Department of Chemistry, Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden.
Methods Mol Biol. 2021;2262:259-267. doi: 10.1007/978-1-0716-1190-6_15.
Small GTPases cycle between active GTP bound and inactive GDP bound forms in live cells. They act as molecular switches and regulate diverse cellular processes at different times and locations in the cell. Spatiotemporal visualization of their activity provides important insights into dynamics of cellular signaling. Conformational sensors for GTPase activity (COSGAs) are based on the conserved GTPase fold and have been used as a versatile approach for imaging small GTPase activity in the cell. Conformational changes upon GDP/GTP binding can be visualized directly in solution, on beads, or in live cells using COSGA by fluorescence lifetime imaging microscopy (FLIM) technique. Herein, we describe the construction of COSGA for imaging K-Ras GTPase activity in live cells.
在活细胞中，小分子 GTP 酶在活性 GTP 结合和非活性 GDP 结合形式之间循环。它们作为分子开关，在细胞的不同时间和位置调节各种细胞过程。对其活性的时空可视化提供了对细胞信号转导动力学的重要见解。GTP 酶活性构象传感器 (COSGAs) 基于保守的 GTP 酶折叠，已被用作在细胞中成像小分子 GTP 酶活性的通用方法。在溶液中、在珠上或在活细胞中，通过 COGSAs 可以使用荧光寿命成像显微镜 (FLIM) 技术直接可视化 GDP/GTP 结合引起的构象变化。在此，我们描述了用于在活细胞中成像 K-Ras GTP 酶活性的 COSGA 的构建。
