Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.
Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.
复制标题
DOI：
10.1038/cdd.2009.120
复制
发表时间：
2010-01
影响因子：
12.4
通讯作者：
Kornbluth, S.
中科院分区：
文献类型：
--
作者：
Johnson, C. E.;Freel, C. D.;Kornbluth, S.
在无细胞系统和完整胚胎中，通过分析静态样品中单个的凋亡调节因子或caspase的激活，研究了影响脊椎动物卵和早期胚胎凋亡的因素。本文介绍了一种监测非洲爪哇活卵母细胞和早期胚胎半胱氨酸氨基转移酶活性的新方法。这种方法利用显微注射近红外caspase底物，只有在其被活性效应半胱氨酸天冬氨酸酶切割后才发出荧光，从而能够阐明其他神秘的细胞凋亡调控方面。特别是，我们证明了短暂的caspase活性(10分钟)足以导致该系统中的凋亡性死亡。我们说明了卵母细胞中的细胞色素c剂量阈值，该阈值由Smac降低，Smac是一种结合从而中和凋亡抑制蛋白的蛋白质。我们发现减数分裂的卵母细胞对细胞色素c产生了抗性，减数分裂过程中受阻的卵母细胞的最终死亡是不依赖于caspase的。最后，通过对非洲爪哇胚胎中caspase活性进行成像获得的数据表明，在非常早期的发育中，细胞凋亡不是细胞自主的。这些研究既验证了该方法是研究细胞凋亡的有用工具，也揭示了早期发育过程中细胞死亡程序的微妙之处。此外，该方法为识别新的凋亡调控因子提供了一种潜在的有价值的筛选方式。
Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. Described here is a novel method for monitoring caspase activity in living Xenopus oocytes and early embryos. The approach, utilizing microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we demonstrate that brief caspase activity (ten minutes) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggests that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators.
登录
查看更多内容
影响因子：
64.5
作者：
Mahrus S;Trinidad JC;Barkan DT;Sali A;Burlingame AL;Wells JA
通讯作者：
Wells JA
影响因子：
2.9
作者：
Peng, Xinzhan;Chen, Huaxian;Olive, D. Michael
通讯作者：
Olive, D. Michael
影响因子：
12.4
作者：
Bhuyan, AK;Varshney, A;Mathew, MK
通讯作者：
Mathew, MK
影响因子：
64.5
作者：
Nutt, LK;Margolis, SS;Kornbluth, S
通讯作者：
Kornbluth, S
影响因子：
2.5
作者：
Arnault, Emilie;Tosca, Lucie;Lefevre, Brigitte
通讯作者：
Lefevre, Brigitte