Microfluidic Analysis of Oscillatory Signaling Pathways Using Phase Locking

使用锁相对振荡信号通路进行微流控分析

基本信息

批准号：
8665981
负责人：
SHUICHI TAKAYAMA
金额：
$ 28.85万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-30 至 2016-05-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：振荡信号调节多种整体生理和细胞过程，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，但即使是最知名和最常研究的通路也可能存在争议并且电路架构缺乏清晰度。这是因为使用传统分子或遗传工具进行的通路扰动研究只能提供有限的信息，从而导致多种可能的机制。该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，以仅用传统分子或遗传扰动无法实现的方式剖析此类振荡路径。具体来说，我们将使用微流体向细胞施加周期性化学输入，并使用细胞内信号传导的实时荧光读数观察锁相细胞反应。将使用信号通路的计算机模型来评估观察到的频率响应特性。将剖析信号通路架构以及抑制剂、激动剂和调节剂的作用模式和机制。虽然该方法应该适用于任何振荡信号通路，但我们首先关注两条 GPCR 信号通路（M3 毒蕈碱乙酰胆碱受体和 5 型代谢型谷氨酸受体），它们具有非常不同的振荡机制，并且在生理和药理学上都很重要（糖尿病）和精神分裂症）。目标 1. 分析碱性条件下细胞的锁相响应：用受体配体对活细胞进行微流体脉冲刺激。使用钙和 IP3 的基因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，完善电路架构和参数以匹配目标 1 中的观察结果。目标 3. 通过锁相分析描绘调节剂的作用机制：研究细胞的锁相反应在抑制剂、激动剂和调节剂存在下如何变化。使用数学模型的实验观察来描述作用机制。目标 4. 传播自调节芯片，使任何人都可以进行微流体锁相研究。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Microfluidic automation using elastomeric valves and droplets: reducing reliance on external controllers.

DOI：
10.1002/smll.201200456
发表时间：
2012-10-08
期刊：
SMALL
影响因子：
13.3
作者：
Kim, Sung-Jin;Lai, David;Park, Joong Yull;Yokokawa, Ryuji;Takayama, Shuichi
通讯作者：
Takayama, Shuichi

Control of soft machines using actuators operated by a Braille display.

DOI：
10.1039/c3lc51083b
发表时间：
2014-01-07
期刊：
Lab on a chip
影响因子：
6.1
作者：
Mosadegh B;Mazzeo AD;Shepherd RF;Morin SA;Gupta U;Sani IZ;Lai D;Takayama S;Whitesides GM
通讯作者：
Whitesides GM

Label-free direct visual analysis of hydrolytic enzyme activity using aqueous two-phase system droplet phase transitions.