Abstract
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.
Highlights
Liquid phase separation has emerged as an organizing principle in biology and is thought to underlie the formation of various membrane-less cellular organelles (Banani et al (2017))
We focus on fluorescence recovery after photobleaching (FRAP) dynamics at thermodynamic equilibrium, where the total volume fraction φtot (x) depends on space only
After photobleaching, unbleached molecules diffuse into the condensate leading to FRAP dynamics of unbleached molecules inside (Fig. 2b,c)
Summary
Liquid phase separation has emerged as an organizing principle in biology and is thought to underlie the formation of various membrane-less cellular organelles (Banani et al (2017)). Hallmark properties of such organelles are their rapid formation and dissolution, their fusion, and their wetting to membranes (Hyman et al (2014)). To probe the dynamics of condensates, biomolecules are typically labelled with fluorescent tags. In systems with tagged molecules, various methods exist to characterize molecular properties such as binding rates and diffusion coefficients, including fluorescence correlation spectroscopy (FCS) (Ries and Schwille (2012); Rigler and Elson (2012), single-particle tracking (SPT)
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