Bardet Pierre-Luc, Kolahgar Golnar, Mynett Anita, Miguel-Aliaga Irene, Briscoe James, Meier Pascal, Vincent Jean-Paul
Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):13901-5. doi: 10.1073/pnas.0806983105. Epub 2008 Sep 8.
There is a growing interest in the mechanisms that control the apoptosis cascade during development and adult life. To investigate the regulatory events that trigger apoptosis in whole tissues, we have devised a genetically encoded caspase sensor that can be detected in live and fixed tissue by standard confocal microscopy. The sensor comprises two fluorophores, mRFP, monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (eGFP), that are linked by an efficient and specific caspase-sensitive site. Upon caspase activation, the sensor is cleaved and eGFP translocates to the nucleus, leaving mRFP at membranes. This is detected before other markers of apoptosis, including anti-cleaved caspase 3 immunoreactivity. Moreover, the sensor does not perturb normal developmental apoptosis and is specific, as cleavage does not occur in Drosophila embryos that are unable to activate the apoptotic cascade. Importantly, dying cells can be recognized in live embryos, thus opening the way for in vivo imaging. As expected from the high conservation of caspases, it is also cleaved in dying cells of chick embryos. It is therefore likely to be generally useful to track the spatiotemporal pattern of caspase activity in a variety of species.
在发育和成年期控制凋亡级联反应的机制方面，人们的兴趣日益浓厚。为了研究在整个组织中触发凋亡的调控事件，我们设计了一种基因编码的半胱天冬酶传感器，通过标准共聚焦显微镜可在活组织和固定组织中检测到。该传感器由两个荧光团组成，单体红色荧光蛋白（mRFP）和增强型绿色荧光蛋白（eGFP），它们通过一个高效且特异的半胱天冬酶敏感位点相连。半胱天冬酶激活后，传感器被切割，eGFP转位至细胞核，mRFP留在细胞膜上。这在凋亡的其他标志物（包括抗切割的半胱天冬酶3免疫反应性）出现之前就能被检测到。此外，该传感器不会干扰正常的发育性凋亡且具有特异性，因为在无法激活凋亡级联反应的果蝇胚胎中不会发生切割。重要的是，在活胚胎中可以识别正在死亡的细胞，从而为体内成像开辟了道路。正如半胱天冬酶高度保守所预期的那样，它在鸡胚胎的死亡细胞中也会被切割。因此，追踪多种物种中半胱天冬酶活性的时空模式可能具有普遍用途。
