Bastiaens P I, Jovin T M
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8407-12. doi: 10.1073/pnas.93.16.8407.
We have devised a microspectroscopic strategy for assessing the intracellular (re)distribution and the integrity of the primary structure of proteins involved in signal transduction. The purified proteins are fluorescent-labeled in vitro and reintroduced into the living cell. The localization and molecular state of fluorescent-labeled protein kinase C beta I isozyme were assessed by a combination of quantitative confocal laser scanning microscopy, fluorescence lifetime imaging microscopy, and novel determinations of fluorescence resonance energy transfer based on photobleaching digital imaging microscopy. The intensity and fluorescence resonance energy transfer efficiency images demonstrate the rapid nuclear translocation and ensuing fragmentation of protein kinase C beta I in BALB/c3T3 fibroblasts upon phorbol ester stimulation, and suggest distinct, compartmentalized roles for the regulatory and catalytic fragments.
我们设计了一种显微光谱策略，用于评估参与信号转导的蛋白质的细胞内（重新）分布及其一级结构的完整性。纯化后的蛋白质在体外进行荧光标记，然后重新导入活细胞中。通过定量共聚焦激光扫描显微镜、荧光寿命成像显微镜以及基于光漂白数字成像显微镜的新型荧光共振能量转移测定方法相结合，对荧光标记的蛋白激酶CβI同工酶的定位和分子状态进行了评估。强度和荧光共振能量转移效率图像显示，在佛波酯刺激下，BALB/c3T3成纤维细胞中的蛋白激酶CβI迅速发生核转位并随后碎片化，这表明调节片段和催化片段具有不同的、分区化的作用。
