Maes Margaret E, Schlamp Cassandra L, Nickells Robert W
Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, Wisconsin, United States of America.
Cellular and Molecular Pathology Graduate Program, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, Wisconsin, United States of America.
PLoS One. 2017 Sep 7;12(9):e0184434. doi: 10.1371/journal.pone.0184434. eCollection 2017.
The pro-apoptotic BCL2 gene family member, BAX, plays a pivotal role in the intrinsic apoptotic pathway. Under cellular stress, BAX recruitment to the mitochondria occurs when activated BAX forms dimers, then oligomers, to initiate mitochondria outer membrane permeabilization (MOMP), a process critical for apoptotic progression. The activation and recruitment of BAX to form oligomers has been studied for two decades using fusion proteins with a fluorescent reporter attached in-frame to the BAX N-terminus. We applied high-speed live cell imaging to monitor the recruitment of BAX fusion proteins in dying cells. Data from time-lapse imaging was validated against the activity of endogenous BAX in cells, and analyzed using sigmoid mathematical functions to obtain detail of the kinetic parameters of the recruitment process at individual mitochondrial foci. BAX fusion proteins behave like endogenous BAX during apoptosis. Kinetic studies show that fusion protein recruitment is also minimally affected in cells lacking endogenous BAK or BAX genes, but that the kinetics are moderately, but significantly, different with different fluorescent tags in the fusion constructs. In experiments testing BAX recruitment in 3 different cell lines, our results show that regardless of cell type, once activated, BAX recruitment initiates simultaneously within a cell, but exhibits varying rates of recruitment at individual mitochondrial foci. Very early during BAX recruitment, pro-apoptotic molecules are released in the process of MOMP, but different molecules are released at different times and rates relative to the time of BAX recruitment initiation. These results provide a method for BAX kinetic analysis in living cells and yield greater detail of multiple characteristics of BAX-induced MOMP in living cells that were initially observed in cell free studies.
促凋亡的BCL2基因家族成员BAX在内在凋亡途径中起关键作用。在细胞应激下，当活化的BAX形成二聚体，然后形成寡聚体时，BAX会募集到线粒体，从而启动线粒体外膜通透性改变（MOMP），这是凋亡进程的关键过程。二十年来，人们一直使用在BAX N端框内连接荧光报告基因的融合蛋白来研究BAX的激活和募集以形成寡聚体。我们应用高速活细胞成像技术来监测垂死细胞中BAX融合蛋白的募集情况。来自延时成像的数据与细胞内源性BAX的活性进行了验证，并使用S形数学函数进行分析，以获得单个线粒体灶募集过程动力学参数的详细信息。BAX融合蛋白在凋亡过程中的行为与内源性BAX相似。动力学研究表明，在缺乏内源性BAK或BAX基因的细胞中，融合蛋白的募集也受到最小影响，但融合构建体中不同荧光标签的动力学存在中度但显著的差异。在测试3种不同细胞系中BAX募集的实验中，我们的结果表明，无论细胞类型如何，一旦激活，BAX募集在细胞内同时启动，但在单个线粒体灶处的募集速率各不相同。在BAX募集的非常早期，促凋亡分子在MOMP过程中释放，但相对于BAX募集启动时间，不同分子在不同时间和速率释放。这些结果为活细胞中BAX动力学分析提供了一种方法，并更详细地揭示了活细胞中BAX诱导的MOMP的多种特征，这些特征最初是在无细胞研究中观察到的。
