Genetically-Encoded Voltage Probe Development

基因编码电压探针的开发

• 批准号: 8825541
• 负责人: THOMAS E HUGHES
• 金额: $ 55.16万
• 依托单位: JOHN B. PIERCE LABORATORY, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-15 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8825541
• 关键词: Action Potentials; Behavioral; Brain; Calcium; Calcium Signaling; Cells; Ciona intestinalis; Circadian Rhythms; Collaborations; Color; Complex; Computers; Cytophotometry; Development; Disease; Drosophila genus; Electrodes; Electrophysiology (science); Electroporation; Engineering; Event; Evolution; Fluorescence; Fluorescent Probes; Funding; Genes; Glare; Grant; Green Fluorescent Proteins; Health; Hippocampus (Brain); Image; Imaging Techniques; Kinetics; Label; Laboratories; Length; Libraries; Life; Light; Mammalian Cell; Membrane Potentials; Metabolic; Methods; Modification; Molecular; Molecular Biology; Monitor; Mus; Mutagenesis; Mutate; Mutation; Nervous System Physiology; Nervous system structure; Neurobiology; Neuromodulator; Neurons; Optical Methods; Oranges; Orthologous Gene; Patients; Phosphoric Monoester Hydrolases; Point Mutation; Property; Protein Inhibition; Proteins; Publishing; Reading; Recovery; Reporter; Reporting; Rhodopsin; Robotics; Rodent; Science; Signal Transduction; Site; Smell Perception; Specificity; Speed; Spinal cord damage; Staging; Surrogate Markers; Techniques; Testing; Time; Tissues; Translating; Validation; Work; Zebrafish; awake; barrel cortex; base; brain cell; combinatorial; design; electrical potential; fictional works; improved; in utero; in vivo; insight; member; millisecond; neural circuit; neurophysiology; optogenetics; patch clamp; prototype; relating to nervous system; research study; response; screening; sensor; somatosensory; tau Proteins; technique development; tool; voltage

DESCRIPTION (provided by applicant): Neuronal electrical activity is the central underpinning of nervous system function. While understood as essential for over a century, the tools to study circuit level neurophysiology have remained largely unchanged in 50 years. The advent of molecular biology has dramatically advanced neurobiology by allowing molecular characterization of the nervous system but has not translated into significant gains in neural electrophysiology. Opto-molecular methods have revolutionized our study of neuronal connectivity, development, gene distribution, calcium signaling and recently, targeted neuronal activation (i.e. optogenetics). A glaring exception to this light-based revolution is the use of optical methods to monitor electrical activity. Intracellular calcium levels and metabolic signals are often used as a surrogate marker of electrical activity, however they are temporally delayed, do not detect subthreshold events and more often than not fail to capture the relevant suprathreshold activity. The PIs laboratories, as part of a multi laboratory collaboration have been developing genetically encoded voltage sensors based on fusions of green fluorescent protein orthologs and voltage sensing domains. Our grant members have published most of the significant advances in genetically-encoded voltage sensors in recent years. Our most recent probes, Arclight and ElectricPK significantly improved the signal size and response speed of fluorescent voltage probes. The current application will continue this successful collaborative search for voltage probes. We are seeking probes which combine large F/ V signal sizes, a range of useful response speeds and red-shifted fluorescence spectra. During this previous funded period time, we discovered that by altering the voltage sensor domain, the linker length, the fluorescent protein and by introducing point mutations in the fluorescent protein, we could develop probes with vastly superior signal size and response kinetics. We also confirmed, however, that a purely empirical step (i.e. large scale screening of single, incrementally-modified constructs) is required to make dramatic improvements in response properties. We will employ a staged evolution approach involving successive rounds of directed and random sequence modification followed by direct testing in mammalian cells. The current experiments will be an advance over all previous studies in two important ways: i) we will create vastly greater numbers (20x) of potential probe (thousands) using domain swapping and site directed / random mutagenesis and ii) the larger numbers of constructs will be prescreened by an automated, robotic microfluorimetry method which evaluates the fluorescence signal size and speed in electrically-active mammalian cells. Finally, all successful candidates will be validated for in vio functionality in Drosophila circadian neurons and rodent somatosensory/barrel cortex.

描述（由申请人提供）：神经元电活动是神经系统功能的核心基础。尽管一个多世纪以来研究电路级神经生理学的工具一直被认为是必不可少的，但 50 年来基本没有变化。分子生物学的出现通过对神经系统进行分子表征，极大地推进了神经生物学的发展，但尚未转化为神经电生理学的重大进展。光分子方法彻底改变了我们对神经元连接、发育、基因分布、钙信号传导以及最近的靶向神经元激活（即光遗传学）的研究。这场基于光的革命的一个明显的例外是使用光学方法来监测电活动。细胞内钙水平和代谢信号通常用作电活动的替代标记，但它们会暂时延迟，无法检测阈下事件，并且通常无法捕获相关的阈上活动。作为多实验室合作的一部分，PI 实验室一直在开发基于绿色荧光蛋白直系同源物和电压传感域融合的基因编码电压传感器。近年来，我们的资助成员发表了基因编码电压传感器的大部分重大进展。我们最新的探针 Arclight 和 ElectricPK 显着提高了荧光电压探针的信号大小和响应速度。当前的应用将继续这种对电压探头的成功合作搜索。我们正在寻找结合了大 F/V 信号大小、一系列有用的响应速度和红移荧光光谱的探针。在之前的资助期间，我们发现通过改变电压传感器结构域、接头长度、荧光蛋白以及在荧光蛋白中引入点突变，我们可以开发出具有极其优越的信号大小和响应动力学的探针。然而，我们也证实，纯粹的经验步骤（即大规模筛选单一的、增量修改的 结构）需要显着改善响应特性。我们将采用分阶段进化方法，包括连续几轮定向和随机序列修饰，然后在哺乳动物细胞中进行直接测试。当前的实验将在两个重要方面比以前的所有研究取得进展：i）我们将使用域交换和定点/随机诱变创建更多数量（20x）的潜在探针（数千），ii）更多数量的构建体将通过自动机器人微荧光测定方法进行预筛选，该方法评估电活性哺乳动物细胞中的荧光信号大小和速度。最后，所有成功的候选者都将在果蝇昼夜节律神经元和啮齿动物体感/桶状皮质中进行体外功能验证。