Normal tissue cells are generally not viable when suspended in a fluid and are therefore said to be anchorage dependent. Such cells must adhere to a solid, but a solid can be as rigid as glass or softer than a baby's skin. The behavior of some cells on soft materials is characteristic of important phenotypes; for example, cell growth on soft agar gels is used to identify cancer cells. However, an understanding of how tissue cells—including fibroblasts, myocytes, neurons, and other cell types—sense matrix stiffness is just emerging with quantitative studies of cells adhering to gels (or to other cells) with which elasticity can be tuned to approximate that of tissues. Key roles in molecular pathways are played by adhesion complexes and the actinmyosin cytoskeleton, whose contractile forces are transmitted through transcellular structures. The feedback of local matrix stiffness on cell state likely has important implications for development, differentiation, disease, and regeneration.

Directional cell locomotion is critical in many physiological processes, including morphogenesis, the immune response, and wound healing. It is well known that in these processes cell movements can be guided by gradients of various chemical signals. In this study, we demonstrate that cell movement can also be guided by purely physical interactions at the cell-substrate interface. We cultured National Institutes of Health 3T3 fibroblasts on flexible polyacrylamide sheets coated with type I collagen. A transition in rigidity was introduced in the central region of the sheet by a discontinuity in the concentration of the bis-acrylamide cross-linker. Cells approaching the transition region from the soft side could easily migrate across the boundary, with a concurrent increase in spreading area and traction forces. In contrast, cells migrating from the stiff side turned around or retracted as they reached the boundary. We call this apparent preference for a stiff substrate "durotaxis." In addition to substrate rigidity, we discovered that cell movement could also be guided by manipulating the flexible substrate to produce mechanical strains in the front or rear of a polarized cell. We conclude that changes in tissue rigidity and strain could play an important controlling role in a number of normal and pathological processes involving cell locomotion.

Although myosin II is known to play an important role in cell migration, little is known about its specific functions. We have addressed the function of one of the isoforms of myosin II, myosin IIB, by analyzing the movement and mechanical characteristics of fibroblasts where this protein has been ablated by gene disruption. Myosin IIB null cells displayed multiple unstable and disorganized protrusions, although they were still able to generate a large fraction of traction forces when cultured on flexible polyacrylamide substrates. However, the traction forces were highly disorganized relative to the direction of cell migration. Analysis of cell migration patterns indicated an increase in speed and decrease in persistence, which were likely responsible for the defects in directional movements as demonstrated with Boyden chambers. In addition, unlike control cells, mutant cells failed to respond to mechanical signals such as compressing forces and changes in substrate rigidity. Immunofluorescence staining indicated that myosin IIB was localized preferentially along stress fibers in the interior region of the cell. Our results suggest that myosin IIB is involved not in propelling but in directing the cell movement, by coordinating protrusive activities and stabilizing the cell polarity. INTRODUCTIONThe functional roles of myosin II in nonmuscle cells have been an important topic of investigation. Although its involvement in cytokinesis has been investigated in detail (Robinson and Spudich, 2000), there is also strong evidence that myosin II plays a role in cell migration. For example, although myosin II null mutants of Dictyostelium are capable of migration, they display a lower migration speed and a loss of forward bias in protrusion compared with wild-type cells (Wessels et al., 1988), particularly on surfaces of increased adhesiveness (Jay et al., 1995).Fibroblast migration involves a number of controlled and coordinated processes, including protrusion, adhesion, translocation, and detachment (Lauffenburger and Horwitz, 1996;Mitchison and Cramer, 1996;Sheetz et al., 1998). It is commonly accepted that translocation of the cell body and detachment from the substrate require contractile forces (Jay et al., 1995;Wolenski, 1995;Anderson et al., 1996;Svitkina et al., 1997). Consistent with the idea, treatment of cells with myosin II inhibitors causes relaxation of traction forces and impairment of cell migration (Pelham and Wang, 1999). Equally important is a guidance mechanism in response to environmental cues. In addition to chemotaxis, fibroblasts show profound responses in morphology, traction forces, and motility rates, to physical signals (Pelham and Wang, 1997;Lo et al., 2000). They are also able to steer their migration toward substrates of high rigidity (Lo et al., 2000). Because the detection of such physical characteristics as rigidity cannot be achieved through purely chemical means, the cell must invoke a contractile mechanism that probes the environment. Myosin II may be involved in such a sensing stru...

