Development of nanobody-based POLArIS orientation probes enabled multi-color/multi-target orientation imaging in living cells
Development of nanobody-based POLArIS orientation probes enabled multi-color/multi-target orientation imaging in living cells
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基于纳米体的 POLArIS 定向探针的开发实现了活细胞中的多色/多目标定向成像
DOI：
10.1016/j.bbrc.2021.05.088
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发表时间：
2021
影响因子：
3.1
通讯作者：
Terada Sumio.
中科院分区：
文献类型：
--
作者：
Nakai Nori;Sato Keisuke;Tani Tomomi;Kawagishi Masahiko;Ka Hiromasa;Saito Kenta;Terada Sumio.
荧光偏振显微镜（FPM）可以显示荧光分子的偶极取向，并已用于分析生物分子（包括细胞骨架蛋白）的结构动力学。为了通过FPM监测靶分子的取向，靶分子需要以空间受限的方式用荧光团标记，使得荧光团不会自由旋转。最近，报道了一种使用荧光蛋白进行这种标记的通用探针，POLArIS（用于定向和定位评估的探针，识别感兴趣的特定细胞内结构）。POLArIS是一种融合蛋白，由非免疫球蛋白基重组结合剂Affimer和绿色荧光蛋白（GFP）组成，其中Affimer和GFP彼此刚性连接。可以通过噬菌体展示筛选Affimer来开发用于感兴趣分子的POLArIS探针。该筛选之后是荧光蛋白与所选Affimer的刚性连接。然而，基于Affimer的POLArIS不能与动物免疫文库一起用于选择特异性结合子克隆。此外，由于POLArIS缺乏颜色变化，因此无法使用POLArIS的多色FPM。在这项研究中，我们已经开发了具有纳米抗体的新版本的POLArIS，其与动物免疫库兼容，并扩展了具有青色/绿色/黄色/红色荧光蛋白的POLArIS的颜色变化，从而能够对多个目标进行多色定向成像。使用基于纳米抗体的POLArIS定向探针，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，我们制作了基于纳米抗体的POLArIS探针，其具有不同的偶极取向，用于调节相对于靶分子的荧光偏振取向。这些基于纳米抗体的POLArIS具有颜色和偶极方向的选择，将增强该探针的性能，用于活细胞、组织和整个生物体中荧光偏振成像的更广泛应用。
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.