DataSheet1_Diffusion State Transitions in Single-Particle Trajectories of MET Receptor Tyrosine Kinase Measured in Live Cells.pdf

Single-particle tracking enables the analysis of the dynamics of biomolecules in living cells with nanometer spatial and millisecond temporal resolution. This technique reports on the mobility of membrane proteins and is sensitive to the molecular state of a biomolecule and to interactions with other biomolecules. Trajectories describe the mobility of single particles over time and provide information such as the diffusion coefficient and diffusion state. Changes in particle dynamics within single trajectories lead to segmentation, which allows to extract information on transitions of functional states of a biomolecule. Here, mean-squared displacement analysis is developed to classify trajectory segments into immobile, confined diffusing, and freely diffusing states, and to extract the occurrence of transitions between these modes. We applied this analysis to single-particle tracking data of the membrane receptor MET in live cells and analyzed state transitions in single trajectories of the un-activated receptor and the receptor bound to the ligand internalin B. We found that internalin B-bound MET shows an enhancement of transitions from freely and confined diffusing states into the immobile state as compared to un-activated MET. Confined diffusion acts as an intermediate state between immobile and free, as this state is most likely to change the diffusion state in the following segment. This analysis can be readily applied to single-particle tracking data of other membrane receptors and intracellular proteins under various conditions and contribute to the understanding of molecular states and signaling pathways.

单粒子追踪（single-particle tracking）技术可实现纳米级空间分辨率与毫秒级时间分辨率下活细胞内生物分子动力学的分析。该技术可反映膜蛋白的迁移特性，且对生物分子的分子状态及其与其他生物分子的相互作用具有高敏感性。轨迹可描述单粒子随时间的迁移行为，并提供扩散系数、扩散状态等关键信息。单条轨迹内粒子动力学的变化可触发轨迹分割，借此可提取生物分子功能状态转变的相关信息。本研究开发了均方位移分析（mean-squared displacement analysis）方法，可将轨迹片段划分为静止、受限扩散及自由扩散三种状态，并提取这三种扩散模式间的转变发生频率。我们将该分析方法应用于活细胞内膜受体MET的单粒子追踪数据，分析了未激活受体以及与配体内化素B（internalin B）结合的受体的单条轨迹中的状态转变。研究发现，与未激活的MET相比，结合内化素B的MET从自由扩散与受限扩散状态向静止状态的转变频率显著提升。受限扩散充当了静止与自由扩散状态间的中间态，因为该状态最容易在后续轨迹片段中发生扩散状态的转变。该分析方法可便捷地推广应用于不同实验条件下其他膜受体与细胞内蛋白的单粒子追踪数据，有助于增进对生物分子状态与信号通路的理解。