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G.A. Abrams, J.D. Foley, P.F. Nealey, C.J. Murphy; Differential Activation of the Small GTPase, Rho, in Corneal Epithelial Cells Plated on Nanopatterned and Smooth Substrates . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1343.
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Purpose: To determine Rho-activation status for primary-cultured human corneal epithelial cells (HCECs) plated on nanopatterned substrates and whether the activation status of Rho correlates with cellular behaviors on these surfaces. Methods: 250,000 HCECs were plated onto 3cm2 silicon wafers that were patterned with 200nm ridges and grooves, or flat control surfaces. Cellular orientation analysis: Cells were fixed at 2 and 6 hours, stained for F-actin, and random fields were imaged. The data were analyzed using KS300 software (Zeiss) to determine the orientation of each cell and the percentage of cells within 10 degree intervals. Cellular activity studies: Multiple fields were imaged at 5 minute intervals between 5 and 7 hours and cellular outlines were traced and the centroid for each cell was determined using the NIH Image J software. "Activity" was defined as a centroid shift of > 1 cell diameter, or significant morphological spreading in response to cell:cell contact during the 2 hour period. Rho activation status: Cell lysate was incubated with agarose-conjugated rhotekin. GTP-Rho/rhotekin complexes were collected by centrifugation, washed, separated by PAGE and blotted onto nitrocellulose membranes for quantitative western blot analysis (STORM). Results: HCECs plated on patterned surfaces were randomly oriented with respect to the substratum at 2 hours, but were highly aligned (> 35% within 10 degrees of alignment) by 6 hours. Cells on flat surfaces were randomly oriented at both 2 and 6 hours. GTP-Rho levels were higher on flat than on patterned surfaces at 6 hours. Cellular activity was approximately two-fold greater on flat surfaces at 6 hours. Conclusions: GTP-Rho concentration is correlated with cellular activity at 6 hours as cells on flat surfaces showed both higher levels of GTP-Rho and cellular movement. In addition, cellular alignment on patterned surfaces at 6 hours was found to be significantly different than would be expected for a uniform distribution of cells within any given 10 degree interval (~11%). These results suggest that higher levels of GTP-Rho are needed for movement of HCECs. Cellular alignment on substrates with nanotopographic features is not correlated with the activation status of Rho.
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