A Toolkit for Light-Control of Molecular Processes in Living Cells

活细胞中分子过程的光控制工具包

• 批准号: 8431348
• 负责人: CHRISTOPHER A VOIGT
• 金额: $ 44.35万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8431348
• 关键词: Actins; Area; Bacteria; Biochemistry; Biological Models; Biomedical Research; Cell model; Cell physiology; Cells; Cellular Morphology; Cellular biology; Communities; Complex; Cytoskeleton; Development; Dictyostelium; Disease; Engineering; Family; Feedback; Fibroblasts; Generations; Generic Drugs; Green Fluorescent Proteins; Guanosine Triphosphate Phosphohydrolases; Image; Individual; Information Systems; Kinetics; Label; Libraries; Life; Light; Light Cell; Location; Mammalian Cell; Measurement; Measures; Membrane; Microscopy; Modeling; Molecular; Mouse-ear Cress; Mus; Optical reporter; Organism; Pathway Analysis; Pattern; Phytochrome; Play; Postdoctoral Fellow; Process; Protein Engineering; Proteins; Relative (related person); Research; Research Personnel; Resolution; Resources; Saccharomycetales; Signal Pathway; Signal Transduction; Site; System; Technology; Time; Work; Yeasts; base; biological systems; chromophore; flexibility; in vivo; innovation; instrumentation; interdisciplinary collaboration; interest; light gated; mathematical model; novel; protein protein interaction; research study; rho; sensor; synthetic biology; tool

DESCRIPTION (provided by applicant): Genetically-encodable optical reporters, such as Green Fluorescent Protein, have revolutionized the observation and measurement of cellular states. In principle, it would be equally revolutionary to be able to control and precisely manipulate diverse cellular processes using light. Most light-regulated proteins, however, either require engineering of precisely tethered semi-synthetic chromophores or control specific functions, such as channel opening. Thus our ability to use light to specifically perturb biological systems is impeded by the lack of a generic, genetically-encodable light-sensitive protein equivalent to GFP. We have recently demonstrated the use of a new genetically encoded light-control protein-protein interaction switch, derived from the phytochrome signaling network of Arabidopsis thaliana. Because protein-protein interactions are one of the most general currencies of cellular information, this system can in principle be generically used to control diverse functions. We have shown that this system can be used to precisely and reversibly translocate target proteins to the membrane with micrometer spatial resolution and second time resolution. By translocating Rho family GTPases and their upstream activators, which control the actin cytoskeleton, we can use light to precisely reshape and direct the cell morphology of mammalian cells. We have demonstrated that this system works in yeast, Dictyostelium, and mammalian cells. Here we propose to develop the phytochrome system as a plug-and-play module that can be used for light-gated control of labeled molecules in space and time. We aim to optimize the light control instrumentation and to develop a molecular toolkit of single cell light control modules in a number of model systems. This highly flexible light-gated protein-protein interaction will provide remote control "dials" that will enable a new generation of perturbative, quantitative experiments in cell biology.

描述(申请人提供)：可遗传编码的光学记者，如绿色荧光蛋白，已经彻底改变了对细胞状态的观察和测量。原则上，能够利用光控制和精确操纵不同的细胞过程也将是同样具有革命性的。然而，大多数光调控蛋白要么需要精确连接的半合成发色团，要么需要控制特定的功能，如通道开放。因此，由于缺乏一种等同于GFP的通用的、可遗传编码的光敏蛋白，我们利用光来特定干扰生物系统的能力受到了阻碍。我们最近已经证明了一种新的遗传编码的光控制蛋白质-蛋白质相互作用开关的使用，该开关来自拟南芥的光敏色素信号网络。由于蛋白质-蛋白质相互作用是细胞信息最通用的货币之一，该系统原则上可以普遍用于控制不同的功能。我们已经证明，该系统可以以微米级的空间分辨率和二次分辨率将目标蛋白精确和可逆地转移到膜上。通过移位Rho家族GTP酶及其上游控制肌动蛋白细胞骨架的激活物，我们可以利用光来精确重塑和指导哺乳动物细胞的形态。我们已经证明了这个系统在酵母、网柄基菌和哺乳动物细胞中起作用。在这里，我们建议将光敏色素系统开发为一个即插即用模块，可以用于在空间和时间上对标记分子进行光门控制。我们的目标是优化光控制仪器，并开发一种用于多个模型系统的单细胞光控制模块的分子工具包。这种高度灵活的光门蛋白质-蛋白质相互作用将提供远程控制“拨盘”，使细胞生物学中的新一代微扰、定量实验成为可能。