A Protocol for Studying Transcription Factor Dynamics Using Fast Single-Particle Tracking and Spot-On Model-Based Analysis.
A Protocol for Studying Transcription Factor Dynamics Using Fast Single-Particle Tracking and Spot-On Model-Based Analysis.
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使用快速单粒子跟踪和基于模型的定点分析研究转录因子动力学的协议。
DOI：
10.1007/978-1-0716-2140-0_9
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Hansen,AndersS
中科院分区：
文献类型：
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作者：
Jha,Asmita;Hansen,AndersS
单粒子跟踪技术（SPT）可以直接观察活细胞中单个蛋白随时间的扩散动态。因此，SPT已成为量化核蛋白（如转录因子（tf））动力学的有力方法。在这里，我们提供了一个进行和分析SPT实验的方案，重点是在哺乳动物细胞中快速跟踪tf（“fastSPT”）。首先，我们探索了如何设计和制备用于SPT实验的细胞。接下来，我们研究了如何通过低密度成像来优化SPT实验，以最小化跟踪误差，并通过频闪激励来最小化运动模糊。接下来，我们将讨论如何将原始SPT数据转换为单粒子轨迹。最后，我们说明如何使用动力学建模包Spot-On分析这些轨迹。我们讨论了如何使用Spot-On来拟合位移直方图，并提取有用的信息，如被束缚和自由扩散的tf的比例，以及它们相关的扩散系数。
Single-particle tracking (SPT) makes it possible to directly observe single protein diffusion dynamics in living cells over time. Thus, SPT has emerged as a powerful method to quantify the dynamics of nuclear proteins such as transcription factors (TFs). Here, we provide a protocol for conducting and analyzing SPT experiments with a focus on fast tracking (“fastSPT”) of TFs in mammalian cells. First, we explore how to engineer and prepare cells for SPT experiments. Next, we examine how to optimize SPT experiments by imaging at low densities to minimize tracking errors and by using stroboscopic excitation to minimize motion-blur. Next, we discuss how to convert raw SPT data into single-particle trajectories. Finally, we illustrate how to analyze these trajectories using the kinetic modeling package Spot-On. We discuss how to use Spot-On to fit histograms of displacements and extract useful information such as the fraction of TFs that are bound and freely diffusing, and their associated diffusion coefficients.