Abstract
Cells undergoing apoptosis show a plethora of time-dependent changes. The available tools for imaging apoptosis in live cells rely either on the detection of the activity of caspases, or on the visualization of exposure of phosphatidyl serine in the outer leaflet of the cell membrane. We report here a novel method for the detection of mitochondrial events during apoptosis, namely translocation of Bax to mitochondria and release of cytochrome c (Cyt c) using bimolecular fluorescence complementation. Expression of split yellow fluorescent protein (YFP) fragments fused to Bax and Cyt c, resulted in robust induction of YFP fluorescence at the mitochondria of apoptotic cells with very low background. In vivo expression of split YFP protein fragments in liver hepatocytes and intra-vital imaging of subcutaneous tumor showed elevated YFP fluorescence upon apoptosis induction. Thus, YFP complementation could be applied for high-throughput screening and in vivo molecular imaging of mitochondrial events during apoptosis.
Highlights
Apoptotic cells can be recognized by defined morphological changes: the cell shrinks, its chromatin condenses, the plasma membrane shows blebbing, and the cell is fragmented into compact membrane-enclosed structures
When the yellow fluorescent protein (YFP) fragments were separated to different cellular compartments by fusing the N-YFP to the mitochondrial leader sequence cytochrome oxidase subunit-VIII, the levels of the YFP signal were significantly lowered (Figures 1a and b)
The ability to monitor apoptosis is important for the study of cancer progression and response to therapy
Summary
Apoptotic cells can be recognized by defined morphological changes: the cell shrinks, its chromatin condenses, the plasma membrane shows blebbing, and the cell is fragmented into compact membrane-enclosed structures. These morphological changes are a consequence of characteristic molecular and biochemical events occurring in an apoptotic cell, including activation of cysteine-dependent, aspartate-specific proteases (caspases) that coordinate and execute the process. Some rely on the structural changes occurring in an apoptotic tissue, which can be detected by ultrasound[2] or diffusion-weighted MRI.[3] Other methods include reagents developed for monitoring activation of caspase proteases by fluorescence,[4] bioluminescence[5] or radiolabeled probes.[6]. A proapoptotic member of the family, resides in the cytosol in an inactive state, in which its N- and C-terminal parts are folded within the protein core.[13,14]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
