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 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 due to difficulties in the rational three-dimensional design of protein connection, 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 and can target arbitrary biomolecules by combining with phage-display screening. As an initial test case of POLArIS, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, 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 expressed in a cell-type/tissue specific manner.

中文翻译：

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POLArIS，一种用于分子定向的多功能探针，揭示了早期胚胎中与微管紫苑相关的肌动蛋白丝

生物分子组装体控制细胞的生理。它们的功能通常取决于成分的分子排列在位置和方向上的变化。尽管包括超高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，但是实时监测活细胞内荧光标记分子的方向仍是一项挑战。荧光偏振显微镜（FPM）报告了发射偶极子的方向，因此是一种很有前途的解决方案。为了使用FPM进行成像，目标蛋白需要以空间受限的方式用荧光探针标记，但是由于蛋白质连接的合理三维设计困难，没有通用的方法来限制荧光团标记。在这里，我们报告POLArIS，一种用于分子定向成像的遗传编码且用途广泛的探针。代替使用直接标记方法，我们使用了以空间受限方式连接到荧光蛋白的重组结合剂，并且可以通过结合噬菌体展示筛选来靶向任何生物分子。作为POLArIS的最初测试案例，我们开发了POLArISact，它与活细胞中的F-肌动蛋白特异性结合。我们证实，可以通过观察用FPM表达POLArISact的细胞来监测F-肌动蛋白的方向。在表达POLArISact的活海星早期胚胎中，我们发现在有丝分裂期间，肌动蛋白丝从中心体放射状延伸，并伴有微管翠菊。利用遗传密码的本质，