High Throughput of Protein-based Voltage Probes

基于蛋白质的电压探针的高通量

• 批准号: 9769176
• 负责人: Vincent A Pieribone
• 金额: $ 78.94万
• 依托单位: JOHN B. PIERCE LABORATORY, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9769176
• 关键词: Action Potentials; Amino Acids; Animals; BRAIN initiative; Biological Assay; Butterflies; Characteristics; Computers; Development; Electrodes; Exhibits; Fluorescence; Funding; Hour; Imagery; Imaging Techniques; In Situ; Individual; Kinetics; Laboratories; Libraries; Measurement; Measures; Membrane Potentials; Methods; Microelectrodes; Microscopy; Monitor; Motivation; Mutagenesis; Mutation; Neurons; Neurosciences; Optical Methods; Output; Patients; Photons; Post-Translational Protein Processing; Process; Production; Property; Protein Engineering; Proteins; Reporting; Research Personnel; Rest; Robot; Robotics; Scheme; Signal Transduction; Spinal cord damage; System; Techniques; Time; Tissues; Translating; Work; base; brain cell; brain electrical activity; calcium indicator; computerized data processing; density; design; improved; information processing; novel; operation; optical spectra; response; scale up; screening; voltage

A significant motivation of the BRAIN Initiative is the desire to understand information processing in neuronal tissues in situ. Towards this end a steadily growing number of neuroscience investigators are turning to optical methods to monitor neuronal activity as opposed to more traditional methods that rely on electrodes. The most commonly used method involves genetically encoded, fluorescent calcium indicators (i.e. GCaMPs) combined with multiphoton or wide field microscopy. These methods allow activity measurements in large numbers of identified neurons from intact animals. Advancement of these studies rely on the development of improved, genetically encoded, protein-based indicators. Fluorescent intracellular calcium indicators produce robust signals in response to neuronal activity, however they i) exhibit very slow kinetics relative to action potentials, ii) have poor individual action potential reporting fidelity and iii) cannot enable visualization of hyperpolarizations of membrane potential. Fluorescence voltage indicators provide signals which are richer in information, more temporally relevant and offer a more direct measure of neuronal electrical activity. A number of new and more practically useful fluorescent voltage indicators have been developed over the past decade with improved properties. During a previous funding period our laboratory developed a high throughput workflow to create and screen protein-based, voltage sensitive indicators. Using this platform we have discovered several novel indicator templates and new indicators. With the steady increase in throughput of our screening workflow we have seen, as expected, more rapid improvements in indicator properties which has translated to greater practical use. However, the protein design and modification space is enormous hence we propose further scaling up of the screening throughput and widening the template design space. We propose to screen upwards of 1150 novel constructs per day or ~8000 per week. We will include random mutagenesis in the process given the newly developed screening capacity. With this new platform we are seeking to develop : i) indicators with maximum total “burst” photon output in response to action potentials, ii) fluorescence output increases from a weak resting level lasting of between 2-40 ms in response to action potential-type voltage transients, iii) positive voltage/fluorescence output slope relationship indicators, iv) indicators with green, red and near IR emission spectrum, v) indicators targeted to subcellular regions of the neuron, and vi) probes with reduced bleach rates.

大脑计划的一个重要动机是希望了解信息处理过程 神经组织原位观察。为此，越来越多的神经科学研究人员正在转向 到光学方法来监测神经元活动，而不是更传统的依赖电极的方法。 最常用的方法涉及基因编码的荧光钙指示剂(即GCaMP) 结合多光子或广域显微镜。这些方法允许在很大程度上测量活动 从完整的动物身上识别出的神经元的数量。这些研究的进展有赖于 改良的、基因编码的、基于蛋白质的指标。荧光细胞内钙指示剂产生 对神经元活动作出反应的强健信号，然而它们i)相对于动作表现出非常缓慢的动力学 潜力，ii)个人行动潜力报告保真度较差，以及iii)无法实现可视化 膜电位的超极化。荧光电压指示器提供更丰富的信号 信息，在时间上更相关，并提供了更直接的神经元电活动测量。一个 在过去的几年里，已经开发了许多新的、更实用的荧光电压指示器 十年，性能得到改善。 在之前的资助期内，我们的实验室开发了高吞吐量的工作流来创建和 筛选以蛋白质为基础的电压敏感指示剂。利用这个平台，我们发现了几个新颖的 指标模板和新指标。随着我们筛选工作流程吞吐量的稳步增长，我们 正如预期的那样，指标属性得到了更快的改善，这转化为更大的 实用型。然而，蛋白质的设计和修饰空间是巨大的，因此我们建议进一步 扩大筛选吞吐量，拓宽模板设计空间。 我们建议每天筛选1150个以上的新构建物，或每周~8000个。我们将包括 在给予新开发的筛选能力的过程中进行随机突变。有了这个新平台，我们 正在寻求开发：i)具有响应于动作电位的最大总“突发”光子输出的指示器， 二)荧光输出从持续2-40毫秒的微弱静息水平增加 电位型电压瞬变，iii)正电压/荧光输出斜率关系指示器，iv) 具有绿色、红色和近红外发射光谱的指示器，v)指示器定位于 神经元和vi)具有较低漂白率的探针。