Quantifying Dynamics in Phase-Separated Condensates Using Fluorescence Recovery after Photobleaching

Abstract

Cells contain numerous membraneless organelles that assemble by intracellular liquid-liquid phase separation. The viscous properties and associated biomolecular mobility within these condensed phase droplets, or condensates, are increasingly recognized as important for cellular function and also dysfunction, for example, in protein aggregation pathologies. Fluorescence recovery after photobleaching (FRAP) is widely used to assess condensate fluidity and to estimate protein diffusion coefficients. However, the models and assumptions utilized in FRAP analysis of protein condensates are often not carefully considered. Here, we combine FRAP experiments on both in vitro reconstituted droplets and intracellular condensates with systematic examination of different models that can be used to fit the data and evaluate the impact of model choice on measured values. A key finding is that model boundary conditions can give rise to widely divergent measured values. This has important implications, for example, in experiments that bleach subregions versus the entire condensate, two commonly employed experimental approaches. We suggest guidelines for determining the appropriate modeling framework and highlight emerging questions about the molecular dynamics at the droplet interface. The ability to accurately determine biomolecular mobility both in the condensate interior and at the interface is important for obtaining quantitative insights into condensate function, a key area for future research.

Identifying Sequence Perturbations to an Intrinsically Disordered Protein that Determine Its Phase Separation Behavior

“…5D,E). By fitting the FRAP recovery curves to a 3D infinite model, we find diffusion coefficients ranging from D = 0.01 μm 2 /s to 0.025 μm 2 /s, approximately one order of magnitude faster than that for full-length LAF-1 58 . There are modest differences, notably that the construct with deletion of residues 21-30 (ie lower T sat that WT) exhibited faster fusion and FRAP recovery compared to that of RGGshuff-pres (ie highest T sat of all constructs we tested).…”

Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences

“…Recent work on FRAP specifically in LLPS systems addresses some of the above concerns, particularly with an eye to in vitro FRAP experiments (Taylor et al 2019). Although Taylor and colleagues explicitly ignore the role of long binding events in modeling FRAP recovery-an aspect that is almost certainly not valid for many instances of LLPS in cells-they nevertheless raise many useful points.…”

Charge-Driven Condensation of RNA and Proteins Suggests Broad Role of Phase Separation in Cytoplasmic Environments

“…This prediction remains to be tested experimentally. Other studies have employed fluorescence recovery after photobleaching (FRAP) to establish a liquid character of condensates (35) but there has been significant criticism of this approach, primarily because the observed range of recovery times is enormous and cannot be solely attributed to diffusion. (74)(75)(76).…”

“…Multivalent interactions, the presence of intrinsically disordered peptides (IDPs) (25), and electrostatic interactions between highly charged molecules such as RNA (25,26) are generally considered the key factors that promote phase separation (PS) (8,10,18,(27)(28)(29)(30)(31)(32)(33). Experiments such as fluorescence recovery after photobleaching (FRAP) suggest that these condensates retain liquid-like behavior (34,35) and therefore can fully support biological function. However, diffusion inside condensates (10,18,36) may be retarded and transitions from the liquid to gel or solid phases including aggregation to fibrils may occur (37)(38)(39).…”