Abstract：
Time-lapse imaging of the subcellular localization and dynamic behavior of proteins is critical to understand their biological functions in cells. With the advent of various methodologies and computational tools, the precise tracking and quantification of protein spatiotemporal dynamics have become feasible. Kymograph analysis, in particular, has been extensively adopted for the quantitative assessment of proteins, vesicles, and organelle movements. However, conventional kymograph analysis, which is based on a single linear trajectory, may not comprehensively capture the complexity of proteins that alter their course during intracellular transport and activity. In this chapter, we introduced an advanced protocol for whole-cell kymograph analysis that allows for three-dimensional (3D) tracking of protein dynamics. This method was validated through the analysis of tip-focused endocytosis and exocytosis processes in growing tobacco pollen tubes by employing both the advanced whole-cell and classical kymograph methods. In addition, we enhanced this method by integrating pseudo-colored kymographs that enables the direct visualization of changes in protein fluorescence intensity with fluorescence recovery after photobleaching to advance our understanding of protein localization and dynamics. This comprehensive method offers a novel insight into the intricate dynamics of protein activity within the cellular context.
摘要：
蛋白质的亚细胞定位和动态行为的延时成像对于了解其在细胞中的生物学功能至关重要。随着各种方法和计算工具的出现，蛋白质时空动力学的精确跟踪和定量已经变得可行。Kymograph分析,特别是,已被广泛用于蛋白质的定量评估，囊泡，和细胞器运动。然而,常规测绘仪分析，它基于单一的线性轨迹，可能无法全面捕获在细胞内运输和活动过程中改变其过程的蛋白质的复杂性。在这一章中,我们引入了一种用于全细胞kymograph分析的高级方案,该方案允许对蛋白质动力学进行三维(3D)跟踪.通过采用先进的全细胞和经典的测花机方法，通过分析生长中的烟草花粉管中尖端集中的内吞和胞吐过程来验证该方法。此外,我们通过整合伪彩色kymograps来增强这种方法，使光漂白后的荧光恢复与蛋白质荧光强度的变化直接可视化，以提高我们对蛋白质定位和动力学的理解。这种全面的方法为细胞环境中蛋白质活性的复杂动态提供了新的见解。
