Fibronectin fragmentation promotes alpha4beta1 integrin-mediated contraction of a fibrin-fibronectin provisional matrix.
ABSTRACT In injured tissues, the fibrin-fibronectin (FN) provisional matrix provides a framework for cell adhesion, migration, and repair. Effective repair and remodeling require a proper balance between extracellular matrix (ECM) deposition, contraction, and turnover. We utilized a three-dimensional (3D) fibrin-FN provisional matrix model to determine the contributions of the FN-binding integrin receptors alpha5beta1 and alpha4beta1 to matrix contraction. CHOalpha5 cells expressing alpha5beta1, a receptor for FN's RGD cell-binding domain, were highly contractile, and cells were well spread on a 3D fibrin-FN matrix. In contrast, CHOalpha4 cells expressing the alpha4beta1 receptor for FN's alternatively spliced V region attached less efficiently to FN and were deficient in fibrin-FN matrix contraction. Surprisingly, cell adhesion and matrix contraction by CHOalpha4 cells were dramatically enhanced, to levels equivalent to CHOalpha5 cells, when proteolyzed FN was used in place of intact FN in the fibrin-FN matrix. Similar enhancement was observed when ligand binding by alpha4beta1 integrins was activated by treatment with Mn(++), but not by stimulation of actin organization with LPA. Therefore, alpha4beta1-dependent cell responses to the provisional matrix are modulated by cleavage of matrix components.
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ABSTRACT: Branched actin assembly is critical for both cell motility and membrane trafficking. The branched actin regulator cortactin is generally considered to promote cell migration by controlling leading-edge lamellipodial dynamics. However, recent reports indicate that lamellipodia are not required for cell movement, suggesting an alternate mechanism. Because cortactin also regulates membrane trafficking and adhesion dynamics, we hypothesized that altered secretion of extracellular matrix (ECM) and/or integrin trafficking might underlie motility defects of cortactin-knockdown (KD) cells. Consistent with a primary defect in ECM secretion, both motility and lamellipodial defects of cortactin-KD cells were fully rescued by plating on increasing concentrations of exogenous ECM. Furthermore, cortactin-KD cell speed defects were rescued on cell-free autocrine ECM produced by control cells, but not on ECM produced by cortactin-KD cells. Investigation of the mechanism revealed that whereas endocytosed fibronectin (FN) is redeposited at the basal cell surface by control cells, cortactin-KD cells exhibit defective FN secretion and abnormal FN retention in a late endocytic/lysosomal compartment. Cortactin-KD motility and FN deposition defects were phenocopied by KD in control cells of the lysosomal fusion regulator synaptotagmin-7. Rescue of cortactin-KD cells by expression of cortactin-binding domain mutants revealed that interaction with the Arp2/3 complex and actin filaments is essential for rescue of both cell motility and autocrine ECM secretion phenotypes, whereas binding of SH3-domain partners is not required. Efficient cell motility, promoted by cortactin regulation of branched actin networks, involves processing and resecretion of internalized ECM from a late endosomal/lysosomal compartment.Current biology: CB 08/2011; 21(17):1460-9. · 10.99 Impact Factor