Computational protocol: Dynamic actin cycling through mitochondrial subpopulations locally regulates the fission–fusion balance within mitochondrial networks

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Protocol publication

[…] All images were acquired on a spinning-disk confocal (UltraVIEW VoX; Perkin Elmer) on a Nikon Eclipse Ti microscope using an Apochromat × 100 1.49 numerical aperture oil-immersion objective (Nikon) in a temperature-controlled chamber (37 °C). Digital images were acquired with an EM charge-coupled device camera (C9100; Hamamatsu Photonics) using Volocity software (PerkinElmer) at 1 frame every 1–30 s or 1 frame per minute. Specifically, after the plates are mounted on the microscope, we selected the first observed cell with identifiable actin recruitment to mitochondria. The cell was imaged for no longer than 15–20 min with <10% laser power and then immediately a second cell was imaged in a separate area of the plate. No more than four cells were imaged from a single plate. Cells were excluded from analysis if they displayed signs of phototoxicity such as blebbing or vacuolization. All experiments were performed on at least three independent occasions. Time-lapse maximum intensity projection movies were created by acquiring nine z-slices separated by 0.5 μm step size every 30 s for 15–20 min. To induce ROS production, we photobleached a region or the entire HeLa cell expressing pKillerRed-dMito with a 561-nm laser at 100% for 100 iterations.Images of mitochondria (both confocal slices and maximum intensity projections) were segmented using the pixel classification feature of the interactive learning and segmentation toolkit (Ilastik 1.1.5) and analysed by Fiji (NIH). Segmentation of TOM20 labelled mitochondria was carried out using Volocity (PerkinElmer). Automated image analysis was manually verified by comparing the segmentation mask to the original image files. Measures of mitochondrial area, number, circularity and fractional occupancy were generated using the ‘analyse particles' function in Fiji (NIH) with a minimum area of 0.25 μm2. Measures of mitochondrial length, junctions (voxels with three or more neighbors) and branches (slab segments connecting end points to either junctions or other endpoints) were determined using the ‘skeletonize' and ‘analyse skeleton' plugins in Fiji (NIH). Analyses were carried out on either whole cells or square ∼100 μm2 ROIs around subpopulations of actin-positive mitochondria.For actin recruitment analyses in and , time 0 is defined as 1 min before the first observable actin recruitment to >25% of mitochondria within the ROI. For actin disassembly analyses in and , time 4 is defined as 1 min subsequent to the disassembly of actin from >75% of mitochondria within the ROI. For , segmentation of mitochondria from both control and Drp1-K38A cells was carried out on maximum intensity projections and mitochondrial length and number was normalized per cell. All other analyses were performed on single plane images. Line scans were generated using ImageJ (NIH) and normalized per fluorescent tag. Three-dimensional renderings were generated using Volocity (PerkinElmer). All images were assembled using Fiji (NIH) and Illustrator (Adobe). Statistics and graphing were performed using Prism (GraphPad) software. Comparisons of two data sets were performed using unpaired two-tailed Student's t-test for normally distributed data sets and Mann–Whitney test for non-normally distributed data sets. Comparisons for multiple data sets were performed using one-way analysis of variance with Tukey's post-hoc test for normally distributed data sets and Kruskal–Wallis test with Dunn's multiple comparison test for non-normally distributed data sets. […]

Pipeline specifications

Software tools ilastik, ImageJ
Applications SPIM, Microscopic phenotype analysis