PKA phosphorylation and T55D mutation prolong the lifetime of mitochondrial Drp1 foci.

(A, B) HeLa cells transfected with GFP-Drp1 (green), dsRed2/mito (COX8 matrix targeting sequence, red), ± PKA catalytic subunit were imaged for ≥1 h at 37°C, capturing images every 30 s. (A) Representative frames of time lapse series (see Video S3). Blue lines connect GFP-Drp1 punctae that could be tracked for at least 5 min; x symbols denote mitochondrial fission events. Note that GFP-Drp1 punctae often split with the fragmenting mitochondrion. (B) Cumulative frequency plot of Drp1 punctae lifetimes and average lifetimes (bar graph inset; means ± s.e.m. of 35–62 cells and 11,000 to 40,000 punctae per condition from two independent experiments). (C) Model of mitochondrial fusion by PKA/AKAP1. GTP-bound Drp1 translocates to mitochondria to assemble into oligomeric complexes. Drp1 assembly stimulates GTP hydrolysis, leading to mitochondrial fission and release of Drp1 into the cytosol to complete the cycle. OMM-anchored PKA/AKAP1 phosphorylates Drp1 at SerPKA, stabilizing the GTP-bound state to promote growth of Drp1 complexes to a size that is incompatible with membrane scission. Protein phosphatases (PP), including calcineurin, dephosphorylate Drp1 SerPKA to return Drp1 into its active, rapidly cycling state.(A, B) 转染了GFP-Drp1（绿色荧光）、dsRed2/线粒体（COX8基质靶向序列，红色荧光）的HeLa细胞（HeLa cells），分别施加蛋白激酶A（PKA）催化亚基与未施加该亚基，于37℃下成像时长≥1小时，每30秒采集一幅图像。(A) 为延时序列的代表性帧（详见补充视频S3）。蓝色线条连接可被追踪至少5分钟的GFP-Drp1斑点；叉号（x符号）代表线粒体分裂事件。值得注意的是，GFP-Drp1斑点常伴随线粒体片段化而发生分裂。(B) 为Drp1斑点寿命的累积频率分布图，内嵌柱状图展示平均寿命（均值±标准误[standard error of the mean, s.e.m.]，数据来自2次独立实验，每个实验条件下包含35~62个细胞、11000~40000个Drp1斑点）。(C) 为PKA/AKAP1介导的线粒体融合模型。结合三磷酸鸟苷（GTP）的Drp1转位至线粒体，组装为寡聚复合体。Drp1的组装会刺激GTP水解，进而引发线粒体分裂，并将Drp1释放至细胞质以完成循环。锚定在线粒体外膜（outer mitochondrial membrane, OMM）的PKA/AKAP1会在SerPKA位点磷酸化Drp1，稳定GTP结合状态，促进Drp1复合体生长至无法介导膜切割的尺寸。包括钙调神经磷酸酶（calcineurin）在内的蛋白磷酸酶（PP）可对Drp1的SerPKA位点进行去磷酸化，使Drp1回到活跃的快速循环状态。