Although many of the regulators of actin assembly are known, we do not understand how these components act together to organize cell shape and movement. To address this question, we analyzed the spatial dynamics of a key actin regulator—the Scar/WAVE complex—which plays an important role in regulating cell shape in both metazoans and plants. We have recently discovered that the Hem-1/Nap1 component of the Scar/WAVE complex localizes to propagating waves that appear to organize the leading edge of a motile immune cell, the human neutrophil. Actin is both an output and input to the Scar/WAVE complex: the complex stimulates actin assembly, and actin polymer is also required to remove the complex from the membrane. These reciprocal interactions appear to generate propagated waves of actin nucleation that exhibit many of the properties of morphogenesis in motile cells, such as the ability of cells to flow around barriers and the intricate spatial organization of protrusion at the leading edge. We propose that cell motility results from the collective behavior of multiple self-organizing waves.

We examine the relationships of three variables (projected area, migration speed, and traction force) at various type I collagen surface densities in a population of fibroblasts. We observe that cell area is initially an increasing function of ligand density, but that above a certain transition level, increases in surface collagen cause cell area to decline. The threshold collagen density that separates these two qualitatively different regimes, approximately 160 molecules/ microm(2), is approximately equal to the cell surface density of integrin molecules. These results suggest a model in which collagen density induces a qualitative transition in the fundamental way that fibroblasts interact with the substrate. At low density, the availability of collagen binding sites is limiting and the cells simply try to flatten as much as possible by pulling on the few available sites as hard as they can. The force per bond under these conditions approaches 100 pN, approximately equal to the force required for rupture of integrin-peptide bonds. In contrast, at high collagen density adhesion, traction force and motility are limited by the availability of free integrins on the cell surface since so many of these receptors are bound to the surface ligand and the force per bond is very low.

Much experimental data exist on the mechanical properties of neutrophils, but so far, they have mostly been approached within the framework of liquid droplet models. This has two main drawbacks: 1), It treats the cytoplasm as a single phase when in reality, it is a composite of cytosol and cytoskeleton; and 2), It does not address the problem of active neutrophil deformation and force generation. To fill these lacunae, we develop here a comprehensive continuum-mechanical paradigm of the neutrophil that includes proper treatment of the membrane, cytosol, and cytoskeleton components. We further introduce two models of active force production: a cytoskeletal swelling force and a polymerization force. Armed with these tools, we present computer simulations of three classic experiments: the passive aspiration of a neutrophil into a micropipette, the active extension of a pseudopod by a neutrophil exposed to a local stimulus, and the crawling of a neutrophil inside a micropipette toward a chemoattractant against a varying counterpressure. Principal results include: 1), Membrane cortical tension is a global property of the neutrophil that is affected by local area-increasing shape changes. We argue that there exists an area dilation viscosity caused by the work of unfurling membrane-storing wrinkles and that this viscosity is responsible for much of the regulation of neutrophil deformation. 2), If there is no swelling force of the cytoskeleton, then it must be endowed with a strong cohesive elasticity to prevent phase separation from the cytosol during vigorous suction into a capillary tube. 3), We find that both swelling and polymerization force models are able to provide a unifying fit to the experimental data for the three experiments. However, force production required in the polymerization model is beyond what is expected from a simple short-range Brownian ratchet model. 4), It appears that, in the crawling of neutrophils or other amoeboid cells inside a micropipette, measurement of velocity versus counterpressure curves could provide a determination of whether cytoskeleton-to-cytoskeleton interactions (such as swelling) or cytoskeleton-to-membrane interactions (such as polymerization force) are predominantly responsible for active protrusion.

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.

Native proteins are often substituted by short peptide sequences. These peptides can recapitulate key, but not all biofunctional properties of the native proteins. Here, we quantify the similarities and differences in spread area, contractile activity, and migration speed for balb/c 3T3 fibroblasts adhered to fibronectin- (FN) and Arg-Gly-Asp (RGD)-modified substrata of varying surface density. In both cases spread area has a biphasic dependence on surface ligand density (sigma) with a maximum at sigma approximately 200 molecules/microm2, whereas the total traction force increases and reaches a plateau as a function of sigma. In addition to these qualitative similarities, there are significant quantitative differences between fibroblasts adhered to FN and RGD. For example, fibroblasts on FN have a spread area that is on average greater by approximately 200 microm2 over a approximately 40-fold change in sigma. In addition, fibroblasts on FN exert approximately 3-5 times more total force, which reaches a maximum at a value of sigma approximately 5 times less than for cells adhered to RGD. The data also indicate that the differences in traction are not simply a function of the degree of spreading. In fact, fibroblasts on FN (sigma approximately 2000 microm(-2)) and RGD (sigma approximately 200 microm(-2)) have both similar spread area (approximately 600 microm2) and migration speed (approximately 11 microm/h), yet the total force production is five times higher on FN than RGD (approximately 0.05 dyn compared to approximately 0.01 dyn). Thus, the specific interactions between fibroblasts and FN molecules must inherently allow for higher traction force generation in comparison to the interactions between fibroblasts and RGD.

