10779/rcsi.10793063.v1 Manus W. Ward Manus W. Ward Markus Rehm Markus Rehm Heiko Düssmann Heiko Düssmann Slavomir Kacmar Slavomir Kacmar Caoimhin G. Concannon Caoimhin G. Concannon Jochen HM Prehn Jochen HM Prehn Real time single cell analysis of Bid cleavage and Bid translocation during caspase-dependent and neuronal caspase-independent apoptosis. Royal College of Surgeons in Ireland 2019 Animals Apoptosis BH3 Interacting Domain Death Agonist Protein Caspases Cells Cultured Fluorescence Resonance Energy Transfer Fluorescent Dyes Glutamic Acid Humans Membrane Potentials Mitochondria Neurons Rats Receptors Glutamate Tumor Necrosis Factor Recombinant Fusion Proteins Physiology Medical Physics 2019-11-22 17:04:27 Journal contribution https://repository.rcsi.com/articles/journal_contribution/Real_time_single_cell_analysis_of_Bid_cleavage_and_Bid_translocation_during_caspase-dependent_and_neuronal_caspase-independent_apoptosis_/10793063 <p>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.</p>