RhoC over-expression altered paxillin distribution and accelerated focal adhesion turnover.

<div><p>(A-B) Z-stacked confocal images showed differential distribution of paxillin staining (green) in ‘non-spreading’ (i) and ‘spreading’ (ii), Capan1 (A) and nRhoC (B) cells, 60 minutes after plating on the fibronectin-coated surface. Main images presented are in the XY plane with Z axis at the basal (ventral) aspects of cells as shown in the XZ and YZ sections at the top and right of composite images respectively (the cross-section of images in X, Y and Z axes are denoted by green, red and blue lines respectively). The obvious focal staining at the cell protrusion end of ‘spread’ Capan1 cells (Aii) was not present in the ‘spread’ nRhoC cells (Bii, arrowheads: cell protrusion ends). In the few ‘non-spreading’ cells, there were greater amounts of paxillin staining along the basal membrane of Capan1 cells (Ai) than that of nRhoC (Aii) cells. Scale bar: 5μm.</p> <p>(C-E) Focal adhesion ‘disassembly assay’ (NOC: nocodazole) showed that, compared to Capan 1, nRhoC cells had accelerated disassembly and re-assembly of mature focal adhesions (FA: large 4-5 μm) and small focal adhesion (1μm). Mean Intensity of peripheral paxillin staining showed similar levels 30 minutes after washout of the nocodazole treatment in Capan1 and nRhoC cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s001" target="_blank">Methods S1</a>). However, while nRhoC cells had decreased the paxillin intensity levels significantly by 15 minutes after recovery from nocodazole, Capan1 cells took 30 minutes to achieve similar reduction, suggesting a quicker turnover of new focal adhesions in nRhoC cells. The size of focal adhesions after nocodazole treatment also demonstrates a quicker recovery/ re-assembly of stable large focal adhesions in nRhoC cells as compared to Capan1 cells. This could well explain the rapid movement and the increased cell spreading and decreased adhesions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone-0081575-g001" target="_blank">Figures 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone-0081575-g002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s006" target="_blank">Videos S1, S2</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081575#pone.0081575.s008" target="_blank">S3</a>), as a result of introduction of RhoC in nRhoC cells. *p<0.05, Mann-Whitney test. Summary statistics in the box-whisker plot represented median ± inter-quartile range. </p>