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N. Lakshman, A. Kim, L. Ma, K.J. Bayless, G.E. Davis, W.M. Petroll; Rho Plays a Central Role in Fibroblast Mediated Matrix Contraction and Alignment in 3–D Culture . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2988.
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© ARVO (1962-2015); The Authors (2016-present)
To assess quantitatively the role of Rho on the pattern and amount of cell–induced matrix reorganization in 3–D culture.
Human corneal fibroblasts were infected with adenoviruses that express green fluorescent protein (GFP) or GFP–N19Rho (dominant negative Rho). One day later these cells were plated at low densities inside 100 µm thick type I collagen matrices and allowed to spread for 24 hours. Cells were fixed and f–actin was labeled using Alexa Fluor 546 phalloidin. Fluorescent (for f–actin) and reflected light (for collagen fibrils) 3–D optical section images were acquired using laser confocal microscopy. Fourier transform (FT) analysis was used to assess local collagen fibril alignment, and changes in cell morphology and collagen density were measured using MetaMorph. Decrease in matrix height was used as an indicator of global matrix contraction.
Culture in S+ induced significant global matrix contraction by GFP control cells; this was inhibited by N19Rho (32.1 + 14.1% vs. 48.6 + 0.6%, p < 0.05). Local collagen fibril density was greatest at the ends of cells in GFP S+, and these fibrils were aligned nearly parallel to the pseudopodial tips (|Δθ| = 8.4 ± 4.8°), where Δθ is the difference between collagen and pseudopodial alignment measured from FT analysis. In contrast, fibrils were aligned more perpendicular to the cell axis along the cell body (|Δθ| = 59.0 ± 15.1°, p<0.05). Fibrils were more randomly oriented at the ends of cells expressing N19Rho, as indicated by a decrease in the FT–derived Orientation Index (12.6 ± 7.4% vs. 30.9 ± 9.2%, p < 0.05). Cells expressing N19Rho had fewer stress fibers and longer, thinner extensions than controls; the height of the cells (projection along z–axis) was also increased (27.1 + 9.8 µm vs. 14.9 + 4.1 µm, p < 0.05).
Rho plays a key role in regulating both the morphology and mechanical behavior of corneal fibroblasts in 3–D culture. Rho–dependent force generation leads to compaction of collagen fibrils into specific patterns of alignment, and this process contributes to global matrix contraction.
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