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

Areum Kim, Neema Lakshman, W. Matthew Petroll

Research output: Contribution to journalArticle

87 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3683-3692
Number of pages10
JournalExperimental Cell Research
Volume312
Issue number18
DOIs
StatePublished - Nov 1 2006

Keywords

  • Actin
  • Cell mechanics
  • Collagen matrices
  • Confocal microscopy
  • Corneal fibroblasts
  • Cytoskeleton
  • Fourier transform
  • Myosin
  • Rho kinase

ASJC Scopus subject areas

  • Cell Biology

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