Microfluidic Analysis of Oscillatory Signaling Pathways Using Phase Locking

描述(申请人提供)：振荡信号调节多种完整的生理和细胞过程，从G蛋白偶联受体(GPCR)信号到昼夜节律。虽然是一个活跃的研究领域，但即使是最著名和最普遍研究的路径也可能存在争议，对电路结构缺乏清晰度。这是因为使用传统的分子或遗传工具进行的途径扰动研究只能提供有限的信息，导致了多种可信的机制。这项提议将开发基于非线性频率和波形响应分析的工具和方法，以剖析这种振荡路径，而这种方式是仅靠传统的分子或遗传扰动是不可能的。具体地说，我们将使用微流体向细胞施加周期性的化学输入，并使用细胞内信号的实时荧光读数来观察锁相细胞反应。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。信号电路的结构，以及作用模式和作用机制的抑制剂，激动剂和调节剂将被剖析。虽然该方法应该适用于任何振荡信号通路，但我们首先将重点放在两条GPCR信号通路上(M3 M胆碱型乙酰胆碱受体和5型代谢型谷氨酸受体)，它们具有非常不同的振荡机制，并且具有重要的生理和药理作用(糖尿病和精神分裂症)。目的1.分析碱性条件下细胞的锁相反应：利用受体配体对活细胞进行微流控脉冲刺激。使用钙和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。目的2.构建信号电路的数学模型：首先，根据已公布的数据构建可信的数学模型。然后，在不确定度和灵敏度分析结果的指导下，改进电路结构和参数，以与目标1中的观测结果相匹配。目的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下的锁相反应如何变化。利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，使任何人都可以访问微流控锁相研究。

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.