Fluorescent labeling techniques for investigation of fibronectin fibrillogenesis (labeling fibronectin fibrillogenesis).
ABSTRACT Fibronectin fibrillogenesis is a cell-mediated, step-wise process that converts soluble fibronectin into insoluble fibronectin matrix. The deposition of fibronectin fibrils occurs at specific sites on the cell surface and depends on the unfolding of the fibronectin dimer. Fibronectin matrix provides positional information for cell migration during early embryogenesis and plays an important role in cell growth, differentiation, survival, and oncogenic transformation. Here we present simple techniques, based on the use of fluorescently labeled fibronectin and species-specific antifibronectin antibodies that allow determination of the fibronectin fibril growth in conventional in vitro cell cultures and in three-dimensional matrix environment.
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ABSTRACT: Cellular interactions with extracellular matrices (ECM) through the application of mechanical forces mediate numerous biological processes including developmental morphogenesis, wound healing and cancer metastasis. They also play a key role in the cellular repopulation and/or remodeling of engineered tissues and organs. While 2-D studies can provide important insights into many aspects of cellular mechanobiology, cells reside within 3-D ECMs in vivo, and matrix structure and dimensionality have been shown to impact cell morphology, protein organization and mechanical behavior. Global measurements of cell-induced compaction of 3-D collagen matrices can provide important insights into the regulation of overall cell contractility by various cytokines and signaling pathways. However, to understand how the mechanics of cell spreading, migration, contraction and matrix remodeling are regulated at the molecular level, these processes must also be studied in individual cells. Here we review the evolution and application of techniques for imaging and assessing local cell-matrix mechanical interactions in 3-D culture models, tissue explants and living animals.Experimental Cell Research 06/2013; · 3.56 Impact Factor
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ABSTRACT: Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell-matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy.Experimental Cell Research 10/2012; · 3.56 Impact Factor
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ABSTRACT: Fibronectin (FN), an extracellular matrix (ECM) glycoprotein, is a key factor in the compatibility of dental implant materials. Our objective was to determine the optimal dimensions of microgrooves in the transmucosal part of a dental implant, for optimal absorption of plasma FN and expression of cellular FN by human gingival ﬁbroblasts (HGFs). Microgroove titanium surfaces were fabricated by photolithography with parallel grooves: 15μm, 30μm, or 60μm in width and 5μm or 10μm in depth. Smooth titanium surfaces were used as controls. Surface hydrophilicity, plasma FN adsorption and cellular FN expression by HGFs were measured for both microgroove and control samples. We found that narrower and deeper microgrooves amplified surface hydrophobicity. A 15-μm wide microgroove was the most hydrophobic surface and a 60-μm wide microgroove was the most hydrophilic. The latter had more expression of cellular FN than any other surface, but less absorption of plasma FN than 15-μm wide microgrooves. Variation in microgroove depth did not appear to effect FN absorption or expression unless the groove was narrow (∼15 or 30μm). In those instances, the shallower depths resulted in greater expression of cellular FN. Our microgrooves improved expression of cellular FN, which functionally compensated for plasma FN. A microgroove width of 60μm and depth of 5 or 10μm appears to be optimal for the transmucosal part of the dental implant.Journal of dentistry 08/2013; · 3.20 Impact Factor