Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape

ABSTRACT Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 g/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Realtime videomicroscopic studies revealed that addition of 250 M ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 M. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors. GRGDSP had little effect on cell or nuclear shape in the absence of ATP and, hence, in the absence of cytoskeletal tension. These data suggest that large-scale changes in cell and nuclear shape result from the action of mechanical tension that is generated within the cytoskeleton via an actomyosin filament sliding mechanism, transmitted across integrin receptors and physically resisted by immobilized adhesion sites within the extracellular matrix. Rapid and coordinated changes of cell, cytoskeletal and nuclear form result when this cellular force balance is altered.