Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis.
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.
Karbowski
Mariusz
M
Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Arnoult
Damien
D
Chen
Hsiuchen
H
Chan
David C
DC
Smith
Carolyn L
CL
Youle
Richard J
RJ
D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001681:Biological Assay / Q000379:methods; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008561:Membrane Fusion / Q000502:physiology*; D051379:Mice; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein
D000590:Amino Acid Chloromethyl Ketones; C494826:BAX protein, human; C494827:Bax protein, mouse; C494828:Bax protein, rat; D061945:Caspase Inhibitors; D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D049452:Green Fluorescent Proteins; D020169:Caspases