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Form and function in cell motility: from fibroblasts to keratocytes.

ABSTRACT: It is plain enough that a horse is made for running, but similar statements about motile cells are not so obvious. Here the basis for structure-function relations in cell motility is explored by application of a new computational technique that allows realistic three-dimensional simulations of cells migrating on flat substrata. With this approach, some cyber cells spontaneously display the classic irregular protrusion cycles and handmirror morphology of a crawling fibroblast, and others the steady gliding motility and crescent morphology of a fish keratocyte. The keratocyte motif is caused by optimal recycling of the cytoskeleton from the back to the front so that more of the periphery can be devoted to protrusion. These calculations are a step toward bridging the gap between the integrated mechanics and biophysics of whole cells and the microscopic molecular biology of cytoskeletal components.

SUBMITTER: Herant M

PROVIDER: S-EPMC2856179 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Form and function in cell motility: from fibroblasts to keratocytes.

Herant Marc M Dembo Micah M

Biophysical journal 20100401 8

It is plain enough that a horse is made for running, but similar statements about motile cells are not so obvious. Here the basis for structure-function relations in cell motility is explored by application of a new computational technique that allows realistic three-dimensional simulations of cells migrating on flat substrata. With this approach, some cyber cells spontaneously display the classic irregular protrusion cycles and handmirror morphology of a crawling fibroblast, and others the stea ...[more]

PMID: 20409459

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Project description:Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down-regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation is involved in the mechanism of down-regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-beta1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for Western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum-free defined medium, FGF-2 and TGF-beta1 were hypermethylated. Down-regulation of maspin synthesis was also associated with histone H3 dimethylation at lysine 9. Both maspin mRNA and protein were re-expressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down-regulation of maspin synthesis in corneal stromal cells and suggest regulation of genes upon conversion of keratocytes to wound healing fibroblasts can involve promoter and histone methylation.

Dataset Information

Form and function in cell motility: from fibroblasts to keratocytes.

Publications

Form and function in cell motility: from fibroblasts to keratocytes.

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