Real time single cell analysis of Bid cleavage and Bid translocation during caspase-dependent and neuronal caspase-independent apoptosis.
Bcl-2 homology domain (BH) 3-only proteins couple stress signals to evolutionarily conserved mitochondrial apoptotic pathways. Caspase 8-mediated cleavage of the BH3-only protein Bid into a truncated protein (tBid) and subsequent translocation of tBid to mitochondria has been implicated in death receptor signaling. We utilized a recombinant fluorescence resonance energy transfer (FRET) Bid probe to determine the kinetics of Bid cleavage and tBid translocation during death receptor-induced apoptosis in caspase 3-deficient MCF-7 cells. Cells treated with tumor necrosis factor-alpha (200 ng/ml) showed a rapid cleavage of the Bid-FRET probe occurring 75.4 +/- 12.6 min after onset of the tumor necrosis factor-alpha exposure. Cleavage of the Bid-FRET probe coincided with a translocation of tBid to the mitochondria and a collapse of the mitochondrial membrane potential (DeltaPsim). We next investigated the role of Bid cleavage in a model of caspase-independent, glutamate-induced excitotoxic apoptosis. Rat cerebellar granule neurons were transfected with the Bid-FRET probe and exposed to glutamate for 5 min. In contrast to death receptor-induced apoptosis, neurons showed a translocation of full-length Bid to the mitochondria. This translocation occurred 5.6 +/- 1.7 h after the termination of the glutamate exposure and was also paralleled with a collapse of the DeltaPsim. Proteolytic cleavage of the FRET probe also occurred, however, only 25.2 +/- 3.5 min after its translocation to the mitochondria. Subfractionation experiments confirmed a translocation of full-length Bid from the cytosolic to the mitochondrial fraction during excitotoxic apoptosis. Our data demonstrate that both tBid and full-length Bid have the capacity to translocate to mitochondria during apoptosis.
Ward
Manus W
MW
Department of Physiology and Medical Physics and RCSI Neuroscience Research Centre, Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin 2, Ireland.
Rehm
Markus
M
Duessmann
Heiko
H
Kacmar
Slavomir
S
Concannon
Caoimhin G
CG
Prehn
Jochen H M
JH
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D018698:Glutamic Acid / Q000633:toxicity; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017470:Receptors, Glutamate / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D051023:BH3 Interacting Domain Death Agonist Protein; D005456:Fluorescent Dyes; D017470:Receptors, Glutamate; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D020169:Caspases