RESUMO
The goal of this study was to determine the morphological and sub-cellular mechanical effects of Rac activation on fibroblasts within 3-D collagen matrices. Corneal fibroblasts were plated at low density inside 100 microm thick fibrillar collagen matrices and cultured for 1-2 days in serum-free media. Time-lapse imaging was then performed using Nomarski DIC. After an acclimation period, perfusion was switched to media containing PDGF. In some experiments, Y-27632 or blebbistatin were used to inhibit Rho-kinase (ROCK) or myosin II, respectively. PDGF activated Rac and induced cell spreading, which resulted in an increase in cell length, cell area, and the number of pseudopodial processes. Tractional forces were generated by extending pseudopodia, as indicated by centripetal displacement and realignment of collagen fibrils. Interestingly, the pattern of pseudopodial extension and local collagen fibril realignment was highly dependent upon the initial orientation of fibrils at the leading edge. Following ROCK or myosin II inhibition, significant ECM relaxation was observed, but small displacements of collagen fibrils continued to be detected at the tips of pseudopodia. Taken together, the data suggests that during Rac-induced cell spreading within 3-D matrices, there is a shift in the distribution of forces from the center to the periphery of corneal fibroblasts. ROCK mediates the generation of large myosin II-based tractional forces during cell spreading within 3-D collagen matrices, however residual forces can be generated at the tips of extending pseudopodia that are both ROCK and myosin II-independent.
Assuntos
Movimento Celular/fisiologia , Matriz Extracelular , Fibroblastos/citologia , Proteínas rac de Ligação ao GTP/metabolismo , Fenômenos Biomecânicos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Forma Celular/fisiologia , Colágeno , Córnea/citologia , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Miosina Tipo II/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Proteínas rac de Ligação ao GTP/efeitos dos fármacosRESUMO
Transformed rat fibroblasts expressing two variants of green fluorescent protein, each fused to beta-actin, were used to study actin dynamics during cell protrusion. The recently developed FLAP (fluorescence localization after photobleaching) method permits the tracking of one fluorophore after localized photobleaching by using the other as a colocalized reference. Here, by visualizing the ratio of bleached to total molecules, we found that actin was delivered to protruding zones of the leading edge of the cell at speeds that exceeded 5 micrometers per second. Monte Carlo modeling confirmed that this flow cannot be explained by diffusion and may involve active transport.