A quantitative assay for mitochondrial fusion using Renilla luciferase complementation.
Mitochondria continuously undergo fusion and fission, the relative rates of which define their morphology. Large mitochondria produce energy more efficiently, whereas small mitochondria translocate better to subcellular sites where local production of ATP is acutely required. Mitochondrial fusion is currently assayed by fusing together cells expressing GFP or RFP in their mitochondria and then scoring the frequency of cells with yellow mitochondria (representing fused green and red mitochondria). However, this assay is labor-intensive and only semi-quantitative. We describe here a reporter system consisting of split fragments of Renilla luciferase and YFP fused to mitochondrial matrix-targeting sequences and to leucine zippers to trigger dimerization. The assay enables fusion to be quantitated both visually for individual cells and on a population level using chemiluminescence, laying the foundation for high throughput small molecule and RNAi screens for modulators of mitochondrial fusion. We use the assay to examine cytoskeletal roles in fusion progression.
Huang
Huiyan
H
Department of Pharmacology, Center for Developmental Genetics, Stony Brook University, Stony Brook, NY 11794-5140, USA.
Choi
Seok-Yong
SY
Frohman
Michael A
MA
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003584:Cytological Techniques / Q000379:methods*; D017930:Genes, Reporter; D006801:Humans; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D008928:Mitochondria / Q000502:physiology*
D049410:Luciferases, Renilla