Real-Time Detection of Caspase-3-Like Protease Activation in Vivo Using Fluorescence Resonance Energy Transfer during Plant Programmed Cell Death Induced by Ultraviolet C Overexposure1[C]
AUTOR(ES)
Zhang, Lingrui
FONTE
American Society of Plant Biologists
RESUMO
Caspase-like proteases have been demonstrated to be involved in plant programmed cell death (PCD). Here, the time scale of caspase-3-like protease activation was investigated in single living plant cells undergoing PCD induced by ultraviolet C (UV-C) overexposure. The real-time detection of caspase-3-like protease activation was achieved by measuring the degree of fluorescence resonance energy transfer (FRET) within a recombinant substrate containing enhanced cyan fluorescent protein (ECFP) linked by a peptide possessing the caspase-3 cleavage sequence, DEVD, to enhanced yellow fluorescent protein (EYFP; i.e. ECFP-DEVD-EYFP). Microscopic observations demonstrated that the ECFP-DEVD-EYFP fusion protein could be expressed correctly and the FRET from ECFP to EYFP could be found in transfected Arabidopsis (Arabidopsis thaliana) protoplasts. At 30 min after exposure to UV-C, caspase-3-like protease activation indicated by the decrease in FRET ratio occurred, taking about 1 h to reach completion in single living protoplasts. Mutation in the DEVD tag or a caspase-3 inhibitor could prevent the changes in FRET ratio induced by UV-C treatment, confirming that the decrease in FRET ratio was due to the cleavage of fusion protein as a result of caspase-3-like protease activation. This activation was further confirmed by in vitro caspase-3 substrate assay and western-blot analysis, showing the occurrence of cleavage in ECFP-DEVD-EYFP protein but not in the protein with a mutant DEVD tag. In summary, these results represent direct evidence for the activation of caspase-3-like protease in UV-C-induced PCD, and the FRET technique is a powerful tool for monitoring key events of PCD in living cells in real time.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2719143Documentos Relacionados
- Real-time monitoring of in vitro transcriptional RNA synthesis using fluorescence resonance energy transfer
- Detection of Mycobacterium bovis in Bovine Clinical Specimens Using Real-Time Fluorescence and Fluorescence Resonance Energy Transfer Probe Rapid-Cycle PCR
- Detection, Differentiation, and Quantitation of Pathogenic Leishmania Organisms by a Fluorescence Resonance Energy Transfer-Based Real-Time PCR Assay
- Rapid caspase-3 activation during apoptosis revealed using fluorescence-resonance energy transfer
- Detection of programmed cell death using fluorescence energy transfer.