Figure 3.

PKA activation promotes MAP2 association with actin. HeLa cells expressing wt GFP-MAP2c (green) were incubated with vehicle alone (A) or with 20 μM rolipram plus 10 μM forskolin for 10 h (B) as described in MATERIALS AND METHODS. Cells were fixed and immunolabeled for actin (red). Stacks of images were collected and 3-D reconstructions were created as described in MATERIALS AND METHODS. Shown are maximal projections of a series of single plane (x-y) images collected at 0.2-μm intervals in the z-dimension. The boxed regions are shown in magnified views for the actin (red), GFP-MAP2c (green), and merged images as indicated. Bars, 10 μm. (A) In control, unstimulated cells MAP2c is highly colocalized with microtubules and absent from peripheral regions enriched in actin. MAP2 is absent from actin structures within the ruffle. Video supplement OzerFig 3A.mov shows a revolving 3-D reconstruction of the merged image. (B) PKA activation promotes MAP2c–actin colocalization. Neither protein fills the cytoplasmic volume; however, GFP-MAP2c is partially colocalized within specific structures where actin is enriched (arrowheads). Video supplement OzerFig 3B.mov shows a revolving 3-D reconstruction of the merged image.