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J. Biol. Chem., Vol. 281, Issue 9, 5837-5844, March 3, 2006

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Real Time Single Cell Analysis of Bid Cleavage and Bid Translocation during Caspase-dependent and Neuronal Caspase-independent Apoptosis^*

From the 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

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- (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- exposure. Cleavage of the Bid-FRET probe coincided with a translocation of tBid to the mitochondria and a collapse of the mitochondrial membrane potential (m). 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 m. 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.

Received for publication, October 25, 2005 , and in revised form, December 15, 2005.

^* This work was supported by a grant from Science Foundation Ireland (03/RP/B344) (to J. H. M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Physiology and Medical Physics, Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin 2, Ireland. Tel.: 353-1-402-2261; Fax: 353-1-402-2447; E-mail: Prehn{at}rcsi.ie.

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