Dataset Information

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 hours. 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.

SUBMITTER: Zhou C

PROVIDER: S-EPMC2995278 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Rho Kinase Regulation of Fibroblast Migratory Mechanics in Fibrillar Collagen Matrices.

Zhou Chengxin C Petroll W Matthew WM

Cellular and molecular bioengineering 20100301 1

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 b ...[more]

PMID: 21132118

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Project description:Previous studies have shown that platelet derived growth factor (PDGF) can stimulate corneal keratocyte spreading and migration within 3-D collagen matrices, without inducing transformation to a contractile, fibroblastic phenotype. The goal of this study was to investigate the role of matrix metalloproteinases (MMPs) in regulating PDGF-induced changes in keratocyte motility and mechanical differentiation. Rabbit corneal keratocytes were isolated and cultured in serum-free media (S-) to maintain their quiescent phenotype. A nested collagen matrix construct was used to assess 3-D cell migration, and a standard collagen matrix model was used to assess cell morphology and cell-mediated matrix contraction. In both cases constructs were cultured in S- supplemented with PDGF, with or without the broad spectrum MMP inhibitors GM6001 or BB-94. After 4 days, f-actin, nuclei and collagen fibrils were imaged using confocal microscopy. To assess sub-cellular mechanical activity (extension and retraction of cell processes), time-lapse DIC imaging was also performed. MT1-MMP expression and MMP-mediated collagen degradation were also examined. Results demonstrated that neither GM6001 nor BB-94 affected corneal keratocyte viability or proliferation in 3-D culture. PDGF stimulated elongation and migration of corneal keratocytes within type I collagen matrices, without causing a loss of their dendritic morphology or inducing formation of intracellular stress fibers. Treatment with GM6001 and BB-94 inhibited PDGF-induced keratocyte spreading and migration. Relatively low levels of keratocyte-induced matrix contraction were also maintained in PDGF, and the amount of PDGF-induced collagen degradation was similar to that observed in S- controls. The collagen degradation pattern was consistent with membrane-associated MMP activity, and keratocytes showed positive staining for MT1-MMP, albeit weak. Both matrix contraction and collagen degradation were reduced by MMP inhibition. For most outcome measures, the inhibitory effect of BB-94 was significantly greater than that of GM6001. Overall, the data demonstrate for the first time that even under conditions in which low levels of contractility and extracellular matrix proteolysis are maintained, MMPs still play an important role in mediating cell spreading and migration within 3-D collagen matrices. This appears to be mediated at least in part by membrane-tethered MMPs, such as MT1-MMP.

Project description:Cell motility under physiological and pathological conditions including malignant progression of cancer and subsequent metastasis are founded on environmental confinements. During the last two decades, three-dimensional cell migration has been studied mostly by utilizing biomimetic extracellular matrix models. In the majority of these studies, the in vitro collagen scaffolds are usually assumed to be homogenous, as they consist commonly of one specific type of collagen, such as collagen type I, isolated from one species. These collagen matrices should resemble in vivo extracellular matrix scaffolds physiologically, however, mechanical phenotype and functional reliability have been addressed poorly due to certain limitations based on the assumption of homogeneity. How local variations of extracellular matrix structure impact matrix mechanics and cell migration is largely unknown. Here, we hypothesize that local inhomogeneities alter cell movement due to alterations in matrix mechanics, as they frequently occur in in vivo tissue scaffolds and were even changed in diseased tissues. To analyze the effect of structural inhomogeneities on cell migration, we used a mixture of rat tail and bovine dermal collagen type I as well as pure rat and pure bovine collagens at four different concentrations to assess three-dimensional scaffold inhomogeneities. Collagen type I from rat self-assembled to elongated fibrils, whereas bovine collagen tended to build node-shaped inhomogeneous scaffolds. We have shown that the elastic modulus determined with atomic force microscopy in combination with pore size analysis using confocal laser scanning microscopy revealed distinct inhomogeneities within collagen matrices. We hypothesized that elastic modulus and pore size govern cancer cell invasion in three-dimensional collagen matrices. In fact, invasiveness of three breast cancer cell types is altered due to matrix-type and concentration indicating that these two factors are crucial for cellular invasiveness. Our findings revealed that local matrix scaffold inhomogeneity is another crucial parameter to explain differences in cell migration, which not solely depended on pore size and stiffness of the collagen matrices. With these three distinct biophysical parameters, characterizing structure and mechanics of the studied collagen matrices, we were able to explain differences in the invasion behavior of the studied cancer cell lines in dependence of the used collagen model.

Dataset Information

Rho Kinase Regulation of Fibroblast Migratory Mechanics in Fibrillar Collagen Matrices.

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Rho Kinase Regulation of Fibroblast Migratory Mechanics in Fibrillar Collagen Matrices.

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