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

描述（由申请人提供）：遗传编码的光学报告分子，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，能够利用光来控制和精确操纵各种细胞过程同样具有革命性。然而，大多数光调节蛋白要么需要精确拴系的半合成发色团的工程化，要么控制特定的功能，如通道开放。因此，我们利用光来特异性干扰生物系统的能力受到缺乏与GFP等同的通用的、遗传上可编码的光敏蛋白的阻碍。我们最近展示了一个新的基因编码的光控制蛋白质-蛋白质相互作用开关，来自拟南芥光敏色素信号网络的使用。因为蛋白质-蛋白质相互作用是细胞信息的最普遍的货币之一，所以这个系统原则上可以被普遍地用于控制不同的功能。我们已经表明，该系统可以用于精确和可逆地将靶蛋白转位到膜上，具有微米级的空间分辨率和秒级的时间分辨率。通过易位Rho家族GTP酶及其上游激活剂，控制肌动蛋白细胞骨架，我们可以使用光来精确重塑和指导哺乳动物细胞的细胞形态。我们已经证明，该系统在酵母，网柄藻，和哺乳动物细胞。在这里，我们建议开发光敏色素系统作为一个即插即用的模块，可用于标记的分子在空间和时间的光门控控制。我们的目标是优化光控制仪器，并在一些模型系统中开发单细胞光控制模块的分子工具包。这种高度灵活的光门控蛋白质-蛋白质相互作用将提供远程控制“拨号盘”，这将使新一代的微扰，定量实验在细胞生物学。

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.