Figure 7.

Affixin^249–272–expressing cells showed blockade of cell spreading and abnormal distribution of F-actin, α-actinin, and Mena. (A) CHO-K1 cells transfected with GFP-affixin^249–272 or GFP vector were cultured on FN-coated plates and video microscopy data were collected from 24 to 48 h after transfection at 37°C in a humidified atmosphere of 5% CO₂. Fluorescence microscopic data obtained 38 h after transfection to only visualize GFP-expressing cells under low magnification (a and b; Videos 1 and 2) are shown, and DIC images obtained at 24 h (c and d) or 30 h (e and f) demonstrate cell morphology under enhanced magnification. In c–f, arrows indicate GFP-expressing cells that were confirmed by fluorescence microscopy (not depicted) and arrowheads indicate GFP-negative cells that divided during time-lapse observation (see Videos 3 and 4). Note that cells transfected with GFP-affixin^249–272 were arrested at the early stage of cell spreading with peripheral blebs. Bars: 20 μm (A, in a and b), 10 μm (C). An animated time-lapse version of this figure is available at http://www.jcb.org/cgi/content/full/jcb.200308141/DC1. (B) The percentage of round cells among GFP-positive cells were estimated 24 h after transfection. The values provided represent mean values (±SD) of three independent experiments. (C) GFP-affixin^249–272 was overexpressed in CHO-K1 cells, and 24 h later the cells were fixed with 100% cold methanol (a, b, e, and f) or 2% PFA (c and d) and stained with the anti-α-actinin antibody (a) or rhodamine phalloidin (c). In e and f, GFP-expressing cells were doubly stained with anti-affixin (e, Cy3) and Mena (f, Cy5). Note that in GFP-affixin^249-272–overexpressing cells, α-actinin demonstrated punctate staining at cell periphery, whereas F-actin formed a weak peripheral staining with a high concentration in a limited number of blebs. Colocalization of affixin and Mena was not observed.