Ligation of integrins with extracellular matrix molecules induces the clustering of actin and actin-binding proteins to focal adhesions, which serves to mechanically couple the matrix with the cytoskeleton. During wound healing and development, matrix deposition and remodeling may impart additional tensile forces that modulate integrin-mediated cell functions, including cell migration and proliferation. We have utilized the ability of cells to contract floating collagen gels to determine the effect of fibronectin polymerization on mechanical tension generation by cells. Our data indicate that fibronectin polymerization promotes cell spreading in collagen gels and stimulates cell contractility by a Rho-dependent mechanism. Fibronectin-stimulated contractility was dependent on integrin ligation; however, integrin ligation by fibronectin fragments was not sufficient to induce either tension generation or cell spreading. Furthermore, treatment of cells with polyvalent RGD peptides or pre-polymerized fibronectin did not stimulate cell contractility. Fibronectin-induced contractility was blocked by agents that inhibit fibronectin polymerization, suggesting that the process of fibronectin polymerization is critical in triggering cytoskeletal tension generation. These data indicate that Rho-mediated cell contractility is regulated by the process of fibronectin polymerization and suggest a novel mechanism by which extracellular matrix fibronectin regulates cytoskeletal organization and cell function.Fibronectin is a high molecular mass, multidomain glycoprotein that circulates in a soluble form in the plasma and is also found in an insoluble, multimeric form within the extracellular matrix (1). The polymerization of soluble fibronectin into insoluble fibrils within the extracellular matrix is a dynamic, celldependent process that is mediated by a series of events involving the actin cytoskeleton and integrin receptors (2). The adhesion of cells to fibronectin via integrin receptors has been shown to be important in the regulation and coordination of such complex processes as cell growth, differentiation, and migration (1). Ligation of integrins with extracellular matrix molecules, including fibronectin, induces the clustering of actin and actin-binding proteins to focal adhesions, which serves to mechanically couple the extracellular matrix with the actin cytoskeleton (3). Recent studies suggest that the interaction of cells with the extracellular matrix form of fibronectin triggers changes in cell cycle progression (4 -7), cell migration (8), and actin filament organization (6, 9) that are distinct from those generated by the interaction of cells with nonpolymerized fibronectin. The mechanism by which the matrix form of fibronectin gives rise to cellular phenotypes distinct from protomeric fibronectin is unknown.Studies aimed at understanding the effect of extracellular matrix on cell function indicate that cells respond to fibronectin-coated beads with a local reinforcement of cytoskeletal links that are proportional t...

A simple yet versatile method was developed to prepare a low-density polymerization initiator gradient, which was combined with surface-initiated atom transfer radical polymerization (ATRP) to produce a well-defined poly(2-hydroxyethyl methacrylate) (HEMA) gradient substrate. A smooth variation in film thickness was measured across the gradient, ranging from 20 A to over 80 A, but we observed a nonmonotonic variation in water contact angle. Fits of X-ray reflectivity profiles suggested that at the low graft density end, the polymer chain structure was in a "mushroom" regime, while the polymer chains at high graft density were in a "brush" regime. It was found that the "mushroom" region of the gradient could be made adhesive to cells by adsorbing adhesion proteins, and cell adhesion could be tuned by controlling the density of the polymer grafts. Fibroblasts were seeded on gradients precoated with fibronectin to test cellular responses to this novel substrate, but it was found that cell adhesion did not follow the expected trend; instead, saturated cell adhesion and spreading was found at the low grafting density region.