Fibroblasts in 2D cultures differ dramatically in behavior from those in the 3D environment of a multicellular organism. However, the basis of this disparity is unknown. A key difference is the spatial arrangement of anchored extracellular matrix (ECM) receptors to the ventral surface in 2D cultures and throughout the entire surface in 3D cultures. Therefore, we asked whether changing the topography of ECM receptor anchorage alone could invoke a morphological response. By using polyacrylamide-based substrates to present anchored fibronectin or collagen on dorsal cell surfaces, we found that well spread fibroblasts in 2D cultures quickly changed into a bipolar or stellate morphology similar to fibroblasts in vivo. Cells in this environment lacked lamellipodia and large actin bundles and formed small focal adhesions only near focused sites of protrusion. These responses depend on substrate rigidity, calcium ion, and, likely, the calcium-dependent protease calpain. We suggest that fibroblasts respond to both spatial distribution and mechanical input of anchored ECM receptors. Changes in cell shape may in turn affect diverse cellular activities, including gene expression, growth, and differentiation, as shown in numerous previous studies.adhesion ͉ cell migration ͉ integrin ͉ morphology C ells in the tissues of multicellular organisms show a wide spectrum of sizes and shapes, reflective of their diverse functions. For example, a neuron is highly polarized and elongated, as required for the transmission of information over long distances, whereas epithelial cells, also highly polarized, are generally columnar or cubical in shape, reflective of their barrier functions. It has long been recognized that such variability in cell shape and behavior is coupled to differences in cell growth, differentiation, and other important functions (1, 2).A long-standing question is how the diverse cell shapes are derived. It is generally recognized that chemical signals play an important role in defining the cell shape and migration, as demonstrated by chemotaxis. However, it is also becoming clear that physical and topographical parameters may have equally significant effects (3-5). A dramatic example is the shape change that occurs when a cell is removed from the tissue and is grown on glass or plastic surfaces (6, 7). These 2D cultures, as conveniently used for the maintenance of cells and for biological studies, impose highly unnatural geometric and mechanical constraints to many types of cells (8, 9). Fibroblasts, which are normally bipolar or stellate in shape when embedded in flexible, fibrous networks of the extracellular matrix (ECM), adhere to these stiff, nonpermeable surfaces and adopt a dramatic spread morphology (10).A key aspect that distinguishes fibroblasts in 2D and 3D cultures is the degree and spatial distribution of receptors anchored to the ECM. Integrins, the primary receptors interacting with the ECM, are known to induce the formation of focal adhesions (11,12) and to modulate multiple signaling pathways that ...

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Through association with CDK1, cyclin B accumulation and destruction govern the G2/M/G1 transitions in eukaryotic cells. To identify CDK1 inactivation-dependent events during late mitosis, we expressed a nondestructible form of cyclin B (cyclin BΔ90) by microinjecting its mRNA into prometaphase normal rat kidney cells. The injection inhibited chromosome decondensation and nuclear envelope formation. Chromosome disjunction occurred normally, but anaphase-like movement persisted until the chromosomes reached the cell periphery, whereupon they often somersaulted and returned to the cell center. Injection of rhodamine-tubulin showed that this movement occurred in the absence of a central anaphase spindle. In 82% of cells cytokinesis was inhibited; the remainder split themselves into two parts in a process reminiscent of Dictyostelium cytofission. In all cells injected, F-actin and myosin II were diffusely localized with no detectable organization at the equator. Our results suggest that a primary effect of CDK1 inactivation is on spindle dynamics that regulate chromosome movement and cytokinesis. Prolonged CDK1 activity may prevent cytokinesis through inhibiting midzone microtubule formation, the behavior of proteins such as TD60, or through the phosphorylation of myosin II regulatory light chain.

The migration of vascular endothelial cells in vivo occurs in a fluid dynamic environment due to blood flow, but the role of hemodynamic forces in cell migration is not yet completely understood. Here we investigated the effect of shear stress, the frictional drag of blood flowing over the cell surface, on the migration speed of individual endothelial cells on fibronectin-coated surfaces, as well as the biochemical and biophysical bases underlying this shear effect. Under static conditions, cell migration speed had a bell-shaped relationship with fibronectin concentration. Shear stress significantly increased the migration speed at all fibronectin concentrations tested and shifted the bell-shaped curve upwards. Shear stress also induced the activation of Rho GTPase and increased the traction force exerted by endothelial cells on the underlying substrate, both at the leading edge and the rear, suggesting that shear stress enhances both the frontal forward-pulling force and tail retraction. The inhibition of a Rho-associated kinase, p160ROCK, decreased the traction force and migration speed under both static and shear conditions and eliminated the shear-enhancement of migration speed. Our results indicate that shear stress enhances the migration speed of endothelial cells by modulating the biophysical force of tractions through the biochemical pathway of Rho-p160ROCK.