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Miguel Miron Mendoza, Eric Graham, Matthew Petroll; Fibronectin as a Regulator of Collective Migration and Clustering of Corneal Fibroblasts in 3D Fibrin Matrices. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2043.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported that cells interacting with collagen matrices develop dendritic processes and move independently, whereas cells interacting with fibrin matrices develop stress fibers, move more slowly and form an interconnected meshwork. In this study, we investigate the role of fibronectin in mediating fibrin-induced collective cell migration and clustering using multidimensional time-lapse imaging.
To assess cell spreading, corneal fibroblasts were plated on top of fibrillar collagen or fibrin matrices. To assess 3-D cell migration, compacted cell-populated collagen matrices were embedded inside cell-free fibrin matrices. Some experiments had fluorescent fibronectin in media to allow direct assessment of cell-induced fibronectin reorganization. Constructs were cultured in serum-free media containing PDGF, with or without the RGD peptide or alpha5beta 1 antibody (to block cell-fibronectin binding). 3-D and 4-D imaging were used to assess cell mechanical behavior, connectivity, f-actin organization, and extracellular fibronectin.
Corneal fibroblasts migrating into 3-D fibrin matrices extended into the fibrin while maintaining connection to cells in the inner matrix at the rear. Other cells followed along the same paths, producing long lines of interconnected cells. As migration into fibrin proceeded, adjacent cells having lateral protrusions became interconnected, resulting in formation of a mesh-like structure. Fluorescent labeling demonstrated that this meshwork was lined with fibronectin, as were the junctions between cells. Fibroblasts migrated through these tube-like fibronectin structures one after the other. In some cases, when cells become temporarily separated, residual FN was found in areas where cells were no longer present. Interfering with fibronectin attachment using RGD peptide or alpha5beta1 antibody blocked cell attachment and spreading.
Since fibronectin facilitates cell adhesion to fibrin, we propose a model in which leading edge cells secrete fibronectin to facilitate binding to fibrin ECM during migration. The secreted fibronectin is reorganized, and tracks or conduits are formed. Other cells prefer to stay within these fibronectin conduits, resulting in interconnected lines of cells. Fibronectin may also play a role in mediating cell-cell adhesion, by serving as a bridge for integrin-integrin interactions between cells.
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