Adaptation of cell spreading to varying fibronectin densities and topographies is facilitated by β1 integrins
Enrico Domenico LemmaZhongxiang JiangFranziska KleinTanja LandmannKai WeißenbruchSarah BertelsMarc HipplerBernhard Wehrle‐HallerMartin Bastmeyer
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Abstract:
Cells mechanical behaviour in physiological environments is mediated by interactions with the extracellular matrix (ECM). In particular, cells can adapt their shape according to the availability of ECM proteins, e.g., fibronectin (FN). Several in vitro experiments usually simulate the ECM by functionalizing the surfaces on which cells grow with FN. However, the mechanisms underlying cell spreading on non-uniformly FN-coated two-dimensional substrates are not clarified yet. In this work, we studied cell spreading on variously functionalized substrates: FN was either uniformly distributed or selectively patterned on flat surfaces, to show that A549, BRL, B16 and NIH 3T3 cell lines are able to sense the overall FN binding sites independently of their spatial arrangement. Instead, only the total amount of available FN influences cells spreading area, which positively correlates to the FN density. Immunocytochemical analysis showed that β1 integrin subunits are mainly responsible for this behaviour, as further confirmed by spreading experiments with β1-deficient cells. In the latter case, indeed, cells areas do not show a dependency on the amount of available FN on the substrates. Therefore, we envision for β1 a predominant role in cells for sensing the number of ECM ligands with respect to other focal adhesion proteins.Keywords:
Matrix (chemical analysis)
Fibronectins
Fibronectins isolated fro early-passage and late-passage (in vitro aged) human fibroblasts were shown to differ in their ability to support cell adhesion and to influence cell morphology. Because fibroblast adhesion requires interactions between fibronectin, the cell surface, and the component of the extracellular matrix, we examined those functions in isolated cellular fibronectin. In comparison to fibronectin isolated from early-passage cells, fibronectin from late-passage cells bound poorly to native collagen types I and II. No differences were observed in the binding of the two fibronectins to denatured collagen. The binding of both fibronectins to native collagen was similarly promoted by heparin. Cell binding activity was evaluated by using a Boyden chamber assay to measure chemotaxis in response to either fibronectin. No differences were detected in cell binding. Comparisons of molecular weights by NaDodSO4/polyacrylamide gel electrophoresis reveals that fibronectin from late-passage cells is larger than that from early-passage cells. That difference is observed both in fibronectins isolated from conditioned media and in fibronectins isolated from the cell layer. These data support the hypothesis that late-passage cells produce a structurally and functionally distinct fibronectin. The defective binding to native collagen may account for some aspects of the aged phenotype.
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Abstract Late passage fibroblasts show decreased cell‐substrate adhesion. We provide evidence that the reduced adhesion is due to a defect in the adhesive glycoprotein fibronectin. Late passage cells become more adhesive in culture media that has been conditioned by the growth of early passage cells. Analysis of fibronectins purified from early and late passage cell conditioned media indicates that there are striking differences in their abilities to promote cell adhesion. Young cell fibronectin supports the maximal adhesion of both young and old cells. However, old cells require quantitatively more fibronectin. In contrast, old cell fibronectin is less effective in supporting the adhesion of either cell type. In addition, neither cell type achieves a normal morphology in the presence of old cell fibronectin. The results support the conclusion that the fibronectin released by late passage cells is defective and does not support normal cell‐substrate interactions.
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Abstract Using a previously described model system for the incorporation of plasma fibronectin into the extracellular matrix (McKeown‐Longo, P.J. and Mosher, D.F., 1985. J. Cell Biol., 100 : 364–374), we compared the binding of cell‐derived and plasma‐derived fibronectins to human fibroblast cell layers. Binding was measured in time course experiments using metabolically labeled cell‐derived, iodinated cell‐derived, and iodinated plasma‐derived fibronectins. The kinetics of matrix assembly of cell‐ and plasma‐derived fibronectins were the same. Competitive binding curves using intact fibronectin or the 70‐kD amino‐terminal fragment of fibronectin suggested that cell surface binding sites have equal affinity for cell‐ and plasma‐derived fibronectins. Iodinated fibronectins did not bind to isolated matrices containing collagen type I, fibronectin, and thrombospondin. These results suggest that fibroblasts do not distinguish between cell‐derived and plasma‐derived fibronectins when assembling exogenous fibronectin into extracellular matrix.
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Fibronectins isolated from the conditioned medium produced by cultures of undifferentiated (monolayer) and differentiated (nodular) swine vascular smooth muscle cells are similar but not identical. In general, the nodular-cell fibronectin has a smaller molecular mass than monolayer-cell fibronectin and appears to lack the COOH-terminal interchain disulfide linkage. We studied the incorporation of cellular and plasma fibronectins into the cell layer. Smooth muscle cells bound 2.5 times more monolayer-cell fibronectin than nodular-cell fibronectin. Polypeptide fragments of human plasma fibronectin were used as a model system to investigate fibronectin incorporation into the cell layer. Only intact molecules were incorporated into the cell layer and subsequently organized into fibers. Polypeptide fragments of molecular mass 205 kDa and 185 kDa were not incorporated even though they retained the collagen-, cell-, and heparin-binding regions. Incorporation appears to require an activity associated with either the NH2-terminal or COOH-terminal domains. We propose that fibronectin activity is lost during differentiation of smooth muscle cells.
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Fibronectin molecules are dimers composed of subunits whose primary structures may differ. This is due to alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA. Using two monoclonal antibodies specific for two different epitopes of domain 5 (high affinity for heparin), we have quantitatively analyzed the expression of the IIICS sequence in human fibronectins from different sources. The results demonstrated that the percentage of fibronectin subunits containing the IIICS is higher in fibronectins from tumor-derived or simian virus 40-transformed human cells than in fibronectins from human plasma or normal human fibroblasts. Furthermore, we observed that 45-65% of fibronectin subunits from transformed cells or normal embryonic fibroblasts are sialylated on the heparin-binding domain 5, whereas this occurs in only 24-28% of fibronectin subunits from normal adult fibroblasts. On the contrary, no sialylation was observed on domain 5 in fibronectin from human plasma.
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Fibronectin, a structural glycoprotein on cell surfaces, appears in significant concentrations too in human blood plasma. Fibronectin influences cell interactions and cell growth and is an essential part of the extracellular matrix. The following review summarizes the biochemistry and function of fibronectin--or rather of fibronectins, bearing in mind the difference in protein structure between plasma fibronectin and cell surface fibronectin. Fibronectin is identical with one opsonin (alpha 2-opsonic glycoprotein). During shock and septicemia it undergoes a marked decrease. The diminished concentration in the plasma indicates a reduced function of the reticulo-endothelial system. A report is given on initial substitution trials.
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Fibronectins are a group of closely related proteins that are found in body fluids and tissue extracellular matrices. Fibronectins play important roles in the maintenance of hemostasis and in the organization of developing tissues. The interaction of cells with fibronectin probably involves specific cell-sunface molecules. Two distinct receptors for fibronectin havebeen postulated. One receptor, a 140-kilodalton complex, has been identified in a variety of cell types and is believed to mediate the attachment of cells to fibronectin-coated substrata. The existence of a second receptor has been proposed but it has not been identified. This receptor may function in the assembly of soluble fibronectin into the extracellular matrix. In this paper some of the recent developments in the identification and characterization of fibronectin-binding molecules on the surfaces of eukaryotic cells are outlined.
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