POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos
POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos
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DOI：
10.1073/pnas.2019071118
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发表时间：
2021-03-16
影响因子：
11.1
通讯作者：
Terada, Sumio
中科院分区：
文献类型：
--
作者：
Sugizaki, Ayana;Sato, Keisuke;Terada, Sumio
生物分子组合控制着细胞的生理机能。它们的功能通常取决于组分分子排列的变化，包括位置和方向的变化。虽然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方向是具有挑战性的。荧光偏振显微镜（FPM）报告了发射偶极子的取向，因此是一个很有前途的解决方案。FPM成像时，需要用荧光探针对靶蛋白进行空间约束标记，但由于蛋白质连接的合理三维设计存在困难，因此没有一种通用的荧光团约束标记方法。在这里，我们报告POLArIS，一个基因编码和多功能探针分子取向成像。我们没有使用直接标记方法，而是使用了一种重组粘合剂，以一种空间受限的方式连接到荧光蛋白上，通过结合噬菌体展示筛选，可以靶向感兴趣的特定生物分子。作为最初的测试案例，我们开发了POLArISact，它可以特异性地与活细胞中的F-actin结合。我们证实，通过观察用FPM表达POLArISact的细胞，可以监测F-actin的取向。在表达POLArISact的活海星早期胚胎中，我们发现在有丝分裂过程中，肌动蛋白丝与微管紫晶一起从中心体呈放射状延伸。利用基因编码的特性，POLArIS可以用于各种活体标本，包括发育中的胚胎和动物的整个身体，也可以以细胞类型/组织特定的方式表达。
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 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 be expressed in a cell type/tissue specific manner.