Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: Fluorescent-labeled protein kinase C beta I
Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: Fluorescent-labeled protein kinase C beta I
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DOI：
10.1073/pnas.93.16.8407
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发表时间：
1996-08-06
影响因子：
11.1
通讯作者：
Jovin, TM
中科院分区：
文献类型：
--
作者：
Bastiaens, PIH;Jovin, TM
我们设计了一种显微光谱策略，用于评估细胞内（再）分布和参与信号转导的蛋白质的一级结构的完整性，纯化的蛋白质在体外被荧光标记并重新引入活细胞。荧光标记的蛋白激酶C β I同工酶的定位和分子状态进行了评估的组合，定量共聚焦激光扫描显微镜，荧光寿命成像显微镜，和新的测定荧光共振能量转移的基础上光漂白数字成像显微镜。强度和荧光共振能量转移效率的图像表明快速核转位和随后的片段化的蛋白激酶C β I在BALB/c 3 T3成纤维细胞佛波醇酯刺激后，并建议不同的，区室化的作用的监管和催化片段。
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/c 3T3 fibroblasts upon phorbol ester stimulation, and suggest distinct, compartmentalized roles for the regulatory and catalytic fragments.