title: High-throughput detection and quantification of mitochondrial fusion through imaging flow cytometry.
abstract: Mitochondria are highly dynamic organelles whose fusion and fission play an increasingly important role in a number of both normal and pathological cellular functions. Despite the increased interest in mitochondrial dynamics, robust, and quantitative methods to analyze mitochondrial fusion and fission activity in intact cells have not been developed. The current state-of-the art method to measure mitochondrial fusion activity is the polyethylene glycol (PEG) fusion assay in which cells expressing distinct mitochondrially-targeted fluorescent proteins (FPs) are fused together and mitochondrial fusion activity is determined by the rate at which color mixing occurs. Although this assay is useful, cell-cell fusion events are rare, and finding the number of fused cells required to generate statistically rigorous data is both tedious and time-consuming. Furthermore, the data-collection methods available for fluorescence microscopy lead to inherent selection biases that are difficult to control for. To that end, we have developed an unbiased and high-throughput method to detect, image, and analyze fused cells using the Amnis ImagestreamX™ MKII. With IDEAS™ software, we developed algorithms for identifying the fused cells (two nuclei within a single cell), distinguishing them from cell aggregates. Additionally, using the fluorescence localization of the mitochondrially-targeted fluorescent proteins (YFP and DsRed), we applied a modified co-localization algorithm to identify those cells that had a high co-localization score indicating mitochondrial fusion activity. These algorithms were tested using negative controls (FPs associated with fusion deficient mitochondria) and positive controls (cells expressing both FPs in the same mitochondria). Once validated these algorithms could be applied to test samples to evaluate the degree of mitochondrial fusion in cells with various genetic mutations. Ultimately, this new method is the first robust, high-throughput way to directly measure mitochondrial fusion in intact cells. Given how many cellular processes are being linked mitochondrial dynamics, this technique will provide a powerful new tool in the study of this important organelle. © 2016 International Society for Advancement of Cytometry.
authors.affiliation: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia.
authors.forename: Aldo
authors.initials: A
authors.lastname: Nascimento
authors.affiliation: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia.|Flow Cytometry Core, University of Virginia School of Medicine, Charlottesville, Virginia.
authors.forename: Joanne
authors.initials: J
authors.lastname: Lannigan
authors.affiliation: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia.
authors.forename: David
authors.initials: D
authors.lastname: Kashatus
mesh_terms: D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000235:genetics*; D063154:Mitochondrial Dynamics / Q000235:genetics*
chemical_list: D049452:Green Fluorescent Proteins
keywords: PEG assay; high-throughput imaging; imaging flow cytometry; mitochondrial dynamics; mitochondrial fission; mitochondrial fusion; mitochondrial morphology