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M. Vishwanath, J.V. Jester, L. Ma, C.A. Otey, W.M. Petroll; Modulation of Corneal Fibroblast Contractility Within Fibrillar Collagen Matrices . Invest. Ophthalmol. Vis. Sci. 2003;44(13):898.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the effects of serum on the mechanical activity of isolated corneal fibroblasts in a collagen matrix model, and to investigate whether cellular force generation is associated with stress fiber contraction in these cells. Methods: A telomerase-infected, extended life-span human corneal fibroblast cell line (THK) was transfected using a vector for enhanced green fluorescent protein (EGFP)-α-actinin. Cells were plated at low density on top or inside 100 µm thick fibrillar collagen lattices and cultured for 3 to 7 days in media containing 10% fetal calf serum (S+). Time-lapse imaging was then performed at 1 to 10 minute intervals for 4-5 hours. At each time interval, EGFP and Nomarski DIC images were acquired in rapid succession. S+ media was used initially for perfusion. After 2 hours, perfusion was switched to serum free media (S-) for 1-2 hour, then back to S+ media for 1 hour. Finally, cytochalasin D was added to the culture media (25 µM final concentration). Results: EGFP-α-actinin was localized to focal adhesions and stress fibers of THK cells as confirmed using vinculin and phalloidin counterstaining. The fibrillar ECM structure was clearly visualized using DIC. Fibroblasts in S+ repeatedly extended and retracted pseudopodia. Extending processes generated tractional forces as indicated by pulling in of the collagen fibrils in front of the cell. Switching from S+ to S- induced cell elongation and relaxation of tension on the matrix within 5 minutes. Rapid formation and extension of filipodia was also observed; interestingly, this occurred without generation of tractional forces. Switching back to S+ media caused contraction of the cells, retraction of new filipodia, and compression of the matrix. Changes in the distance between focal adhesions and consecutive α-actinin dense bodies along stress fibers during cell elongation and contraction suggest a direct correlation between stress fiber contraction and cellular force generation. Cytochalasin D induced rapid disassembly of stress fibers and focal adhesions, cell elongation and ECM relaxation without formation of filipodia. Conclusions: The data suggests that removal of serum decreases Rho activity (resulting in cell relaxation) and increases Rac activity (resulting in membrane protrusion and filipodial extension), and that this process is reversible. This study provides the first direct correlation of stress fiber contraction and tension generation in individual corneal fibroblasts.
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