Real time single cell analysis of bid cleavage and bid translocation during caspase-dependent and neuronal caspase-independent apoptosis

Manus W. Ward, Markus Rehm, Heiko Duessmann, Slavomir Kacmar, Caoimhin G. Concannon, Jochen H.M. Prehn*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

73 Scopus citations


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-α (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-α exposure. Cleavage of the Bid-FRET probe coincided with a translocation of tBid to the mitochondria and a collapse of the mitochondrial membrane potential (ΔΨm).We next investigated the role of Bid cleavage in amodel 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 ΔΨm. 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.

Original languageEnglish (US)
Pages (from-to)5837-5844
Number of pages8
JournalJournal of Biological Chemistry
Issue number9
StatePublished - Mar 3 2006
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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