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).(A) 共聚焦显微成像（Confocal micrograph）展示了PC12细胞中GFP-Drp1在胞质与线粒体的双重定位，线粒体由MitoTracker Deep Red（线粒体追踪染料深红）标记。(B–D) 针对PC12细胞的荧光漂白恢复（FRAP, Fluorescence Recovery After Photobleaching）分析显示，蛋白激酶A（PKA, Protein Kinase A）激活与A激酶锚定蛋白1（AKAP1）敲低对GFP-Drp1动力学具有相反调控效应。将共表达GFP-Drp1与AKAP1靶向或对照短发夹RNA（shRNA, short hairpin RNA）的PC12细胞经或不经毛喉素/咯利普兰（25/1 µM，处理1~3小时）处理后，通过在5×5 µm方形区域内光漂白线粒体GFP-Drp1，并以5秒为间隔监测荧光恢复，以此测定Drp1的周转情况。其中(B)展示了代表性细胞的成像帧（对照组：对照shRNA±毛喉素/咯利普兰；shAKAP1组：AKAP1 shRNA #1）；(C)展示了平均荧光恢复曲线；(D)以基于单条恢复曲线的双指数拟合（决定系数R²≈0.99）得到的可移动组分（mobile fraction, mFx）与50%恢复时间（t1/2）的比值，绘制Drp1周转情况，每组数据为8~10个细胞的均值±标准误（s.e.m., standard error of the mean），来自单次代表性实验。(E–F) Drp1的亚细胞分级分离实验。将共表达GFP-Drp1与线粒体外膜（outer mitochondrial, om）靶向PKA（+组）或线粒体外膜GFP（omGFP，-组）的COS细胞经洋地黄皂苷（digitonin，500 µg/ml）透化处理后，分级分离为胞质组分（cyto, cytosolic）与包含线粒体的重膜组分（mito, mitochondrial）。随后对各组分进行免疫印迹检测，靶标包括总Drp1、磷酸化PKA位点丝氨酸的Drp1（pDrp1, phospho-SerPKA Drp1）以及线粒体标志物TOM40，并通过光密度分析法进行定量：(E)为代表性免疫印迹条带；(F)为6次独立实验的汇总数据，以均值±标准误表示。