Abstract
Fluorescent proteins (FP) are frequently used for studying proteins inside cells. In advanced fluorescence microscopy, FPs can report on additional intracellular variables. One variable is the local density near FPs, which can be useful in studying densities within cellular bio-condensates. Here, we show that a reduction in fluorescence lifetimes of common monomeric FPs reports increased levels of local densities. We demonstrate the use of this fluorescence-based variable to report the distribution of local densities within heterochromatin protein 1α (HP1α) in mouse embryonic stem cells (ESCs), before and after early differentiation. We find that local densities within HP1α condensates in pluripotent ESCs are heterogeneous and cannot be explained by a single liquid phase. Early differentiation, however, induces a change towards a more homogeneous distribution of local densities, which can be explained as a liquid-like phase. In conclusion, we provide a fluorescence-based method to report increased local densities and apply it to distinguish between homogeneous and heterogeneous local densities within bio-condensates.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵* Shared first authors
(i) added experimental results that provide tangential validation of our findings: depletion of MSR transcripts in mouse embryonic stem cells (ESCs) do indeed reproduce similar observed results as do retinoic acid (RA) induced early ESC differentiation (ii) reanalyzed all the original data and analyze newly-acquired data and increase sample sizes and statistics, which reproduce the main observations we reported in the original paper, with more statistical power (iii) prepared some of the previous figures as well as new ones, based on the new analyses of the previous and the newly-acquired data: the FLIM-related figures are now clearer to understand and also include quantitative plots (e.g., pixel-wise lifetime histograms) to make it easier for the reader to see the difference in lifetimes and their heterogeneities between undifferentiated and early-differentiated ESCs (iv) added fluorescence anisotropy images of undifferentiated and early-differentiated ESCs, to show that the difference in lifetimes between undifferentiated and early-differentiated ESCs can be recovered also from fluorescence anisotropy imaging, as well as add text to explain why in the case of changing fluorescence lifetimes, we still cannot discern homoFRET contributions from lifetime change contributions to the overall observed fluorescence anisotropy values (v) added more figures and textual explanations to support our response to the reviewers as well as edit the existing text to make it clearer.