Figure 3.
CMS is localized in the cytoplasm and colocalizes with actin and p130Cas to membrane ruffles. (A and B) COS-7 cells grown in medium containing 10% FBS, plated on poly-d-lysine coverslips, were transfected with the flag-tagged CMS constructs CMS, CMS/TH, and CMS/3SH3 as indicated. Cells were fixed and stained with rhodamine-labeled phalloidin (lane 1) or with anti-flag antibody (lanes 3 and 4). Merged images are shown in lane 2. Actin clustering adjacent to the nucleus (B, lane 1 marked with an arrow). Vesicles in CMS cells are indicated by arrows (B, lane 3). (C and D) Membrane ruffle induction in COS-7 cells transfected with flag-tagged CMS or GST-tagged p130Cas. Serum-starved cells were treated with 10 nM phorbol 12-myristate 13-acetate (PMA) for 10 min, fixed, and subjected to immunofluorescence analysis by using the anti-flag or p130Cas antibody (lane 3). Actin filaments were visualized by staining with rhodamine-labeled phalloidin (lane 1). The cells were analyzed by confocal microscopy (×250 and ×630). Yellow-orange color in merged image shows colocalization of CMS and p130Cas with actin (lane 2, marked with arrows). (Bar, 20 μm.)