Purchase this article with an account.
W. Petroll, L. Ma, A. Kim, L. Ly, M. Vishwanath; Differential Regulation of the Dynamic Sub–Cellular Mechanical Activity of Corneal Fibroblasts by Rho and Rac . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2306.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose:The small GTPases Rac and Rho play a central role in regulating spreading and contraction, respectively, of a variety of cell types on 2–D substrates. The goal of this study was to determine the morphological and sub–cellular mechanical effects of Rho and Rac on corneal fibroblasts inside 3–D matrices. Methods:Human corneal fibroblasts were plated at low density inside 100 µm thick fibrillar collagen matrices and cultured for 1 to 3 days in serum free (S–) media. Time–lapse imaging was then performed at 1–2 minute intervals using Nomarski DIC. After 1 hour, perfusion was switched to S– media containing either 1 mM LPA (which activates Rho), 10 ng/ml PDGF (which activates Rac), or the Rho–kinase (ROCK) inhibitor Y–27632 (10 µM) for 1 hour. Perfusion media was then changed to either LPA + Y–27632, or PDGF + Y–27632. In other experiments, time–lapse imaging was performed following microinjection of constitutively active Rac (L61Rac, 800 µg/ml) or wild type Rac (as a control). Changes in cell morphology and extracellular matrix (ECM) deformation were measured using MetaMorph. Results:Cells produced little or no ECM deformation in S– media. Addition of LPA (n=6 cells) induced retraction of cell processes and contraction along the cell body, as indicated by a decrease in cell length (–12.1 + 7.0%, p <0.05) and cell area (–11.8 + 12.2%). Force generation was greatest along the cell body, as indicated by the location of maximum compressive ECM strain (–14.7 + 7.9%, p < 0.05). These effects were reversed after adding Y–27632. In contrast to LPA, stimulation with PDGF (n=7 cells) induced rapid cell spreading, as indicated by an increase in cell length (30.8 + 34.1%, p = 0.054), cell area (45.9 + 27.1%, p<0.05), and the number of pseudopodial processes (10.4 + 2.6 vs. 5.7 + 3.7 processes per cell, p<0.05). Forces were more transient than those observed after LPA treatment, and the pattern of matrix strain was more complex. However, in all cases maximal ECM compression was located at the leading edge of extending pseudopodia (–11.0 + 5.3%, p<0.05). A similar response was induced by microinjection of L61Rac (n= 3 cells). Following ROCK inhibition with Y–27632, PDGF induced a similar, but even more localized response at the tips of extending pseudopodia (n=6 cells). Conclusions: Taken together, the data suggest that during Rac–induced cell spreading, transient forces are generated at the ends of extending pseudopodia via a ROCK–independent mechanism. In contrast, during Rho–induced cell contraction, more sustained forces are generated along the cell body via a ROCK–dependent mechanism.
This PDF is available to Subscribers Only