New approaches for imaging and controlling the molecules of life.

我们利用细胞周期依赖的泛素化调控来开发细胞周期进程的遗传编码指示剂Fucci。这项技术使我们能够可视化实验动物（如小鼠和鱼类）复杂组织中单个细胞的细胞周期行为。我们使用两种新的珊瑚荧光蛋白作为FRET供体和受体，开发了一个电压传感器，美人鱼。这项技术可以直接观察可兴奋细胞的电活动。我们开发了一种远红色荧光蛋白，具有很大的斯托克斯位移，Keima。我们展示了Keima对双色荧光成像技术的有用性，如荧光相互关联光谱和双光子激发显微镜。荧光蛋白Dronpa光致变色的结构基础尚不清楚。我们在环境温度下对溶液中的Dronpa进行了核磁共振分析，以发现蛋白质在暗状态下的结构灵活性。我们还开发了许多Dronpa的变种，它们以不同的速度在明暗状态之间变化。这些突变体被用来提高超分辨率显微镜的性能。KikGR是一种荧光蛋白，通过紫光照射诱导显示绿到红的光可转换性。通过对绿态和红态的晶体学研究，我们获得了控制绿到红光转换的β消除反应遵循E1（单分子）机制的证据。使用KikGR作为光学荧光笔，我们证明了在核重组过程中，跨越核包膜的扩散屏障的限制较小。

We harnessed the regulation of cell-cycle-dependent ubiquitination to develop a genetically encoded indicator for cell-cycle progression, Fucci. This technology permits us to visualize the cell-cycle behavior of individual cells within complex tissues of experimental animals, such as mice and fish. We used two new coral fluorescent proteins as FRET donor and acceptor to develop a voltage sensor, Mermaid. This technology allows for direct visualization of electrical activities in excitable cells. We developed a far-red fluorescent protein endowed with a large Stokes shift, Keima. We show the usefulness of Keima for dual-color fluorescence imaging technologies, such as fluorescence cross-correlation spectroscopy and two-photon excitation microscopy.The structural basis for the photochromism in the fluorescent protein Dronpa is poorly understood. We performed NMR analyses of Dronpa in solution at ambient temperatures to find structural flexibility of the protein in the dark state. We have also developed numerous mutants of Dronpa, which change between bright and dark states with different speeds. These mutants were used to improve performance of super-resolution microscopy. KikGR is a fluorescent protein engineered to display green-to-red photoconvertibility that is induced by irradiation with violet light. Through crystallographic studies on both green and red states, we obtained evidence that the β-elimination reaction governing the green-to-red photoconversion follows an E1 (unimolecular) mechanism. Using KikGR as an optical highlighter, we demonstrate that diffusion barrier across the nuclear envelope is less restrictive during nuclear reassembly.