Yu-li Wang

Tissue Cells Feel and Respond to the Stiffness of Their Substrate

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 babys 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.

Stresses at the Cell-to-Substrate Interface during Locomotion of Fibroblasts

Recent technological improvements in the elastic substrate method make it possible to produce spatially resolved measurements of the tractions exerted by single motile cells. In this study we have applied these developments to produce maps of the tractions exerted by 3T3 fibroblasts during steady locomotion. The resulting images have a spatial resolution of approximately 5 micrometers and a maximum intensity of approximately 10(2) kdyn/cm2 (10(4) pN/micrometers2). We find that the propulsive thrust for fibroblast locomotion, approximately 0.2 dyn, is imparted to the substratum within 15 micrometers of the leading edge. These observations demonstrate that the lamellipodium of the fibroblast is able to generate intense traction stress. The cell body and posterior seem to be mechanically passive structures pulled forward entirely by this action.

A role for the lissencephaly gene LIS1 in mitosis and cytoplasmic dynein function

Mutations in the LIS1 gene cause gross histological disorganization of the developing human brain, resulting in a brain surface that is almost smooth. Here we show that LIS1 protein co-immunoprecipitates with cytoplasmic dynein and dynactin, and localizes to the cell cortex and to mitotic kinetochores, which are known sites for binding of cytoplasmic dynein. Overexpression of LIS1 in cultured mammalian cells interferes with mitotic progression and leads to spindle misorientation. Injection of anti-LIS1 antibody interferes with attachment of chromosomes to the metaphase plate, and leads to chromosome loss. We conclude that LIS1 participates in a subset of dynein functions, and may regulate the division of neuronal progenitor cells in the developing brain.

Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts.

Mechanical interactions between cell and substrate are involved in vital cellular functions from migration to signal transduction. A newly developed technique, traction force microscopy, makes it possible to visualize the dynamic characteristics of mechanical forces exerted by fibroblasts, including the magnitude, direction, and shear. In the present study such analysis is applied to migrating normal and transformed 3T3 cells. For normal cells, the lamellipodium provides almost all the forces for forward locomotion. A zone of high shear separates the lamellipodium from the cell body, suggesting that they are mechanically distinct entities. Timing and distribution of tractions at the leading edge bear no apparent relationship to local protrusive activities. However, changes in the pattern of traction forces often precede changes in the direction of migration. These observations suggest a frontal towing mechanism for cell migration, where dynamic traction forces at the leading edge actively pull the cell body forward. For H-ras transformed cells, pockets of weak, transient traction scatter among small pseudopods and appear to act against one another. The shear pattern suggests multiple disorganized mechanical domains. The weak, poorly coordinated traction forces, coupled with weak cell-substrate adhesions, are likely responsible for the abnormal motile behavior of H-ras transformed cells.

Focal adhesion kinase is involved in mechanosensing during fibroblast migration

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase localized at focal adhesions and is believed to mediate adhesion-stimulated effects. Although ablation of FAK impairs cell movement, it is not clear whether FAK might be involved in the guidance of cell migration, a role consistent with its putative regulatory function. We have transfected FAK-null fibroblasts with FAK gene under the control of the tetracycline repression system. Cells were cultured on flexible polyacrylamide substrates for the detection of traction forces and the application of mechanical stimulation. Compared with control cells expressing wild-type FAK, FAK-null cells showed a decrease in migration speed and directional persistence. In addition, whereas FAK-expressing cells responded to exerted forces by reorienting their movements and forming prominent focal adhesions, FAK-null cells failed to show such responses. Furthermore, FAK-null cells showed impaired responses to decreases in substrate flexibility, which causes control cells to generate weaker traction forces and migrate away from soft substrates. Cells expressing Y397F FAK, which cannot be phosphorylated at a key tyrosine site, showed similar defects in migration pattern and force-induced reorientation as did FAK-null cells. However, other aspects of F397-FAK cells, including the responses to substrate flexibility and the amplification of focal adhesions upon mechanical stimulation, were similar to that of control cells. Our results suggest that FAK plays an important role in the response of migrating cells to mechanical input. In addition, phosphorylation at Tyr-397 is required for some, but not all, of the functions of FAK in cell migration.

Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells.

velocity drops to ~86 nm/sec and individual steps become apparent. The mean size of steps may be determined by taking the power spectrum of the autocorrelation function of the record.18,31,36This spectrum (Fig. 7B, inset) displays a prominent peak at a spatial frequency of 0.121 nm -1, corresponding to a step size of 8.26 nm for this particular record. This value-which involves no elastic corrections-is identical, within experimental error, to the kinesin step size of 8.3 :::t::0.2 nm estimated in earlier work using a fixed trap, to which a 19% adjustment for series compliance had been applied.18 The force clamp enables us, for the first time, to acquire stepping data for kinesin molecules subjected to high loads over distances as great as 200-300 nm, a feat previously impossible with a stationary trap.

Responses of fibroblasts to anchorage of dorsal extracellular matrix receptors

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.

Flexible substrata for the detection of cellular traction forces

By modulating adhesion signaling and cytoskeletal organization, mechanical forces play an important role in various cellular functions, from propelling cell migration to mediating communication between cells. Recent developments have resulted in several new approaches for the detection, analysis and visualization of mechanical forces generated by cultured cells. Combining these methods with other approaches, such as green-fluorescent protein (GFP) imaging and gene manipulation, proves to be particularly powerful for analyzing the interplay between extracellular physical forces and intracellular chemical events.

The regulation of traction force in relation to cell shape and focal adhesions.

Mechanical forces provide critical inputs for proper cellular functions. The interplay between the generation of, and response to, mechanical forces regulate such cellular processes as differentiation, proliferation, and migration. We postulate that adherent cells respond to a number of physical and topographical factors, including cell size and shape, by detecting the magnitude and/or distribution of traction forces under different conditions. To address this possibility we introduce a new simple method for precise micropatterning of hydrogels, and then apply the technique to systematically investigate the relationship between cell geometry, focal adhesions, and traction forces in cells with a series of spread areas and aspect ratios. Contrary to previous findings, we find that traction force is not determined primarily by the cell spreading area but by the distance from cell center to the perimeter. This distance in turn controls traction forces by regulating the size of focal adhesions, such that constraining the size of focal adhesions by micropatterning can override the effect of geometry. We propose that the responses of traction forces to center-periphery distance, possibly through a positive feedback mechanism that regulates focal adhesions, provide the cell with the information on its own shape and size. A similar positive feedback control may allow cells to respond to a variety of physical or topographical signals via a unified mechanism.

Cortical actin turnover during cytokinesis requires myosin II

The involvement of myosin II in cytokinesis has been demonstrated with microinjection, genetic, and pharmacological approaches; however, the exact role of myosin II in cell division remains poorly understood. To address this question, we treated dividing normal rat kidney (NRK) cells with blebbistatin, a potent inhibitor of the nonmuscle myosin II ATPase. Blebbistatin caused a strong inhibition of cytokinesis but no detectable effect on the equatorial localization of actin or myosin. However, whereas these filaments dissociated from the equator in control cells during late cytokinesis, they persisted in blebbistatin-treated cells over an extended period of time. The accumulation of equatorial actin was caused by the inhibition of actin filament turnover, as suggested by a 2-fold increase in recovery half-time after fluorescence photobleaching. Local release of blebbistatin at the equator caused localized accumulation of equatorial actin and inhibition of cytokinesis, consistent with the function of myosin II along the furrow. However, treatment of the polar region also caused a high frequency of abnormal cytokinesis, suggesting that myosin II may play a second, global role. Our observations indicate that myosin II ATPase is not required for the assembly of equatorial cortex during cytokinesis but is essential for its subsequent turnover and remodeling.