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一个Cre-依赖的记者鼠标用于蛋白质激酶A活动动态的定量实时成像

Elizabeth I Tilden^1,2, Aditi Maduskar¹, Anna Oldenborg¹

¹Department of Neuroscience, Washington University in St. Louis, St. Louis, MO, USA.

Scientific reports

|February 6, 2024

概括

我们开发了一个新的记者小鼠,FL-AK,用于跟踪蛋白激酶A (PKA) 活动. 该工具在各种细胞类型中提供了可靠和一致的PKA信号测量.

科学领域:

细胞生物学细胞生物学
分子信号传递是分子信号传递.
神经科学是一个神经科学.

背景情况:

细胞内信号的动态对于细胞功能至关重要.
蛋白激酶A (PKA) 是一个关键的信号分子,涉及新陈代谢,大脑活动,发育和癌症.
以前用于PKA活动成像的方法,如FLIM-AKAR,具有侵入性,并产生可变表达.

研究的目的:

开发一种新的,非侵入性的记者小鼠,用于PKA活动的定量成像.
为了克服基于病毒或电穿孔的PKA记者传递的局限性.
建立一个可靠和一致的方法来监测PKA信号动态.

主要方法:

开发了FL-AK记者鼠标系,以一种来自ROSA26位点的Cre-dependent方式表达FLIM-AKAR.
使用光终身成像显微镜 (FLIM) 和光度测量用于定量PKA活动测量.
在脑切片和体内研究中进行验证,以评估PKA活性对受体激活的反应.

主要成果:

随着时间的推移,FL-AK鼠标系列表现出强大而一致的FLIM-AKAR表达.
在脑切片中,FL-AK成功地报告了在激活Gαs和Gαq结合受体时增加的PKA活性.
在体内实验表明,FL-AK检测到PKA酸化作为对神经调节器受体激活的反应.

结论:

FL-AK 记者鼠标为研究 PKA 信号动态提供了一种定量,强大和灵活的工具.
这种小鼠线允许精确监测不同细胞类型和生理环境中的PKA活性.
FL-AK有助于更深入地了解PKA在各种生物过程中的作用.

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