 193, 161–166
    OpenUrlCrossRefPubMedWeb of Science
    1. Shyy J. Y. and
    2. Chien S.
    (1997). Role of integrins in cellular responses to mechanical stress and adhesion. Curr. Opin. Cell Biol 9, 707–713
    OpenUrlCrossRefPubMedWeb of Science
    1. Simpson D. G.,
    2. Carver W.,
    3. Borg T. K. and
    4. Terracio L.
    (1994). Role of mechanical stimulation in the establishment and maintenance of muscle cell differentiation. Int. Rev. Cytol 150, 69–94
    OpenUrlPubMedWeb of Science
    1. Sims J. R.,
    2. Karp S. and
    3. Ingber D. E.
    (1992). Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape. J. Cell Sci 103, 1215–1222
    OpenUrlAbstract/FREE Full Text
    1. Smith P. G.,
    2. Janiga K. E. and
    3. Bruce M. C.
    (1994). Strain increases airway smooth muscle cell proliferation. Am. J. Respir. Cell Mol. Biol 10, 85–90
    OpenUrlPubMedWeb of Science
    1. Smith P. G.,
    2. Tokui T. and
    3. Ikebe M.
    (1995). Mechanical strain increases contractile enzyme activity in cultured airway smooth muscle cells. Am. J. Physiol 268, 999–.
    OpenUrl
    1. Smith P. G.,
    2. Garcia R. and
    3. Kogerman L.
    (1997). Strain reorganizes focal adhesions and cytoskeleton in cultured airway smooth muscle cells. Exp. Cell Res 232, 127–136
    OpenUrlCrossRefPubMedWeb of Science
    1. Stamenovic D.,
    2. Fredberg J. J.,
    3. Wang N.,
    4. Butler J. P. and
    5. Ingber D. E.
    (1996). A microstructural approach to cytoskeletal mechanics based on tensegrity. J. Theor. Biol 181, 125–136
    OpenUrlCrossRefPubMedWeb of Science
    1. Sumpio B. E. and
    2. Banes A. J.
    (1988). Response of porcine aortic smooth muscle cells to cyclic tensional deformation in culture. J. Surg. Res 44, 696–701
    OpenUrlCrossRefPubMedWeb of Science
    1. Sumpio B. E.,
    2. Banes A. J.,
    3. Link W. G. and
    4. Johnson G.
    (1988). Enhanced collagen production by smooth muscle cells during repetitive mechanical stretching. Arch. Surg 123, 1233–1236
    OpenUrlCrossRefPubMedWeb of Science
    1. Suprenant K. A.
    (1993). Microtubules, ribosomes and RNA: Evidence for cytoplasmic localization and translational regulation. Cell Motil. Cytoskel 25, 1–9
    OpenUrlCrossRefPubMed
    1. Tagawa H.,
    2. Rozich J. D.,
    3. Tsutsui H.,
    4. Narishige T.,
    5. Kuppuswamy D.,
    6. Sato H.,
    7. McDermott P. J.,
    8. Koide M. and
    9. Cooper G.
    (1996). Basis for increased microtubules in pressure-hypertrophied cardiocytes. Circulation 93, 1230–1243
    OpenUrlAbstract/FREE Full Text
    1. Tagawa H.,
    2. Wang N.,
    3. Narishige T.,
    4. Ingber D. E.,
    5. Zile M. R. and
    6. Cooper G.
    (1997). Cytoskeletal mechanics in pressure-overload cardiac hypertrophy. Circ. Res 80, 281–289
    OpenUrlAbstract/FREE Full Text
    1. Tanaka E.,
    2. Ho T. and
    3. Kirschner M. W.
    (1995). The role of microtubule dynamics in growth cone motility and axonal growth. J. Cell Biol 128, 139–155
    OpenUrlAbstract/FREE Full Text
    1. Thoumine O.,
    2. Ziegler T.,
    3. Girard P. R. and
    4. Nerem R. M.
    (1995). Elongation of confluent endothelial cells in culture: the importance of fields of force in the associated alterations of their cytoskeletal structure. Exp. Cell Res 219, 427–441
    OpenUrlCrossRefPubMedWeb of Science
    1. Thrower D.,
    2. Jordan M. and
    3. Wilson L.
    (1991). Quantification of cellular tubulin in microtubules and tubulin pools by a competitive ELISA. J. Immunol. Meth 136, 45–51
    OpenUrlCrossRefPubMedWeb of Science
    1. Van Essen D. C.
    (1997). A tension-based theory of morphogenesis and compact wiring in the central nervous system. Nature 385, 313–318
    OpenUrlCrossRefPubMedWeb of Science
    1. Wang N.,
    2. Butler J. P. and
    3. Ingber D. E.
    (1993). Mechanotransduction across the cell surface and through the cytoskeleton. Science 260, 1124–1127
    OpenUrlAbstract/FREE Full Text
    1. Wang N. and
    2. Ingber D. E.
    (1994). Control of cytoskeletal mechanics by extracellular matrix, cell shape and mechanical tension. Biophys. J 66, 2181–2189
    OpenUrlCrossRefPubMedWeb of Science
    1. Watson P. A.,
    2. Hannan R.,
    3. Carl. L. L. and
    4. Giger K. E.
    (1996). Contractile activity and passive stretch regulate tubulin mRNA and protein content in cardiac myocytes. Am. J. Physiol 271, 684–.
    OpenUrl
    1. Wilson E.,
    2. Sudhir K. and
    3. Ives H. E.
    (1995). Mechanical strain of rat vascular smooth muscle cells is sensed by specific extracellular matrix/integrin interaction. J. Clin. Invest 96, 2364–2372
    OpenUrlCrossRefPubMedWeb of Science
    1. Zheng J.,
    2. Buxbaum R. E. and
    3. Heidemann S. R.
    (1993). Investigation of microtubule assembly and organization accompanying tension-induced neurite initiation. J. Cell Sci 104, 1239–1250
    OpenUrlAbstract/FREE Full Text
Back to top

 Download PDF

Email
Journal Articles
Microtubule assembly is regulated by externally applied strain in cultured smooth muscle cells
A.J. Putnam, J.J. Cunningham, R.G. Dennis, J.J. Linderman, D.J. Mooney
Journal of Cell Science 1998 111: 3379-3387;
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
Alerts

Article navigation

Other journals from The Company of Biologists

Advertisement

2020 at The Company of Biologists

Despite the challenges of 2020, we were able to bring a number of long-term projects and new ventures to fruition. While we look forward to a new year, join us as we reflect on the triumphs of the last 12 months.

Mole – The Corona Files

"This is not going to go away, 'like a miracle.' We have to do magic. And I know we can."

Mole continues to offer his wise words to researchers on how to manage during the COVID-19 pandemic.

Cell scientist to watch – Christine Faulkner

In an interview, Christine Faulkner talks about where her interest in plant science began, how she found the transition between Australia and the UK, and shares her thoughts on virtual conferences.

Read & Publish participation extends worldwide

“The clear advantages are rapid and efficient exposure and easy access to my article around the world. I believe it is great to have this publishing option in fast-growing fields in biomedical research.”

Dr Jaceques Behmoaras (Imperial College London) shares his experience of publishing Open Access as part of our growing Read & Publish initiative. We now have over 60 institutions in 12 countries taking part – find out more and view our full list of participating institutions.

JCS and COVID-19

For more information on measures Journal of Cell Science is taking to support the community during the COVID-19 pandemic, please see here.

If you have any questions or concerns, please do not hestiate to contact the Editorial Office.