cAMP and PKA/AKAP1 decrease mobility and promote mitochondrial translocation of Drp1.

(A) Confocal micrograph showing mixed cytosolic and mitochondrial localization of GFP-Drp1 in PC12 cells (mito, MitoTracker Deep Red). (B–D) FRAP analysis in PC12 cells shows opposite effects of PKA activation and AKAP1 knockdown on GFP-Drp1 dynamics. PC12 cells co-expressing GFP-Drp1 and either AKAP1-directed or control shRNA were treated ± forskolin/rolipram (25/1 µM, 1–3 h) and Drp1 turnover was measured by bleaching mitochondrial GFP-Drp1 in a 5×5 µm square and monitoring fluorescence recovery at 5 s intervals. (B) shows frames from representative cells (control, forsk/roli: control shRNA ± forskolin/rolipram; shAKAP1: AKAP1 shRNA #1), (C) shows averaged fluorescence recovery curves, and (D) plots Drp1 turnover as the ratio of mobile fraction (mFx) and 50% recovery time (t1/2) derived from biexponential fits (R2∼0.99) of individual recovery curves (means ± s.e.m. of 8–10 cells for each condition from a representative experiment). (E–F) Subcellular fractionation of Drp1. COS cells co-expressing GFP-Drp1 with either outer mitochondrial (om) PKA (+) or omGFP (−) were permeabilized with digitonin (500 µg/ml) and fractionated into a cytosolic (cyto) and a heavy membrane fraction containing mitochondria (mito). Fractions were immunoblotted for total Drp1, phospho-SerPKA Drp1 (pDrp1), and the mitochondrial marker TOM40 and analyzed by densitometry (E, representative blot; F, summary showing means ± s.e.m. of 6 independent experiments).