INTRODUCTION Mechanical signals are critical regulators in the development and function of a variety of tissues. Mechanical forces are known to influence organ morphogenesis (Beloussov et al., 1994), bone resorption and formation (Duncan and Turner, 1995), skeletal muscle differentiation and organization (Simpson et al., 1994), and development of the central nervous system (Van Essen, 1997). Increasing evidence suggests that cells mediate the response to such mechanical information, and they can respond by altering their rates of proliferation, phenotype, and extracellular matrix production in response to mechanical stress. For example, smooth muscle cells (SMCs) subjected to mechanical strain in vitro show increased proliferation (Sumpio and Banes, 1988; Smith et al., 1994; Birukov et al., 1995) and increased collagen production (Sumpio et al., 1988), while adherent endothelial cells exposed to shear stresses respond by reorganizing their cytoskeleton, activating ion channels, and altering gene expression (Davies and Tripathi, 1993). These cellular responses to mechanical information have important implications not only for normal development, but also in the pathogenesis of such diseases as atherosclerosis and hypertension. While the mechanisms by which this mechanical information is transmitted intracellularly to alter gene expression remain unclear, a potential target for mechanical signals is the cytoskeleton. A direct linkage between the extracellular matrix (ECM) and the cytoskeleton mediated by integrins that recognize specific amino acids sequences in the ECM (Ruoshlati, 1991; Juliano and Haskill, 1993) may act as one route for the transmission of external mechanical information from the outside to the inside of a cell (Ingber et al., 1993). The nuclear matrix is also structurally interconnected with the cytoskeleton, and this structural 3379 Journal of Cell Science 111, 3379-3387 (1998) Printed in Great Britain © The Company of Biologists Limited 1998 JCS4571 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. Key words: Microtubule, External strain, Tensegrity, Smooth muscle cell, Mechanical signal SUMMARY Microtubule assembly is regulated by externally applied strain in cultured smooth muscle cells Andrew J. Putnam 1 , James J. Cunningham 1 , Robert G. Dennis 2,4 , Jennifer J. Linderman 1 and David J. Mooney 1,2,3 Departments of 1 Chemical Engineering, 2 Biomedical Engineering and 3 Biologic & Materials Sciences, and 4 The Institute of Gerontology, University of Michigan, Ann Arbor, MI 48109-2136, USA *Author for correspondence (e-mail: mooneyd@umich.edu) Accepted 4 September; published on WWW 28 October 1998