Genetically-encoded optical sensors to study purinergic signaling

用于研究嘌呤能信号传导的基因编码光学传感器

• 批准号: 8995713
• 负责人: Mathew Tantama
• 金额: $ 22.96万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8995713
• 关键词: Adenosine; Affinity; Astrocytes; Axon; Behavior; Binding; Biosensor; Blood flow; Brain; Brain Pathology; Calcium; Cell surface; Cells; Communication; Communities; Detection; Electrophysiology (science); Engineering; Epilepsy; Extracellular Space; Feedback; G-Protein-Coupled Receptors; Glutamates; Goals; Health; Image; Immunity; In Vitro; Kinetics; Lead; Life; Locales; Measures; Mediating; Membrane; Methods; Microglia; Nervous system structure; Neurodegenerative Disorders; Neuroglia; Neurons; Neurotransmitters; Nucleotidases; Optics; P2X-receptor; Pain; Photons; Physiological; Physiology; Presynaptic Terminals; Process; Property; Protein Engineering; Protocols documentation; Purinoceptor; Resolution; Role; Signal Transduction; Slice; Stroke; Synapses; Synaptic Transmission; Synaptic plasticity; System; Testing; Time; Tissues; Vertebral column; Vesicle; Work; age related; analytical tool; animal tissue; autocrine; body system; brain cell; cell type; extracellular; in vivo; live cell microscopy; meetings; multi-photon; neuroregulation; optical sensor; paracrine; receptor; response; sensor; tool; transmission process

 DESCRIPTION (provided by applicant): Extracellular ATP mediates purinergic signaling throughout the nervous system, and purinergic signaling mechanisms have been associated with multiple brain pathologies including stroke, aging-related neurodegenerative diseases, and epilepsy. Therefore, methods to accurately and precisely detect extracellular ATP are essential to the study of its physiological role. To this end, our broad goal is to develop a set of quantitative optical tools to image and study the signaling dynamics of extracellular ATP in real-time at central synapses. In particular, evidence in recent decades suggests that astrocytes modulate synaptic transmission and plasticity through activity-dependent release of gliotransmitters that include extracellular ATP. Spillover of neurotransmitters during synaptic activity can activate G-protein coupled receptors on perisynaptic astrocytes. It is proposed that this activation of astrocyte receptors elicits a calcium response that can lead to release of ATP as a gliotransmitter. Subsequently, the released ATP or its metabolite adenosine can bind to and activate neuronal purinergic receptors, causing either homosynaptic or heterosynaptic neuromodulation. Thus, extracellular ATP released from astrocytes might act as a feedback signal to modulate synaptic efficacy and network behavior. However, there is an unmet need for new analytical tools to measure extracellular ATP, and the limitations of current detection methods have impeded the resolution of important questions regarding the mechanisms and physiological relevance of ATP as a gliotransmitter. To meet this need, the central hypothesis of this proposal is that genetically-encoded fluorescent biosensors can be engineered to sense extracellular ATP with sensor properties suitable for studying physiologically relevant purinergic signaling. We will test our hypothesis in two working aims: Aim 1 is to engineer cell surface-tethered biosensors to quantitatively image ATP release and clearance; Aim 2 is to use these new biosensors to measure activity- dependent ATP release and clearance in primary cultures of astrocytes and neurons as well as in brain slices. Upon completion of these aims, we will be able to provide both new optical tools for measuring extracellular ATP and imaging protocols that are of broad use to the purinergic signaling community.

 描述（由申请人提供）：细胞外 ATP 介导整个神经系统的嘌呤能信号传导，并且嘌呤能信号传导机制与多种脑部病理学相关，包括中风、衰老相关的神经退行性疾病和癫痫。因此，需要准确且精确地检测细胞外 ATP 的方法。为此，我们的总体目标是开发一套定量光学工具来实时成像和研究细胞外 ATP 的信号动力学。特别是，近几十年来的证据表明，星形胶质细胞通过神经递质的活动依赖性释放来调节突触传递和可塑性，其中神经递质在突触活动期间的溢出可以激活突触周围星形胶质细胞上的G蛋白偶联受体。星形胶质细胞受体的这种激活会引发钙反应，从而导致 ATP 作为胶质递质释放，随后释放出 ATP 或其物质。代谢物腺苷可以结合并激活神经元嘌呤能受体，引起同突触或异突触神经调节，因此，星形胶质细胞释放的细胞外 ATP 可能充当调节突触功效和网络行为的反馈信号。测量细胞外 ATP，当前检测方法的局限性阻碍了有关 ATP 作为胶质递质的机制和生理相关性的重要问题的解决。为了满足这一需求，该提案的中心假设是，基因编码的荧光生物传感器可以被设计为感测细胞外 ATP，其传感器特性适合研究生理相关的嘌呤能信号传导。我们将在两个工作目标中测试我们的假设：目标 1 是设计细胞表面束缚的生物传感器来定量成像 ATP 释放和清除；目标 2 是使用这些新的生物传感器来测量星形胶质细胞和神经元原代培养物中活性依赖性 ATP 释放和清除；完成这些目标后，我们将能够提供用于测量细胞外 ATP 的新光学工具和广泛用于嘌呤能信号传导领域的成像协议。

项目成果

Dual-Mode FRET and BRET Sensors for Detecting cAMP Dynamics.

用于检测 cAMP 动态的双模式 FRET 和 BRET 传感器。

• 发表时间: 2019-09-24
• 影响因子: 4.1
• 作者: French, Alexander R;Tesmer, Alexander L;Tantama, Mathew
• 通讯作者: Tantama, Mathew

Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP.

使用 ADP 和 ATP 基因编码荧光传感器检测渗透压休克诱导的细胞外核苷酸释放。

• 发表时间: 2019-07-24
• 作者: Trull, Keelan J;Miller, Piper;Tat, Kiet;Varney, S Ashley;Conley, Jason M;Tantama, Mathew
• 通讯作者: Tantama, Mathew

Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor.

使用基因编码的比率荧光传感器对细胞外 ATP 进行成像。

• 发表时间: 2017
• 影响因子: 3.7
• 作者: Conley, Jason M;Radhakrishnan, Saranya;Valentino, Stephen A;Tantama, Mathew
• 通讯作者: Tantama, Mathew

Imaging pH Dynamics Simultaneously in Two Cellular Compartments Using a Ratiometric pH-Sensitive Mutant of mCherry.

使用 mCherry 的比率 pH 敏感突变体同时对两个细胞室中的 pH 动态进行成像。

• 发表时间: 2018-08-31
• 影响因子: 4.1
• 作者: Rajendran, Megha;Claywell, Benjamin;Haynes, Emily P;Scales, Umi;Henning, Chace K;Tantama, Mathew
• 通讯作者: Tantama, Mathew

Imaging Adenosine Triphosphate (ATP).

三磷酸腺苷 (ATP) 成像。

• 发表时间: 2016-08
• 作者: Rajendran, Megha;Dane, Eric;Conley, Jason;Tantama, Mathew
• 通讯作者: Tantama, Mathew