Microtubule assembly is regulated by externally applied strain in culturedsmooth muscle cells

Mechanical forces clearly regulate the development and phenotype of a varie ty of tissues and cultured cells, However, it is not clear how mechanical i nformation is transduced intracellularly to alter cellular function. Thermo dynamic modeling predicts that mechanical forces influence microtubule asse mbly, and hence suggest microtubules as one potential cytoskeletal target f or mechanical signals. In this study, the assembly of microtubules was anal yzed in rat aortic smooth muscle cells cultured on silicon rubber substrate s exposed to step increases in applied strain. Cytoskeletal and total cellu lar protein fractions were extracted from the cells following application o f the external strain, and tubulin levels were quantified biochemically via a competitive ELISA and western blotting using bovine brain tubulin as a s tandard. In the first set of experiments, smooth muscle cells were subjecte d to a step-increase in strain and the distribution of tubulin between mono meric, polymeric, and total cellular pools was followed with time. Microtub ule mass increased rapidly following application of the strain, with a stat istically 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 microtubul e mass decreased after 1 hour to a value of 437 +/- 24 pg/cell, In the seco nd set of experiments, smooth muscle cells were subjected to increasing dos es of externally applied strain using a custom-built strain device. Monomer ic, polymeric, and total tubulin fractions were extracted after 15 minutes of applied strain and quantified as for the earlier experiments. Microtubul e mass increased with increasing strain while total cellular tubulin levels remained essentially constant at all strain levels. These findings are con sistent with a thermodynamic model which predicts that microtubule assembly is promoted as a cell is stretched and compressional loads on the microtub ules are presumably relieved. Furthermore, these data suggest microtubules are a potential target for translating changes in externally applied mechan ical stimuli to alterations in cellular phenotype.