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¹ Biochemistry Section, Surgical Neurology Branch
² Light Imaging Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
³ Division of Biology, Beckman Institute, California Institute of Technology, Pasadena, CA 91125

Address correspondence to R.J. Youle, Building 10, Rm. 5D-37, MSC 1414, 10 Center Dr., Bethesda, MD 20892-1414. Tel.: (301) 496-6628. Fax: (301) 402-0380. email: youler{at}ninds.nih.gov

A dynamic balance of organelle fusion and fission regulates mitochondrial morphology. During apoptosis this balance is altered, leading to an extensive fragmentation of the mitochondria. Here, we describe a novel assay of mitochondrial dynamics based on confocal imaging of cells expressing a mitochondrial matrix–targeted photoactivable green fluorescent protein that enables detection and quantification of organelle fusion in living cells. Using this assay, we visualize and quantitate mitochondrial fusion rates in healthy and apoptotic cells. During apoptosis, mitochondrial fusion is blocked independently of caspase activation. The block in mitochondrial fusion occurs within the same time range as Bax coalescence on the mitochondria and outer mitochondrial membrane permeabilization, and it may be a consequence of Bax/Bak activation during apoptosis.

Key Words: dynamin; fission; Opal; PAGFP; photoactivation

Abbreviations used in this paper: 3D, three-dimensional; ActD, actinomycin D; mito-PAGFP, mitochondria-targeted PAGFP; MOMP, mitochondrial outer membrane permeabilization; PAGFP, photoactivable GFP; ROI, region of interest; STS, staurosporine; WT MEFs, wild-type mouse embryonic fibroblasts.

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