Dystroglycan is a component of the dystrophin-glycoprotein complex (DGC) in muscle and a cell surface receptor for laminin. Numerous muscular dystrophies are the result of disruption of proteins comprising the DGC, but the underlying pathogenetic mechanisms are unknown. Because apoptosis is an early feature of muscular dystrophy in vivo, and perturbation of cell-extracellular matrix associations is known to induce apoptosis, we investigated the role of dystroglycan-laminin interactions in the propagation and maintenance of cell survival signals in muscle cells. We found that disrupting the interaction between alpha-dystroglycan and the extracellular matrix protein laminin induces apoptosis in muscle cells. This increase in apoptosis is mediated in part by caspase activation and can be blocked by a caspase-3 inhibitor. We demonstrate a role for the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway in muscle cell-survival signaling using a pharmacological inhibitor of PI3K. Treatment with this inhibitor resulted in decreased phosphorylation of AKT and its downstream effector glycogen synthase kinase (GSK)-3beta and induced apoptosis in muscle cell cultures. Disruption of dystroglycan-laminin interactions resulted in decreased phosphorylation of AKT and GSK-3beta. Furthermore, activation of AKT prior to the disruption of dystroglycan-laminin protected the muscle cells from the induction of apoptosis. These results support a role for the PI3K/AKT pathway in the propagation of cell-survival signals mediated by the DGC and provide new insight into the molecular pathogenesis associated with the development of muscular dystrophies.

Clinical applications of precision oncology require accurate tests that can distinguish true cancer specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

Assembly and degradation of fibronectin-containing extracellular matrices are dynamic processes that are up-regulated during wound healing, embryogenesis, and metastasis. Although several of the early steps leading to fibronectin deposition have been identified, the mechanisms leading to the accumulation of fibronectin in disulfide-stabilized multimers are largely unknown. Disulfide-stabilized fibronectin multimers are thought to arise through intra-or intermolecular disulfide exchange. Several proteins involved in disulfide exchange reactions contain the sequence Cys-X-X-Cys in their active sites, including thioredoxin and protein-disulfide isomerase. The twelfth type I module of fibronectin (I 12 ) contains a Cys-X-X-Cys motif, suggesting that fibronectin may have the intrinsic ability to catalyze disulfide bond rearrangement. Using an established protein refolding assay, we demonstrate here that fibronectin has protein-disulfide isomerase activity and that this activity is localized to the carboxyl-terminal type I module I 12 . I 12 was as active on an equal molar basis as intact fibronectin, indicating that most of the protein-disulfide isomerase activity of fibronectin is localized to I 12 . Moreover, the protein-disulfide isomerase activity of fibronectin appears to be partially cryptic since limited proteolysis of I 10 -I 12 increased its isomerase activity and dramatically enhanced the rate of RNase refolding. This is the first demonstration that fibronectin contains protein-disulfide isomerase activity and suggests that cross-linking of fibronectin in the extracellular matrix may be catalyzed by a disulfide isomerase activity contained within the fibronectin molecule.

No abstract

No abstract

Using quantitative fluorescence microscopy in conjunction with a method of gradient substrate assembly established in their group, the authors were able to introduce and measure reproducible changes in cellular morphology and cell density by manipulating polymer grafting density. The mechanism behind this change in cellular behavior was explained by a semiempirical, geometric model that describes the effect of the spatial distribution of the polymer on protein attachment. A 10-fold increase in graft density of poly(2-hydroxyethyl methacrylate) [PHEMA] along the surface of a gradient sample, preexposed to bovine fibronectin, caused a change in the size of fibroblasts on the surface (i.e., cell spreading) from (1238 +/- 704) to (377 +/- 216) microm(2). The results were in quantitative agreement with those obtained on three separate gradient samples. Both cellular response and fibronectin adsorption (as measured via ellipsometry) were found to vary sigmoidally with graft density of PHEMA, demonstrating the high degree of correlation between the two phenomena. A simple, rigid-disk model accounting for the surface coverage of PHEMA was able to predict the amount of adsorbed fibronectin with a correlation coefficient of 0.97. Maximal cell adhesion and cell spreading were found to occur at fibronectin surface densities of 50 and 100 ng/cm(2), respectively. The results demonstrate the role of gradient substrate assembly as a method for quantifying the relationship between protein and cellular response to technologically relevant polymeric materials.