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.
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Ikeda, Osaka, 563-8577, Japan.
Biochem Biophys Res Commun. 2021 Aug 6;565:50-56. doi: 10.1016/j.bbrc.2021.05.088. Epub 2021 Jun 2.
Fluorescence polarization microscopy (FPM) can visualize the dipole orientation of fluorescent molecules and has been used for analyzing architectural dynamics of biomolecules including cytoskeletal proteins. To monitor the orientation of target molecules by FPM, target molecules need to be labeled with fluorophores in a sterically constrained manner, so that the fluorophores do not freely rotate. Recently, a versatile probe for such labeling using fluorescent proteins, POLArIS (Probe for Orientation and Localization Assessment, recognizing specific Intracellular Structures of interest), was reported. POLArIS is a fusion protein consisting of a non-immunoglobulin-based recombinant binder Affimer and a green fluorescent protein (GFP), where the Affimer and GFP are rigidly connected to each other. POLArIS probe for molecules of interest can be developed through phage display screening of Affimer. This screening is followed by the rigid connection of fluorescent proteins to the selected Affimers. The Affimer-based POLArIS, however, cannot be used with animal immune libraries for selecting specific binder clones. In addition, multi-color FPM by POLArIS was not available due to the lack of color variations of POLArIS. In this study, we have developed new versions of POLArIS with nanobodies, which are compatible with animal immune libraries, and expanded color variations of POLArIS with cyan/green/yellow/red fluorescent proteins, enabling multi-color orientation imaging for multiple targets. Using nanobody-based POLArIS orientation probes, we performed two-color FPM of F-actin and vimentin in living cells. Furthermore, we made nanobody-based POLArIS probes that have different dipole orientations for adjusting the orientation of fluorescence polarization with respect to the target molecules. These nanobody-based POLArIS with options of colors and dipole orientations will enhance the performance of this probe for broader applications of fluorescence polarization imaging in living cells, tissues, and whole organisms.
荧光偏振显微镜（FPM）可以可视化荧光分子的偶极取向，已被用于分析包括细胞骨架蛋白在内的生物分子的结构动力学。为了通过 FPM 监测目标分子的取向，需要以空间受限的方式用荧光团标记目标分子，以使荧光团不能自由旋转。最近，报道了一种用于此类标记的荧光蛋白的多功能探针，即 POLArIS（用于评估取向和定位的探针，识别特定的感兴趣的细胞内结构）。POLArIS 是一种由非免疫球蛋白的重组结合物 Affimer 和绿色荧光蛋白（GFP）组成的融合蛋白，其中 Affimer 和 GFP 彼此刚性连接。用于感兴趣的分子的 POLArIS 探针可以通过 Affimer 的噬菌体展示筛选来开发。然后，将荧光蛋白刚性连接到所选的 Affimers 上。然而，由于 POLArIS 缺乏颜色变化，因此基于 Affimer 的 POLArIS 不能与动物免疫文库一起用于选择特定的结合物克隆。此外，由于 POLArIS 缺乏颜色变化，因此无法进行多色 FPM。在这项研究中，我们开发了具有纳米体的新型 POLArIS，它与动物免疫文库兼容，并通过 cyan/green/yellow/red 荧光蛋白扩展了 POLArIS 的颜色变化，实现了多个目标的多色取向成像。使用基于纳米体的 POLArIS 取向探针，我们在活细胞中进行了 F-肌动蛋白和波形蛋白的双色 FPM。此外，我们还制备了具有不同偶极取向的基于纳米体的 POLArIS 探针，以调整相对于目标分子的荧光偏振的取向。这些具有颜色和偶极取向选择的基于纳米体的 POLArIS 将增强该探针在活细胞、组织和整个生物体中的荧光偏振成像的更广泛应用中的性能。
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