Detection of mitochondrial caspase activity in real time in situ in live cells.
Detection of mitochondrial caspase activity in real time in situ in live cells.
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实时原位检测活细胞中线粒体半胱天冬酶活性。
DOI：
10.1017/s1431927604040401
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Herman,Brian
中科院分区：
文献类型：
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作者：
Zhang,Yingpei;Haskins,Catherine;Lopez-Cruzan,Marisa;Zhang,Jianhua;Centonze,VictoriaE;Herman,Brian
细胞凋亡在许多生理和病理过程中发挥着重要作用。细胞死亡程序的启动和执行需要以严格的时间顺序激活多个半胱天冬酶。在这里，我们描述了一种方法，该方法允许使用棱镜和反射器成像光谱系统（PARISS）基于荧光共振能量转移（FRET）测量实时观察活细胞中半胱天冬酶的原位激活。当由 CFP 通过中间 caspase 底物连接到 YFP 组成的融合蛋白（已靶向特定的亚细胞位置）被波长与青色荧光蛋白 (CFP) 激发峰匹配的光源激发时，CFP 荧光团吸收的能量不会以荧光形式发射。相反，激发能量被附近的黄色荧光蛋白 (YFP) 荧光团吸收，该荧光团通过含有 caspase 底物的短肽与 CFP 共价连接。由于 CFP 和 YFP 荧光团的分离，半胱天冬酶对接头肽的切割导致 FRET 损失。使用线粒体靶向 CFP-caspase 3 底物-YFP 构建体 (mC3Y)，我们首次证明在细胞凋亡的晚期阶段，一些细胞的线粒体基质中存在 caspase-3 样活性。
Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP–caspase 3 substrate–YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.