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标题：Spatiotemporal imaging of small GTPases activity in live cells
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作者：Stephanie Voss ; Dennis M. Krüger ; Oliver Koch 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：50
页码：14348-14353
DOI：10.1073/pnas.1613999113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceRas-like small GTPases regulate a wide range of cellular processes involving signal transduction, cytoskeleton rearrangement, and membrane trafficking by switching between an active GTP-bound and an inactive GDP-bound form. In this study, we developed conformational sensors for GTPase activity (COSGAs) by combining protein engineering and chemical labeling. The COSGAs enable quantitative detection of the GDP/GTP binding state of Rab1 and K-Ras in living cells with spatiotemporal resolution to gain insights into Rab membrane cycling and Ras signaling at the plasma membrane. This approach paves a new avenue for imaging activity of signaling molecules in the cell, which could deliver valuable information regarding their function and dynamics. Ras-like small GTPases function as molecular switches and regulate diverse cellular events. To examine the dynamics of signaling requires spatiotemporal visualization of their activity in the cell. Current small GTPase sensors rely on specific effector domains that are available for only a small number of GTPases and compete for endogenous regulator/effector binding. Here, we describe versatile conformational sensors for GTPase activity (COSGAs) based on the conserved GTPase fold. Conformational changes upon GDP/GTP exchange were directly observed in solution, on beads, and in live cells by Forster resonance energy transfer (FRET). The COSGAs allow for monitoring of Rab1 and K-Ras activity in live cells using fluorescence lifetime imaging microscopy. We found that Rab1 is largely active in the cytoplasm and inactive at the Golgi, suggesting that the Golgi serves as the terminal of the Rab1 functional cycle. K-Ras displays polarized activity at the plasma membrane, with less activity at the edge of the cell and membrane ruffles.
关键词：small GTPases ; membrane trafficking ; signal transduction ; FLIM/FRET ; protein chemical modification