Quantitative assessment of local collagen matrix remodeling in 3-D Culture: The role of Rho kinase

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• 作者：Areum Kim ; Neema Lakshman and W. Matthew Petroll
• 关键词：Collagen matrices ; Rho kinase ; Corneal fibroblasts ; Cell mechanics ; Confocal microscopy ; Cytoskeleton ; Actin ; Myosin ; Fourier transform
• 刊名：Experimental Cell Research
• 出版年：2006
• 期刊代码：292_00144827
• 类别：med
• 出版时间：1 November 2006
• 卷：312
• 期：18
• 页码：3683-3692
• 文件大小：1213 K

摘要

The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling.

Human corneal fibroblasts were plated inside 100-μm thick fibrillar collagen matrices and cultured for 24 h in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph.

Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase (p < 0.001). Fibroblasts generally had a bipolar morphology and intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. When Rho kinase was inhibited, cells had a more cortical f-actin distribution and dendritic morphology. Both local collagen fibril density and alignment were significantly reduced (p < 0.01).

Overall, the data suggests that Rho kinase-dependent contractile force generation leads to co-alignment of cells and collagen fibrils along the plane of greatest resistance, and that this process contributes to global matrix contraction.