FIGURE 4.

Annexin 2, actin, and phosphorylated components of the focal adhesion colocalize on Rab11-positive perinuclear vesicles when v-Src is active. A, R1LA29 cells were grown at 35 °C for 8 h and stained for annexin 2 (anx 2), Src-pY416, and F-actin. Annexin 2 and a small amount of Src-pY416 are present at the actin-rich cortex of the cell, and there is an accumulation of annexin 2, Src-pY416, and F-actin on vesicular structures in the perinuclear region of the cell. B, cells were prepared as in A and immunostained for annexin 2, Rab11, and F-actin. The merged image shows intense colocalization of annexin 2 at the perinuclear endosomal compartment (pink arrowheads). C, R1LA29 cells were transfected with Rab11-GFP and immunostained for annexin 2 and F-actin. At 41 °C and in cells expressing annexin 2, Rab11-GFP staining was faint, although mainly targeted to the perinuclear endosomal compartment (blue arrowheads). In cells switched to the permissive temperature and partially depleted of annexin 2, the localization and intensity of Rab11-GFP appeared similar to those observed in control cells at 41 °C (white arrowhead). However, as in B, control cells exhibited strong enrichment of annexin 2 and Rab11-GFP at the perinuclear endosomal compartment (pink arrowhead). D, in cells prepared as in A, annexin 2 colocalizes with FAK-pY925 and paxillin-pY118 at the cell cortex, on ruffling membranes (yellow arrowheads), on vesicles in the cytoplasm (white arrowheads), and in the perinuclear recycling compartment (pink arrowheads). Scale bars, 10 and 15 μm, as indicated.