William A. Marganski

Influence of Type I Collagen Surface Density on Fibroblast Spreading, Motility, and Contractility

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.

Rho Mediates the Shear-Enhancement of Endothelial Cell Migration and Traction Force Generation

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.

The Mechanics of Neutrophils: Synthetic Modeling of Three Experiments

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.

Measurements of cell-generated deformations on flexible substrata using correlation-based optical flow.

The optical flow algorithm presented here is a robust method that rapidly yields a high-density field of substrate displacement vectors based on two optical images. We found that one of the limiting factors, at least for inexperienced experimentalists, is the consistency of focusing or the drift in microscope focus. However, with properly collected images the standard error of the measurement was estimated to be on the order of +/- 0.10 pixels. Finally, although the discussion has been focused on the displacement of flexible substrata, a similar method should be applicable for detecting movements on other types of images, as long as the movement involves a certain degree of local coordination.

Traction Stresses and Morphology of 3T3 Fibroblast Cells on Fibronectin-versus RGD- Modified Elastic Substrata

We report a study on cellular traction forces and morphology on fibronectin (FN)- and Arg-Gly-Asp (RGD) modified substrata. We have focused on fibronectin- and RGD- modified substrata because RGD is a primary cell-binding site on fibronectin. In this study we report the traction stresses exerted by NIH (National Institutes of Health) 3T3 fibroblasts on model polyacrylamide hydrogel substrates using the elastic substrata technique. At equal values of input concentration of fibronectin and RGD we find that the values forprojected cell area are similar. However, a significant difference is observed in the traction forces exerted by fibroblasts on fibronectin- compared to RGD-modified surfaces. At equal values of input concentration, the average force exerted by NIH 3T3 fibroblasts is approximately ten fold higher on fibronectin-modified surfaces in comparison to RGD-modified surfaces.

Effects of angiotensin II on cardiac fibroblast contractility

Angiotensin II is known to mediate cardiac remodeling during hypertension by promoting fibrosis and myocyte hypertrophy. This study investigates the effects of angiotensin II on the contractility of the cardiac fibroblast. Using a version of the elastic substratum method, angiotensin II at 1 /spl mu/M was shown to increase the average traction stress exerted by the cardiac fibroblast up to 75% after one hour of stimulation. The enhanced contractility caused by angiotensin II was eliminated by 1 /spl mu/M of irbesartan which is a competitive blocker of the AT/sub 1/-receptor. Irbesartan not only inhibited the effects of angiotensin II, it actually decreased traction compared to untreated controls. This indicates that the effects of angiotensin II on contractility are mediated at least in part by the AT/sub 1/-receptor. Although cardiac fibroblasts are a minor component of the myocardium, a chronic increase in their contractility could be sufficient to increase resistance to ventricular relaxation during diastole, and thereby negatively affect the end-diastolic filling capacity.