Live imaging of extracellular signal-regulated kinase and protein kinase A activities during thrombus formation in mice expressing biosensors based on Forster resonance energy transfer
Live imaging of extracellular signal-regulated kinase and protein kinase A activities during thrombus formation in mice expressing biosensors based on Forster resonance energy transfer
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DOI：
10.1111/jth.13723
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发表时间：
2017-07-01
影响因子：
10.4
通讯作者：
Matsuda, M.
中科院分区：
文献类型：
--
作者：
Hiratsuka, T.;Sano, T.;Matsuda, M.
背景：用传统的视频广角显微镜或共聚焦显微镜观察了活体小鼠血栓形成的动态特征。然而，由于技术限制，细胞内信号分子活动的精确时空调控，已经在体外进行了广泛的研究，在体内仍然难以捉摸。目的：利用双光子激发显微镜观察表达Forster共振能量转移（FRET）细胞外信号调节激酶（ERK）和蛋白激酶A （PKA）生物传感器的转基因小鼠，观察激光损伤皮下小动脉血栓形成过程中ERK和PKA的活性。结果：当致密的血小板核心形成时，靠近损伤小动脉的基底区ERK活性增加。PKA在覆盖血小板核心的不稳定壳的下游被激活。静脉注射MEK抑制剂PD0325901可抑制血小板栓系和血小板聚集物的移位，这表明ERK活性对于血栓的形成和维持都是不可或缺的。cAMP类似物dbcAMP可以抑制血小板栓系，但不能去除预先形成的血小板聚集体，这表明PKA只能在早期阶段拮抗血栓形成。结论：表达FRET生物传感器的转基因小鼠的体内成像将为观察血栓形成过程中信号分子活性的时空变化以及其他血液学疾病提供新的机会。
Background: The dynamic features of thrombus formation have been visualized by conventional video widefield microscopy or confocal microscopy in live mice. However, owing to technical limitations, the precise spatiotemporal regulation of intracellular signaling molecule activities, which have been extensively studied in vitro, remains elusive in vivo. Objectives: To visualize, by the use of two-photon excitation microscopy of transgenic mice expressing Forster resonance energy transfer (FRET) biosensors for extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), ERK and PKA activities during thrombus formation in laser-injured subcutaneous arterioles. Results: When a core of densely packed platelets had developed, ERK activity was increased from the basal region close to the injured arterioles. PKA was activated at the downstream side of an unstable shell overlaying the core of platelets. Intravenous administration of a MEK inhibitor, PD0325901, suppressed platelet tethering and dislodged platelet aggregates, indicating that ERK activity is indispensable for both initiation and maintenance of the thrombus. A cAMP analog, dbcAMP, inhibited platelet tethering but failed to dislodge the preformed platelet aggregates, suggesting that PKA can antagonize thrombus formation only in the early phase. Conclusion: In vivo imaging of transgenic mice expressing FRET biosensors will open a new opportunity to visualize the spatiotemporal changes in signaling molecule activities not only during thrombus formation but also in other hematologic disorders.