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

描述（由申请人提供）：基因可编码的光学报告，如绿色荧光蛋白，已经彻底改变了细胞状态的观察和测量。原则上，利用光来控制和精确操纵不同的细胞过程同样是革命性的。然而，大多数光调节蛋白要么需要精确地连接半合成的发色团，要么需要控制特定的功能，比如通道打开。因此，由于缺乏一种与GFP相当的通用的、基因可编码的光敏蛋白，我们利用光来特异性扰乱生物系统的能力受到了阻碍。我们最近展示了一种新的遗传编码光控制蛋白-蛋白相互作用开关的使用，该开关来源于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普遍的货币之一，因此该系统原则上可用于控制各种功能。我们已经证明，该系统可以精确和可逆地将靶蛋白转移到膜上，具有微米级的空间分辨率和二次分辨率。通过易位Rho家族gtpase及其上游激活因子（控制肌动蛋白细胞骨架），我们可以利用光精确地重塑和指导哺乳动物细胞形态。我们已经证明了这个系统在酵母、盘基骨菌和哺乳动物细胞中起作用。在这里，我们提出开发光敏色素系统作为一个即插即用模块，可用于光门控标记分子的空间和时间。我们的目标是优化光控仪器，并在许多模型系统中开发单细胞光控模块的分子工具包。这种高度灵活的光门控蛋白-蛋白相互作用将提供远程控制“刻度盘”，使新一代细胞生物学中的微扰定量实验成为可能。

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.