The mechanical function of the cytoskeleton in reconstituted tissues

Cells and extracellular matrices (ECMs) form tissue and organs. The major structural constituents of cells, which dominate their mechanical properties, are actin filaments, microtubules, intermediate filaments, and myosin. The roles of these molecules in maintaining cell structure can be observed only by measuring their functions. The mechanics of individual cells have been measured using various techniques, e.g., cell indentation and deformable substrata. A bidirectional relationship of ECMs and cells determines cell mechanics. Therefore, cell mechanics should be measured with cells grown in ECMs. We present a novel method to measure the mechanical properties of fibroblasts in the ECMs in model tissue. Using this method we found that the actin cytoskeleton is a major contributor to the cellular mechanics. The active tension generated by actomyosin was transmitted to the ECM via actin filaments. Therefore, the alteration of actin filaments by biochemical processes can be probed by measuring cell mechanics. To demonstrate this, we observed the mechanics of fibroblasts treated with Cytochalasin D (CD) and Latrunculin B, which disrupt the actin cytoskeleton by different mechanisms. The high concentration of CD needed to disrupt actin filaments in vivo (∼2muM) relative to that needed in vitro (2 nM) can be explained by a simple binding competition model. The mechanical integrity of the fibroblasts populated model tissues increases significantly due to ECM remodeling by the cells, which requires actomyosin. The role of myosin in cell spreading, which is a preliminary step of ECM remodeling, is not yet understood. We found that myosin II activity was not necessary for cell spreading and even impaired its rate. Cell spreading is driven mainly by actin polymerization.;Cardiac tissue models were made by culturing both embryonic cardiac myocytes and fibroblasts in the ECMs. The myocytes' contractility can be separated from that of the fibroblasts by eliminating the non-sarcomeric actin filaments. Although the hyperpolymerization of microtubules has been speculated to interfere with cardiac contractility, we observed no significant contribution of microtubules to the mechanical properties of cardiac model tissue.