Fluorescent protein lifetimes report increased local densities and phases of nuclear condensates during embryonic stem cell differentiation

Abstract

Fluorescent proteins (FP) have revolutionized biology, and are frequently used for studying proteins inside cells. In advanced fluorescence microscopy, FPs might report on additional intracellular variables. One potential variable could be 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 can report 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.

Intermolecular Energy Migration via HomoFRET Captures the Modulation in the Material Property of Phase-Separated Biomolecular Condensates

“…In addition to phase separation, homoFRET via anisotropy imaging can also report on the higher-order species or nanoclusters formed as the precursors of these supramolecular assemblies 59,60 . Previous studies have highlighted the use of fluorescent protein lifetimes as a measure of droplet densities and crowding 61 . In addition to the droplet interior, steady-state anisotropy can distinctly report on the alterations in polypeptide chain clustering and condensate architecture resulting from the intermolecular associations characteristic of the given condensates.…”

Multiparameter-based photosynthetic state transitions of single phytoplankton cells

“…A confocal-based setup (ISS™ USA) assembled on top of a modified Olympus IX71 inverted microscope stand, similar to previously reported (45, 46). Excitation was provided by two picosecond pulsed diode lasers (λ = 488 nm, pulse width of 80 ps FWHM, operating at 20 MHz repetition rate, λ = 642 nm, pulse width of 100 ps FWHM, operating at 20 MHz repetition rate QuixX® 488-60 PS and QuixX® 642-140 PS, Omicron-Laserage, GmbH).…”