MAM-mediated actin rearrangements proceed via the ROCK/LIMK/cofilin signaling axis.

<p>Hela cells were incubated with bead-coupled GST (A, C) or GST-MAM7 (B, D) for 1 hour following mock treatment (A,B) or treatment of cells with 10 mM Y-27632 for 60 minutes (C, D) and F-actin stained with rhodamine-phalloidin (red). % cells with stress fibers (E) were determined from image analysis following mock treatment (NI) or Y-27632 treatment of cells and attachment of bead coupled to GST-MAM7 (+MAM, red bars) or coupled to GST (−MAM, black bars). Data shown are means ± standard deviation from twelve images (4 frames from n = 3). Images are representative of a set of three experiments. Bar, 10 µm. F-actin/G-actin content was determined for the same experimental conditions (C, cont. beads; M, MAM beads), (F). Results are means ± s.e.m. from duplicate experiments. RhoA activation levels were determined under the same experimental conditions and data significantly different from the negative control are marked (*), (G). % activation was determined by densitometry and samples statistically significantly different from the negative control (GDP-lysate, −) are indicated (*). Levels of p-LIMK and total LIMK (H) or p-cofilin and total cofilin (I) were determined by Western Blotting and densitometry. % activation was determined as ratios of band intensities from phosphorylated and total protein and normalized to mock treated cells attached to bead coupled GST-MAM7 (100%). Data shown are means ± standard deviation from at least three independent experiments and data statistically significantly different from the negative control (student's unpaired two-tailed t-test, p<0.05) are indicated (*).