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.

大脑计划的重要动机是渴望了解信息处理 原位神经元组织。为此，稳步增长的神经科学调查人员正在转向 与依赖电极的更传统的方法相比，要监测神经元活性的光学方法。 最常用的方法涉及遗传编码的荧光钙指标（即GCAMPS） 与多光子或宽场显微镜结合。这些方法允许大型活动测量 来自完整动物的鉴定神经元数量。这些研究的进步取决于 改进，一般编码的基于蛋白质的指标。荧光细胞内钙指标产生 响应神经元活性的强大信号，但是它们i）相对于动作暴露了非常慢的动力学 潜力，ii）个人行动潜力报告忠诚度和iii）无法可视化 膜电位的超极化。荧光电压指示器提供的信号更丰富 信息，更临时相关，并提供对神经元电活动的更直接测量。一个 过去已经开发了新的且实际上有用的荧光电压指示器 具有改进特性的十年。 在以前的资金期间，我们的实验室开发了高吞吐量工作流程来创建和 筛选蛋白质，电压敏感指标。使用这个平台，我们发现了几本小说 指标模板和新指标。随着筛查工作流程吞吐量的稳定增加，我们 正如预期的 实际用途。但是，蛋白质设计和修饰空间是巨大的，因此我们进一步提出 扩展筛选吞吐量并扩大模板设计空间。 我们建议每天筛选1150个新颖的结构或每周约8000个。我们将包括 鉴于新开发的筛选能力，该过程中的随机诱变。使用这个新平台我们 正在寻求开发：i）响应动作电位的指标最大的“爆发”光子输出， ii）荧光输出从弱的静息水平持续2-40毫秒来响应于作用 电型电压瞬变，iii）正电压/荧光输出斜率关系指标，iv） 具有绿色，红色和接近IR发射频谱的指标，v）针对针对亚细胞区域的指标 神经元和vi）降低漂白率的问题。