Genetically-Encoded Voltage Probe Development

基因编码电压探针的开发

• 批准号: 8659528
• 负责人: THOMAS E HUGHES
• 金额: $ 59.86万
• 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 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8659528
• 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; 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; public health relevance; 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中枢基础。虽然被认为是一个世纪以来必不可少的，但研究回路水平神经生理学的工具在50年内基本保持不变。分子生物学的出现通过允许对神经系统进行分子表征而极大地推进了神经生物学，但并没有转化为神经电生理学的显著收益。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被用作电活动的替代标记，然而它们是暂时延迟的，不能检测阈下事件，并且通常不能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遗传编码电压传感器。近年来，我们的资助成员发表了遗传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显著改善了荧光电压探针的信号大小和响应速度。目前的申请将继续这种成功的电压探头合作搜索。我们正在寻找结合大F/ V信号大小、一系列有用的响应速度和红移荧光光谱的联合收割机探针。在之前的资助期内，我们发现通过改变电压传感器结构域、接头长度、荧光蛋白以及通过在荧光蛋白中引入点突变，我们可以开发出具有非常优越的上级信号大小和响应动力学的探针。然而，我们也证实了纯经验步骤（即大规模筛选单一的、递增修饰的 构建体）来显著改善响应特性。我们将采用分阶段进化方法，包括连续几轮的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。目前的实验将在两个重要方面优于所有先前的研究：i）我们将使用结构域交换和定点/随机诱变产生大量（20 x）潜在探针（数千），以及ii）大量构建体将通过自动化机器人显微荧光测定法进行预筛选，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，所有成功的候选人将在果蝇昼夜节律神经元和啮齿动物体感/桶皮质中验证体内功能。