Measuring dynamics of caspase-8 activation in a single living HeLa cell during TNFα-induced apoptosis

被引:56
作者
Luo, KQ [1 ]
Yu, VC [1 ]
Pu, YM [1 ]
Chang, DC [1 ]
机构
[1] Hong Kong Univ Sci & Technol, Dept Biol, Hong Kong, Hong Kong, Peoples R China
关键词
FRET; GFP; apoptosis; caspase-8; caspase-3; programmed cell death;
D O I
10.1016/S0006-291X(03)00559-X
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In this study, we reported the first measurement of the dynamics of activation of caspase-8 in a single living cell. This measurement was conducted using a specially developed molecular sensor based on the FRET (fluorescence resonance energy transfer) technique. This sensor was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a linker containing a tandem caspase-8-specific cleavage site. The change of the FRET ratio upon cleavage was larger than 4-fold. Using this sensor, we found that during TNFalpha-induced apoptosis, the activation of caspase-8 was a slower process than that of caspase-3, and it was initiated much earlier than the caspase-3 activation. Inhibition of caspase-9 delayed the full activation of caspase-3 but did not affect the dynamics of caspase-8. Results of these single-cell measurements suggested that caspase-3 was activated by caspase-8 through two parallel pathways during TNFalpha-induced apoptosis in HeLa cells. (C) 2003 Elsevier Science (USA). All rights reserved.
引用
收藏
页码:217 / 222
页数:6
相关论文
共 14 条
[1]   Biochemical pathways of caspase activation during apoptosis [J].
Budihardjo, I ;
Oliver, H ;
Lutter, M ;
Luo, X ;
Wang, XD .
ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, 1999, 15 :269-290
[2]  
Chang D C, 1997, Methods Mol Biol, V62, P307
[3]   Caspases: the executioners of apoptosis [J].
Cohen, GM .
BIOCHEMICAL JOURNAL, 1997, 326 :1-16
[4]   Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer [J].
Hein, R ;
Tsien, RY .
CURRENT BIOLOGY, 1996, 6 (02) :178-182
[5]   Development and application of a GFP-FRET intracellular caspase assay for drug screening [J].
Jones, J ;
Heim, R ;
Hare, E ;
Stack, J ;
Pollok, BA .
JOURNAL OF BIOMOLECULAR SCREENING, 2000, 5 (05) :307-317
[6]   Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa cells [J].
Luo, KQ ;
Yu, VC ;
Pu, YM ;
Chang, DC .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2001, 283 (05) :1054-1060
[7]   Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis [J].
Mahajan, NP ;
Harrison-Shostak, DC ;
Michaux, J ;
Herman, B .
CHEMISTRY & BIOLOGY, 1999, 6 (06) :401-409
[8]   Confirmation by FRET in individual living cells of the absence of significant amyloid β-mediated caspase 8 activation [J].
Onuki, R ;
Nagasaki, A ;
Kawasaki, H ;
Baba, T ;
Uyeda, TQP ;
Taira, K .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2002, 99 (23) :14716-14721
[9]   Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process -: Role of caspase-3 [J].
Rehm, M ;
Düssmann, H ;
Jänicke, RU ;
Tavaré, JM ;
Kögel, D ;
Prehn, JHM .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, 277 (27) :24506-24514
[10]   Maintenance of caspase-3 proenzyme dormancy by an intrinsic "safety catch" regulatory tripeptide [J].
Roy, S ;
Bayly, CI ;
Gareau, Y ;
Houtzager, VM ;
Kargman, S ;
Keen, SLC ;
Rowland, K ;
Seiden, IM ;
Thornberry, NA ;
Nicholson, DW .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2001, 98 (11) :6132-6137