Biomedical subjects

William A Marganski

Publications and source records attributed to William A Marganski.

7 recordsLinked to original sources

PubMed2005

Targeting of a novel Ca+2/calmodulin-dependent protein kinase II is essential for extracellular signal-regulated kinase-mediated signaling in differentiated smooth muscle cells.

Subcellular targeting of kinases controls their activation and access to substrates. Although Ca2+/calmodulin-dependent 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 gamma isoform of CaMKII that contains a potential targeting sequence in its association domain (CaMKIIgamma G-2). Antisense knockdown of CaMKIIgamma G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKIIgamma G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKIIgamma G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKIIgamma G-2 activation, and before CaMKIIgamma 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 CaMKIIgamma G-2 inhibited targeting. Furthermore, prevention of CaMKIIgamma 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.

Actinin↗

PubMed2004

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.

Animals↗

PubMed2004

Direct comparison of the spread area, contractility, and migration of balb/c 3T3 fibroblasts adhered to fibronectin- and RGD-modified substrata.

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.

Adsorption↗

PubMed2003

Demonstration of altered fibroblast contractile activity in hypertensive heart disease.

OBJECTIVE: The aim of this study is to investigate the idea that altered fibroblast contractile activity is involved in the pathogenesis of hypertensive heart disease (HHD). METHODS: Cell area and contraction are quantified using the traction force microscopy technique for cardiac fibroblasts isolated from both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. RESULTS: The data indicate that there are marked phenotypic differences between the two cell types. For instance, WKY fibroblasts exert an average traction stress of approximately 3.3 kPa and have an area of approximately 2640 microm(2). Under identical conditions the SHR fibroblasts have an area approximately 1.45 times larger (p<0.01) and exert an average stress approximately 1.86 times higher (p<0.01). Challenging WKY fibroblasts with 1 micromol/l angiotensin II (Ang II) gradually causes a approximately 2-fold increase in traction after 1 h while simultaneously causing a approximately 28% decrease in area. In contrast, Ang II has no effect on SHR fibroblasts. The data also show that WKY and SHR cells respond in different ways when challenged with irbesartan (Irb). The addition of 1 micromol/l Irb initially causes WKY cells to decrease their average traction output by approximately 50% after approximately 10 min. Subsequently, contractile activity begins to recover and returns to normal after 1 h. The SHR cells also decrease their tractions by approximately 50%, but this decrease requires 30 min for completion and there is no recovery to the initial contractile state. For both cell types, Irb produces no significant effect on area and the combined effect of equimolar Irb and Ang II is the same as Irb alone. CONCLUSION: These in vitro data suggest that among the many factors producing hypertensive heart disease in SHR's are excessive contraction of their cardiac fibroblasts and defective control of fibroblast contraction by Ang II.

Animals↗

PubMed2003

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.

Cell Membrane↗

PubMed2003

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.

Animals↗

PubMed2003

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.

Acrylic Resins↗