Microfluidic Analysis of Oscillatory Signaling Pathways Using Phase Locking

描述（由申请人提供）：振荡信号调节各种完整的生理和细胞过程，从G蛋白偶联受体（GPCR）信号传导到昼夜节律。尽管这是一个积极研究的领域，但即使是最著名和最常见的研究途径，在电路结构上也可能存在争议和缺乏清晰度。这是因为使用传统分子或遗传工具的途径摄动研究只能提供有限的信息，从而产生多种合理的机制。本提案将开发基于非线性频率和波形响应分析的工具和方法，以单独使用常规分子或遗传扰动无法实现的方式解剖这种振荡途径。具体来说，我们将使用微流体将周期性化学输入应用于细胞，并使用细胞内信号的实时荧光读数观察锁相细胞反应。观察到的频率响应特性将使用信号通路的计算机模型进行评估。信号通路结构以及抑制剂、激动剂和调节剂的作用模式和机制将被剖析。虽然该方法应该适用于任何振荡信号通路，但我们将首先关注两种GPCR信号通路（M3毒蕈碱乙酰胆碱受体和5型代谢性谷氨酸受体），它们具有非常不同的振荡机制，并且在生理学和药理学上具有重要意义（糖尿病和精神分裂症）。目的1。分析细胞在基本条件下的锁相反应：用受体配体对活细胞进行微流控脉冲刺激。利用钙和IP3的遗传编码荧光指示剂获得细胞内信号的高时间分辨率实时成像。目标2。构建信号电路的数学模型：首先根据已发表的数据构建合理的数学模型。然后，在不确定性和灵敏度分析结果的指导下，改进电路结构和参数以匹配Aim 1中的观测结果。目标3。通过锁相分析描述调节剂的作用机制：研究细胞在抑制剂、激动剂和调节剂存在时的锁相反应如何变化。用实验观察和数学模型来描述作用机制。目标4。传播自我调节芯片，使任何人都可以进行微流体锁相研究。

DESCRIPTION (provided by applicant): Oscillatory signals regulate a wide variety of integral physiological and cellular processes, from G- protein coupled receptor (GPCR) signaling to circadian rhythms. Although an actively studied area, even the most well-known and commonly studied pathways can have controversy and lack of clarity on circuit architecture. This is because pathway perturbation studies using conventional molecular or genetic tools only provide limited information resulting in multiple plausible mechanisms. This proposal will develop tools and methods based on non-linear frequency and waveform response analysis to dissect such oscillatory pathways in ways that are not possible with conventional molecular or genetic perturbations alone. Specifically, we will use microfluidics to apply a periodic chemical input to cells and observed phase-locked cellular responses using real-time fluorescent readouts of intracellular signaling. The observed frequency response characteristics will be evaluated using computer models of the signaling pathway. Signaling circuit architecture as well as modes of action and mechanisms of inhibitors, agonists, and modulators will be dissected. Although the method should be applicable to any oscillatory signaling pathway, we will first focus on two GPCR signaling pathways (M3 muscarinic acetylcholine receptor and type 5 metabotropic glutamate receptor) that have very different proposed mechanisms of oscillation and that are physiologically and pharmacologically important (diabetes and schizophrenia). Aim 1. Analyze Phase Locking Response of Cells Under Base Conditions: Perform microfluidic pulsed stimulation of live cells with receptor ligands. Obtain high time resolution real-time imaging of intracellular signals using genetically encoded fluorescent indicators of calcium and IP3. Aim 2. Construct Mathematical Models of Signaling Circuitry: First construct plausible mathematical models based on published data. Then refine the circuit architecture and parameters to match observations in Aim 1, guided by results of uncertainty and sensitivity analyses. Aim 3. Delineate Mechanisms of Action of Modulators Through Phase Locking Analysis: Study how phase locking responses of cells change in the presence of inhibitors, agonists, and modulators. Use the experimental observations with mathematical models to delineate mechanisms of action. Aim 4. Disseminate self-regulating chips that make microfluidic phase-locking studies accessible to anyone.