Abstract
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 onto the fluorescence micrograph z-stack. We apply MEL to both control samples as well as cells that have been treated with hydroxychloroquine sulphate (HCQ) and observe that fission and fusion events mostly occur around the terminal branches of the mitochondrial network, with few events detected in the strongly networked areas such as the perinuclear region. Depolarisation was most abundant in the HCQ treated samples, with the majority of the depolarisation events occurring in the cell periphery.
