Max Planck Institute for the Physics of Complex Systems, Dresden, Germany.
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
Elife. 2021 Oct 12;10:e68620. doi: 10.7554/eLife.68620.
Key processes of biological condensates are diffusion and material exchange with their environment. Experimentally, diffusive dynamics are typically probed via fluorescent labels. However, to date, a physics-based, quantitative framework for the dynamics of labeled condensate components is lacking. Here, we derive the corresponding dynamic equations, building on the physics of phase separation, and quantitatively validate the related framework via experiments. We show that by using our framework, we can precisely determine diffusion coefficients inside liquid condensates via a spatio-temporal analysis of fluorescence recovery after photobleaching (FRAP) experiments. We showcase the accuracy and precision of our approach by considering space- and time-resolved data of protein condensates and two different polyelectrolyte-coacervate systems. Interestingly, our theory can also be used to determine a relationship between the diffusion coefficient in the dilute phase and the partition coefficient, without relying on fluorescence measurements in the dilute phase. This enables us to investigate the effect of salt addition on partitioning and bypasses recently described quenching artifacts in the dense phase. Our approach opens new avenues for theoretically describing molecule dynamics in condensates, measuring concentrations based on the dynamics of fluorescence intensities, and quantifying rates of biochemical reactions in liquid condensates.
生物凝聚体的关键过程是与环境的扩散和物质交换。在实验中，通常通过荧光标记来探测扩散动力学。然而，迄今为止，缺乏用于标记凝聚体成分动力学的基于物理的定量框架。在这里，我们基于相分离的物理原理推导出相应的动力学方程，并通过实验对相关框架进行了定量验证。我们表明，通过使用我们的框架，我们可以通过对光漂白后荧光恢复（FRAP）实验的时空分析来精确确定液体凝聚体内部的扩散系数。我们通过考虑蛋白质凝聚体和两种不同的聚电解质共凝聚物系统的空间和时间分辨数据来展示我们方法的准确性和精度。有趣的是，我们的理论还可以用于确定稀相中的扩散系数与分配系数之间的关系，而无需依赖稀相中的荧光测量。这使我们能够研究加盐对分配的影响，并避免在密集相中最近描述的猝灭伪影。我们的方法为在凝聚体中理论描述分子动力学、基于荧光强度动力学测量浓度以及量化液体凝聚体中生化反应的速率开辟了新途径。
