Figure 3

ROCK-1 and ROCK-2 distribution in diabetic retinal pigment epithelial cells (RPE) and ROCK-1 recruitment in membrane blebbing. ROCK1-phalloidin double staining (ROCK-1 in green, phalloidin in red and DAPI in blue) and occludin (green) on: (A,B) Rat retina, (C): Human retina. In non-diabetic WS rats, phalloidin labelled the regular hexagonal cytoskeleton of RPE cells (A,a). In GK rats, cytoskeleton rearrangement with shape and size modifications of RPE resulting in cell constrictions with reduced area and stress fiber formation are observed (Ae,h). Regular occludin stain the WS RPE (b), but focal occluding disruption and opening are observed in GK RPE (f,i). ROCK-1 is cytoplasmic in WS but translocated at the membrane in GK RPE (c,g,j,k) and ROCK-1 positive blebs are observed in GK RPE (A,j). Transversal section of RPE confirms the membrane translocation of ROCK-1 in the diabetic RPE (A,k), demonstrating that ROCK-1 is activated in GK RPE. Similar faint membrane and perinuclear distribution of ROCK-2 is observed in WS and GK rats (d,i). (C) In human RPE, ROCK-1 is recruited at the membrane (white arrows on flat-mounts and on RPE section). Scale bar = 10 µm.