Department of Neuroanatomy and Cellular Neurobiology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Bunkyo-ku, Tokyo 113-8519, Japan.
Center for Brain Integration Research, TMDU, Bunkyo-ku, Tokyo 113-8519, Japan.
Proc Natl Acad Sci U S A. 2021 Mar 16;118(11). doi: 10.1073/pnas.2019071118.
Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArIS, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArIS with FPM. In living starfish early embryos expressing POLArIS, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.
生物分子组装体控制着细胞的生理学功能。它们的功能通常取决于组成分子在位置和方向上的分子排列变化。尽管包括超分辨率显微镜在内的荧光显微镜的最新进展使我们能够以前所未有的精度确定荧光团的位置，但实时监测活细胞内荧光标记分子的取向仍然具有挑战性。荧光偏振显微镜（FPM）报告发射偶极子的取向，因此是一种很有前途的解决方案。对于 FPM 成像，目标蛋白需要以空间受限的方式用荧光探针标记，但由于蛋白质连接的三维设计存在困难，因此没有通用的荧光标记受限标记方法。在这里，我们报告了 POLArIS，这是一种用于分子取向成像的遗传编码且多功能的探针。我们没有使用直接标记方法，而是使用重组结合物以空间受限的方式与荧光蛋白连接，通过与噬菌体展示筛选相结合，可以靶向特定感兴趣的生物分子。作为初步测试案例，我们开发了 POLArIS，它可以特异性地结合活细胞中的 F-肌动蛋白。我们通过用 FPM 观察表达 POLArIS 的细胞证实了可以监测 F-肌动蛋白的取向。在表达 POLArIS 的活海星早期胚胎中，我们发现有丝分裂期间中心体周围的微管星体与中心体周围的肌动蛋白丝呈放射状延伸。利用遗传编码的性质，POLArIS 可用于各种活体标本，包括发育中的胚胎和动物的整个身体，也可以以细胞类型/组织特异性的方式表达。