Abstract-Subcellular targeting of kinases controls their activation and access to substrates. Although Ca 2ϩ /calmodulindependent protein kinase II (CaMKII) is known to regulate differentiated smooth muscle cell (dSMC) contractility, the importance of targeting in this regulation is not clear. The present study investigated the function in dSMCs of a novel variant of the ␥ isoform of CaMKII that contains a potential targeting sequence in its association domain . Antisense knockdown of CaMKII␥ G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKII␥ G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKII␥ G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKII␥ G-2 activation, and before CaMKII␥ G-2 translocation, vimentin is phosphorylated at a CaMKII-specific site. Differential centrifugation demonstrated that phosphorylation of vimentin in dSMCs is not sufficient to cause its disassembly, in contrast to results in cultured cells. Loading dSMCs with a decoy peptide containing the polyproline sequence within the association domain of CaMKII␥ G-2 inhibited targeting. Furthermore, prevention of CaMKII␥ G-2 targeting led to significant inhibition of ERK activation as well as contractility. Thus, for the first time, this study demonstrates the importance of CaMKII targeting in dSMC signaling and identifies a novel targeting function for the association domain in addition to its known role in oligomerization. Key Words: CaMKII Ⅲ smooth muscle Ⅲ contractility Ⅲ targeting Ⅲ extracellular signal-regulated kinase C a 2ϩ /calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase that is expressed as 4 different isoforms (␣, ␤, ␥, and ␦). Each member of this kinase family possesses 3 major domains ( Figure 1A): an N-terminal catalytic/regulatory domain containing a Ser/Thr kinase region and overlapping autoinhibitory and Ca 2ϩ /calmodulin binding regions, a central-linker domain containing variable regions, and a C-terminal association domain that promotes oligomerization. 1 CaMKII is not active until Ca 2ϩ /calmodulin binds to its regulatory domain. This binding allows CaMKII to undergo autophosphorylation at Thr287 (numbering according to the ␥ isoform), which, in turn, increases its affinity for Ca 2ϩ /calmodulin and allows kinase activity even in the absence of Ca 2ϩ /calmodulin. The ␣ and ␤ isoforms of CaMKII are predominantly expressed in neural tissue, 2 where they have been demonstrated to be involved in synaptic plasticity, memory, and learning. 3,4 In contrast, differentiated smooth muscle cells (dSMCs) express mainly the ␥ isoform of CaMKII, 5 which has been linked to contractile activity by the use of antisense and phar...

The coordination of protrusion with retraction is essential for continuous cell movement. In fish keratocytes the activation of stretch-activated calcium channels, and the resulting increase in intracellular calcium, trigger release of the rear cell margin when forward movement is impeded. Although it is likely that retraction involves a calcium-dependent increase in cytoskeletal contractility, it is not known how the timing, magnitude and localization of contractile forces are organized during retraction. We have addressed this question using a new gelatin traction force assay in combination with calcium imaging to determine what changes in cytoskeletal force production accompany calcium-induced retraction. We find that individual calcium transients are followed within seconds by a rapid increase in traction stress that is maintained, or increases in a stepwise manner, until retraction occurs. Increases in traction stress are accompanied by a distinct sequence of changes in the spatial distribution of large traction stresses. Regions of increased traction stress enlarge at the lateral cell margins and expand forward along the cell margin. In particular, rearward facing propulsive' tractions at the leading edge of the cell, which are normally very low, increase several fold. Following retraction, a precipitous drop in traction stress is observed. Such distinct variations in traction stress are not observed in cells when calcium transients are absent. These results suggest a mechanism by which global increases in intracellular calcium can locally regulate contractile force production, in order to maintain a rapid highly directed mode of movement.

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