Department of Electrical and Electronic Engineering, Stellenbosch University, Stellenbosch, Western Cape, South Africa.
Department of Physiological Sciences, Stellenbosch University, Stellenbosch, Western Cape, South Africa.
PLoS One. 2020 Dec 30;15(12):e0229634. doi: 10.1371/journal.pone.0229634. eCollection 2020.
Mitochondrial fission and fusion play an important role not only in maintaining mitochondrial homeostasis but also in preserving overall cellular viability. However, quantitative analysis based on the three-dimensional localisation of these highly dynamic mitochondrial events in the cellular context has not yet been accomplished. Moreover, it remains largely uncertain where in the mitochondrial network depolarisation is most likely to occur. We present the mitochondrial event localiser (MEL), a method that allows high-throughput, automated and deterministic localisation and quantification of mitochondrial fission, fusion and depolarisation events in large three-dimensional microscopy time-lapse sequences. In addition, MEL calculates the number of mitochondrial structures as well as their combined and average volume for each image frame in the time-lapse sequence. The mitochondrial event locations can subsequently be visualised by superposition over the fluorescence micrograph z-stack. We apply MEL to both control samples as well as to cells before and after treatment with hydrogen peroxide (H2O2). An average of 9.3/7.2/2.3 fusion/fission/depolarisation events per cell were observed respectively for every 10 sec in the control cells. With peroxide treatment, the rate initially shifted toward fusion with and average of 15/6/3 events per cell, before returning to a new equilibrium not far from that of the control cells, with an average of 6.2/6.4/3.4 events per cell. These MEL results indicate that both pre-treatment and control cells maintain a fission/fusion equilibrium, and that depolarisation is higher in the post-treatment cells. When individually validating mitochondrial events detected with MEL, for a representative cell for the control and treated samples, the true-positive events were 47%/49%/14% respectively for fusion/fission/depolarisation events. We conclude that MEL is a viable method of quantitative mitochondrial event analysis.
线粒体的分裂和融合不仅在维持线粒体的内稳态方面起着重要作用，而且在维持细胞整体活力方面也起着重要作用。然而，基于这些高度动态的线粒体事件在细胞环境中的三维定位的定量分析尚未完成。此外，在很大程度上仍不确定在线粒体网络中去极化最有可能发生的位置。我们提出了线粒体事件定位器（MEL），这是一种允许高通量、自动化和确定性地定位和量化大三维显微镜延时序列中线粒体分裂、融合和去极化事件的方法。此外，MEL 还计算了每个图像帧的线粒体结构数量及其组合和平均体积。线粒体事件的位置随后可以通过在荧光显微镜 z 堆栈上叠加来可视化。我们将 MEL 应用于对照样本以及用过氧化氢（H2O2）处理前后的细胞。在对照细胞中，每 10 秒观察到平均 9.3/7.2/2.3 个融合/分裂/去极化事件/细胞。用过氧化物处理后，融合的速度最初增加，平均每个细胞有 15/6/3 个事件，然后回到与对照细胞非常接近的新平衡，平均每个细胞有 6.2/6.4/3.4 个事件。这些 MEL 结果表明，预处理和对照细胞都维持着分裂/融合的平衡，并且处理后的细胞去极化程度更高。当单独验证用 MEL 检测到的线粒体事件时，对于对照和处理样本的一个有代表性的细胞，融合/分裂/去极化事件的真实阳性事件分别为 47%/49%/14%。我们得出结论，MEL 是一种可行的定量线粒体事件分析方法。
