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）：功能丧失方法（如基因敲除/敲低）已广泛用于鉴定组织各种细胞行为所必需的蛋白质。然而，常见的方法，如遗传学或RNAi，永久改变细胞环境。经过数天和数周的补偿变化可以积累，改变细胞的生理机能，并混淆结论。目前作用于更快（秒）时间尺度的方法在其特异性方面有局限性，通常是不可逆的，滴定困难，或难以应用于广泛的靶蛋白。此外，在大多数 当一个途径的组成部分已经确定，问题转向时间“在什么阶段是我的蛋白质参与？和水平“细胞行为对蛋白质的浓度敏感吗？“这些问题的答案需要能够高精度地触发，设置，逆转和监测蛋白质失活的程度-这是传统方法失败的领域，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DeLIGHT（用LIGHT耗尽），作为日益增长的单细胞生物化学领域急需的工具。我们最近在开发光诱导蛋白质相互作用模块方面取得了突破，并将在这里将联合收割机与“锚离”策略结合，以可逆地将蛋白质隔离在惰性细胞内位置。我们的技术是可推广的、急性的和可逆的，与现有的灭活方法相比，能够显着更好地控制蛋白质活性。DeLight可以在用户定义的目标蛋白质的中间浓度下测量单细胞反应，其中失活程度可以由用户以交互式方式进行可视化监测和设置。我们建议在3个模型系统中建立和优化这种方法-网骨藻，芽殖酵母和哺乳动物细胞，以测试该系统的模块性，证明其通用性，并提供工具，立即 细胞生物学社区的应用。