The cellular cytosol is a crowded environment. Biomolecular Förster resonance energy transfer (FRET) sensors have been developed to measure crowding in cytosol mimics comprised of synthetic polymers such as polyethylene glycol (PEG) and Ficoll that impart an excluded volume effect. In the current study, we explore the unsolicited role of PEG in driving the phase separation of a protein crowding sensor, AcGFP1/mCherry-FRET crowding helix 2 (CrH2), into fluorescent puncta. In contrast, a DNA-based FRET sensor with an Alexa488/Cy5 FRET pair overcomes the limitations of puncta formation under the same crowding conditions. Using fluorescence recovery after photobleaching (FRAP) imaging, we uncover the liquid-like physical properties of the PEG-induced puncta. Using two-color fluorescence microscopy imaging, we determine the crowder-induced inhomogeneity, concentration variations, and partition coefficient across the dilute and dense phases of the liquid puncta, which remain largely underexplored in bulk fluorometry measurements. Thus, an average sensor response may originate from an aqueous biphasic system, reporting an erroneous average response instead of distinct levels of crowdedness. A comparison of excluded volume effects conferred by Ficoll and PEGs of various molecular-weight ranges reveals the influence of size, concentration, excluded volume, and chemical composition on the CrH2 sensor response. We demonstrate that PEGs not only impart the excluded volume effect but also enable phase separation and change sensor response through a mechanism that could be caused by polymer interactions with the flexible hinge region of CrH2. Overall, the biophysical aspects of the biomolecular sensor, fluorescent reporters, and sample environment need to be carefully considered to sense crowding properly.
Mohapatra et al. (2026) studied this question.