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Rho Kinase Regulation of Fibroblast Migratory Mechanics in Fibrillar Collagen Matrices

Abstract

Migration of activated corneal fibroblasts plays an important role in matrix patterning during embryonic development and wound repopulation following injury or refractive surgery. In this study, we investigate the role of Rho kinase in regulating fibroblast migration mechanics, by modifying a previously described nested collagen matrix model to facilitate dynamic imaging of cell–matrix interactions. Human corneal fibroblasts were cultured in nested matrices with media containing either 1% fetal bovine serum (FBS), or 1% FBS plus the Rho kinase inhibitor Y-27632. Time-lapse DIC imaging of cell and extracellular matrix (ECM) movements was performed for up to 72 h. In addition, static confocal imaging was used to assess 3-D cell morphology and local matrix reorganization. In 1% FBS, significant tractional forces were generated during migration, as indicated by inward displacement and reorganization of collagen in front of cells. When Rho kinase was inhibited, cells became more elongated, and extended dendritic processes into the outer matrix. Interestingly, these dendritic cells were still able to generate tractional forces at their leading edge, whereas cell translocation was substantially reduced. Overall, the data suggests that Rho kinase impacts 3-D fibroblast migration by affecting morphology, polarization, and mechanical coordination between the leading and trailing edges of cells.

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Author information

Correspondence to W. Matthew Petroll.

Additional information

An erratum to this article can be found at http://dx.doi.org/10.1007/s12195-010-0120-4

Electronic supplementary material

Below is the link to the electronic supplementary material.

Time-lapse DIC images of cell migration under 10% FBS culture conditions. Centripetal displacement of the inner matrix due to cellular contraction is observed. Time is displayed as hours:minutes (MOV 1759 kb)

Time-lapse DIC images of cell migration under 1%FBS culture conditions. Time is displayed as days:hours:minutes (MOV 3599 kb)

Time-lapse DIC images of cell migration in media containing 1%FBS plus 10 µM Y-27632. Time is displayed as days:hours:minutes (MOV 3287 kb)

Time-lapse DIC images of a single cell migrating through the outer matrix in 1%FBS plus 10 µM Y-27632. Time is displayed as hours:minutes (MOV 3426 kb)

Supplemental Figure 1. A) 20X confocal maximum intensity projection image of f-actin (green), collagen fibrils and microspheres (red), and PI (blue) following 72 h of culture in 1%FBS plus fasudil (20 µM). B) 63X confocal maximum intensity projection image (10 microns thick) of f-actin (green), collagen fibrils (red), and PI (blue) after 3 days of culture in 1%FBS + fasudil (TIFF 10858 kb)

Movie 1

Time-lapse DIC images of cell migration under 10% FBS culture conditions. Centripetal displacement of the inner matrix due to cellular contraction is observed. Time is displayed as hours:minutes (MOV 1759 kb)

Movie 2

Time-lapse DIC images of cell migration under 1%FBS culture conditions. Time is displayed as days:hours:minutes (MOV 3599 kb)

Movie 3

Time-lapse DIC images of a single cell migrating from the inner to outer matrix in 1%FBS. Time is displayed as hours:minutes (MOV 2382 kb)

Movie 4

Time-lapse DIC images of cell migration in media containing 1%FBS plus 10 µM Y-27632. Time is displayed as days:hours:minutes (MOV 3287 kb)

Movie 5

Time-lapse DIC images of a single cell migrating through the outer matrix in 1%FBS plus 10 µM Y-27632. Time is displayed as hours:minutes (MOV 3426 kb)

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Zhou, C., Petroll, W.M. Rho Kinase Regulation of Fibroblast Migratory Mechanics in Fibrillar Collagen Matrices. Cel. Mol. Bioeng. 3, 76–83 (2010). https://doi.org/10.1007/s12195-010-0106-2

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Keywords

  • Collagen matrix
  • Cell migration
  • Rho kinase
  • Fibroblast