An optogenetic method to rapidly and reversibly titrate protein levels in cells

一种快速可逆滴定细胞中蛋白质水平的光遗传学方法

• 批准号: 8570534
• 负责人: Orion D Weiner
• 金额: $ 18.76万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8570534
• 关键词: Acute; Address; Area; Behavior; Benchmarking; Biochemistry; Biological Assay; Biological Models; Biological Process; Biological Sciences; Biomedical Research; Cataloging; Catalogs; Cell Cycle; Cell Line; Cell Signaling Process; Cell physiology; Cells; Cellular biology; Chemicals; Chemotaxis; Communities; Development; Dictyostelium; Dictyostelium discoideum; Dimerization; Environment; Eukaryotic Cell; Event; Galactose; Genetic; Goals; Heterodimerization; Human; Human Cell Line; Life; Light; Location; Mammalian Cell; Measures; Methods; Modeling; Monitor; Pathway interactions; Performance; Phase; Process; Protein Inhibition; Proteins; RNA Interference; Relative (related person); Research; Saccharomyces cerevisiae; Saccharomycetales; Signal Transduction; Signaling Protein; Specificity; Staging; System; Techniques; Technology; Testing; Time; Zinc Fingers; base; cell behavior; gene function; genetic manipulation; homologous recombination; human disease; improved; interest; knockout gene; light gated; loss of function; nuclease; optogenetics; phyB phytochrome; protein function; public health relevance; response; small hairpin RNA; tool

DESCRIPTION (provided by applicant): Loss-of-function approaches such as gene knockout/knockdown have been widely used to identify the proteins that are necessary to organize various cellular behaviors. However common approaches, such as genetics or RNAi, change the cellular environment permanently. Over days and weeks compensatory changes can accumulate, alter the cell's physiology, and confound conclusions. Current approaches that act on faster (seconds) timescales have limitations in their specificity, are often irreversible, hrd to titrate, or difficult to apply to a wide range of target proteins. Moreover, in cases where most components of a pathway have been identified, questions shift towards timing "At what stage is my protein involved?" and level "Is cell behavior sensitive to the concentration of my protein?" Answers to such questions require the ability to trigger, set, reverse, and monitor the extent of protein inactivation with high precision - an area where traditional methods fail, and new tools are required. Towards this end, we are developing an optogenetics based loss-of-function method termed DeLIGHT (Depletion with LIGHT), as a much-needed tool for the growing field of single-cell biochemistry. We have recently made breakthroughs in developing a light-induced protein interaction module and will here combine this tool with an 'anchor away' strategy to reversibly sequester proteins at inert intracellular locations. Our technique is generalizable, acute, and reversible, enabling significantly greater control over protein activity than existing inactivation methods. DeLight makes it possible to measure single-cell responses at user-defined intermediate concentrations of a protein of interest, where the extent of inactivation can be visually monitored and set by the user in an interactive fashion. We propose to establish and optimize this approach in 3 model systems - Dictyostelium, budding yeast, and mammalian cells to test the modularity of this system, demonstrate its versatility, and provide tools for immediate application by the cell biology community.

描述（由申请人提供）：诸如基因敲除/敲除之类的功能丧失方法已被广泛用于识别组织各种细胞行为所必需的蛋白质。无论遗传学或RNAi等常见方法如何永久改变细胞环境。在几天和几周内，补偿性变化可以积累，改变细胞的生理和混淆结论。当前以更快（秒）时间标准起作用的方法在其特异性上具有局限性，通常是不可逆的，HRD滴定或难以应用于广泛的靶蛋白。而且，如果大多数 已经确定了途径的组成部分，问题向时机转向“我的蛋白质涉及哪个阶段？”水平“细胞行为对我的蛋白质浓度敏感？”此类问题的答案需要能够以高精度触发，设置，逆转和监视蛋白质失活的程度 - 传统方法失败的区域以及需要新的工具。为此，我们正在开发一种基于光遗传学的功能丧失方法，称为“喜悦”（用光耗尽），这是一种急需的单细胞生物化学领域的工具。最近，我们在开发光诱导的蛋白质相互作用模块方面取得了突破，并在这里将该工具与“锚点”策略相结合，以逆转细胞内位置隔离蛋白质。与现有的失活方法相比，我们的技术是可推广，急性和可逆的，可以显着控制蛋白质活性。 Delight使您可以在感兴趣的蛋白质的用户定义的中间浓度下测量单细胞响应，在这种蛋白质中，用户可以通过交互式方式在视觉上监测和设置失活的程度。我们建议在三种模型系统中建立和优化这种方法 - dictyostelium，萌芽的酵母和哺乳动物细胞，以测试该系统的模块化，展示其多功能性，并提供立即的工具 细胞生物学社区的应用。