Nakai Nori, Sato Keisuke, Tani Tomomi, Saito Kenta, Sato Fumiya, Terada Sumio
Department of Neuroanatomy and Cellular Neurobiology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
Center for Brain Integration Research, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
Microscopy (Oxf). 2019 Oct 9;68(5):359-368. doi: 10.1093/jmicro/dfz022.
Fluorescence polarization microscopy, which can visualize both position and orientation of fluorescent molecules, is useful for analyzing architectural dynamics of proteins in vivo, especially that of cytoskeletal proteins such as actin. Fluorescent phalloidin conjugates and SiR-actin can be used as F-actin orientation probes for fluorescence polarization microscopy, but a lack of appropriate methods for their introduction to living specimens especially to tissues, embryos, and whole animals hampers their applications to image the orientation of F-actin. To solve this problem, we have developed genetically encoded F-actin orientation probes for fluorescence polarization microscopy. We rigidly connected circular permutated green fluorescent protein (GFP) to the N-terminal α-helix of actin-binding protein Lifeact or utrophin calponin homology domain (UtrCH), and normal mEGFP to the C-terminal α-helix of UtrCH. After evaluation of ensemble and single particle fluorescence polarization with the instantaneous FluoPolScope, one of the constructs turned out to be suitable for practical usage in live cell imaging. Our new, genetically encoded F-actin orientation probe, which has a similar property of an F-actin probe to conventional GFP-UtrCH, is expected to report the 3D architecture of the actin cytoskeleton with fluorescence polarization microscopy, paving the way for both the single molecular orientation imaging in cultured cells and the sub-optical resolution architectural analysis of F-actin networks analysis of F-actin in various living systems.
荧光偏振显微镜能够可视化荧光分子的位置和方向，对于分析体内蛋白质的结构动态，尤其是肌动蛋白等细胞骨架蛋白的结构动态非常有用。荧光鬼笔环肽缀合物和SiR-肌动蛋白可作为荧光偏振显微镜的F-肌动蛋白方向探针，但缺乏将它们引入活标本尤其是组织、胚胎和整个动物的合适方法，这阻碍了它们用于成像F-肌动蛋白的方向。为了解决这个问题，我们开发了用于荧光偏振显微镜的基因编码F-肌动蛋白方向探针。我们将环状排列的绿色荧光蛋白（GFP）刚性连接到肌动蛋白结合蛋白Lifeact或肌养蛋白钙调蛋白同源结构域（UtrCH）的N端α-螺旋，将正常的mEGFP连接到UtrCH的C端α-螺旋。在用瞬时FluoPolScope评估系综和单颗粒荧光偏振后，其中一种构建体被证明适用于活细胞成像的实际应用。我们新的基因编码F-肌动蛋白方向探针具有与传统GFP-UtrCH类似的F-肌动蛋白探针特性，有望通过荧光偏振显微镜报告肌动蛋白细胞骨架的三维结构，为培养细胞中的单分子方向成像以及各种生命系统中F-肌动蛋白网络的亚光学分辨率结构分析铺平道路。
