Real-time monitoring full length bid interacting with Bax during TNF-alpha-induced apoptosis.
Bid, a member of the pro-apoptotic Bcl-2 protein family, is activated through caspase-8-mediated cleavage into a truncated form (p15 tBid) during TNF-alpha(tumor necrosis factor alpha)-induced apoptosis. Activated tBid can induce Bax oligomerization and translocation to mitochondria, triggering the release of cytochrome c, caspase-3 activation and cell apoptosis. However, it is debatable that whether Bid and tBid can interact directly with Bax in living cells. In this study, we used confocal fluorescence microscope, combined with both FRET (fluorescence resonance energy transfer) and acceptor photobleaching techniques, to study the dynamic interaction between Bid and Bax during TNF-alpha-induced apoptosis in single living cell. In ASTC-a-1 cells, full length Bid induced Bax translocation to mitochondria by directly interacting with Bax transiently in response to TNF-alpha treatment before cell shrinkage. Next, we demonstrated that, in both ASTC-a-1 and HeLa cells, Bid was not cleaved before cell shrinkage even under the condition that caspase-8 had been activated, but in MCF-7 cells Bid was cleaved. In addition, in ASTC-a-1 cells, caspase-3 activation was a biphasic process and Bid was cleaved after the second activation of caspase-3. In summary, these findings indicate that, FL-Bid (full length-Bid) directly regulated the activation of Bax during TNF-alpha-induced apoptosis in ASTC-a-1 cells and that the cleavage of Bid occurred in advanced apoptosis.
Pei
Yihui
Y
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China.
Xing
Da
D
Gao
Xuejuan
X
Liu
Lei
L
Chen
Tongsheng
T
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000502:physiology*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D025941:Protein Interaction Mapping; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein / Q000502:physiology*
D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D014409:Tumor Necrosis Factor-alpha; D051028:bcl-2-Associated X Protein; D053148:Caspase 3