Permeabilization activated reduction in fluorescence: A novel method to measure kinetics of protein interactions with intracellular structures.
Permeabilization activated reduction in fluorescence: A novel method to measure kinetics of protein interactions with intracellular structures.
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透化激活荧光减少：一种测量蛋白质与细胞内结构相互作用动力学的新方法。
DOI：
10.1002/cm.21306
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Quintero,OmarA
中科院分区：
文献类型：
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作者：
Singh,PaliP;Hawthorne,JenciL;Davis,ChristieA;Quintero,OmarA
理解运动信息是理解生物功能的基础。先进的成像技术促进了细胞动力学分析的发展。我们发展了渗透性激活荧光还原(PARF)分析，用于确定表观1/2和固定部分，描述了感兴趣的蛋白质从细胞内结构中解离。为了创造可观察到解离事件的条件，用洋地黄素使表达荧光标记蛋白的细胞通透性，将未结合的蛋白稀释到细胞外介质中。当介质体积比胞液体积大得多时，非结合池的浓度急剧下降，使系统失去平衡，有利于解离事件。结合蛋白的损失可以观察到细胞内结构的荧光损失，并且可以符合指数衰减。我们比较了PARF解离动力学和先前发表的FRAP测定的平衡动力学。PARF解离速率与基于平衡的FRAP分析对这些速率大小的预测一致。当用于研究一组细胞骨架蛋白的结合动力学时，PARF分析表明，细丝稳定导致较慢的荧光损失。此外，常用的“通用”F-肌动蛋白标记物在动力学性质上表现出差异，这表明并不是所有荧光标记的肌动蛋白标记物都以相同的方式与肌动蛋白网络相互作用。我们还观察到GFP-VASP的差示解离动力学，这取决于标记的细胞结构。这些结果表明，非平衡体系的PARF分析揭示了动力学信息，而不需要其他定量方法所需的基础设施投资，如FRAP、光激活或体外重建分析。©2016 Wiley期刊，Inc.
Understanding kinetic information is fundamental in understanding biological function. Advanced imaging technologies have fostered the development of kinetic analyses in cells. We have developed Permeabilization Activated Reduction in Fluorescence (PARF) analysis for determination of apparentt1/2and immobile fraction, describing the dissociation of a protein of interest from intracellular structures. To create conditions where dissociation events are observable, cells expressing a fluorescently‐tagged protein are permeabilized with digitonin, diluting the unbound protein into the extracellular media. As the media volume is much larger than the cytosolic volume, the concentration of the unbound pool decreases drastically, shifting the system out of equilibrium, favoring dissociation events. Loss of bound protein is observed as loss of fluorescence from intracellular structures and can be fit to an exponential decay. We compared PARF dissociation kinetics with previously published equilibrium kinetics as determined by FRAP. PARF dissociation rates agreed with the equilibrium‐based FRAP analysis predictions of the magnitude of those rates. When used to investigate binding kinetics of a panel of cytoskeletal proteins, PARF analysis revealed that filament stabilization resulted in slower fluorescence loss. Additionally, commonly used “general” F‐actin labels display differences in kinetic properties, suggesting that not all fluorescently‐tagged actin labels interact with the actin network in the same way. We also observed differential dissociation kinetics for GFP‐VASP depending on which cellular structure was being labeled. These results demonstrate that PARF analysis of non‐equilibrium systems reveals kinetic information without the infrastructure investment required for other quantitative approaches such as FRAP, photoactivation, or in vitro reconstitution assays. © 2016 Wiley Periodicals, Inc.