Dual Activation of cAMP Production Through Photostimulation or Chemical Stimulation.
Dual Activation of cAMP Production Through Photostimulation or Chemical Stimulation.
复制标题
通过光刺激或化学刺激的cAMP产生的双重激活。
DOI：
10.1007/978-1-0716-0755-8_14
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Altschuler DL
中科院分区：
文献类型：
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作者：
Naim N;Reece JM;Zhang X;Altschuler DL
CAMP是多种细胞信号通路的重要调节因子。这种环核苷酸需要严格的时空控制才能发挥有效的作用。光激活蛋白由于具有快速的开关速率和最小的脱靶效应，已成为研究信号动力学的有力工具。Beggiatoa的光活化腺苷环化酶(BPAC)在蓝光刺激下迅速产生cAMP。然而，光传递并不总是可行的，特别是在体内。因此，我们通过融合BPAC和纳米荧光素(NLuc)来创建一个发光激活的环化酶，以允许cAMP活性的化学激活。这种双激活的腺酰环化酶可以用短时间的光激发，也可以用呋喃西林和其他相关的荧光素进行长期的化学激活。结合起来，这些都可以用来模拟cAMP信号的瞬时、慢性和振荡模式。此外，当这些试剂与隔室特异性靶向结构域结合时，为cAMP时空动力学研究提供了新的有力工具。在这里，我们描述了在哺乳动物细胞中使用BPAC-nLuc的详细方法，用光和荧光素刺激cAMP产生，以及测量总cAMP积累。
cAMP is a crucial mediator of multiple cell signaling pathways. This cyclic nucleotide requires strict spatio-temporal control for effective function. Light-activated proteins have become a powerful tool to study signaling kinetics due to having quick on/off rates and minimal off-target effects. The photoactivated adenylyl cyclase from Beggiatoa (bPAC) produces cAMP rapidly upon stimulation with blue light. However, light delivery is not always feasible, especially in vivo. Hence, we created a luminescence-activated cyclase by fusing bPAC with Nanoluciferace (nLuc) to allow chemical activation of cAMP activity. This dual-activated adenylyl cyclase can be stimulated using short bursts of light or long-term chemical activation with furimazine and other related luciferins. Together these can be used to mimic transient, chronic, and oscillating patterns of cAMP signaling. Moreover, when coupled to compartment-specific targeting domains, these reagents provide a new powerful tool for cAMP spatiotemporal dynamic studies. Here, we describe detailed methods for working with bPAC-nLuc in mammalian cells, stimulating cAMP production with light and luciferins, and measuring total cAMP accumulation.