Single-Molecular Quantification of Flowering Control Proteins Within Nuclear Condensates in Live Whole Arabidopsis...

Here we describe the coupled standardization of two complementary fluorescence imaging techniques and apply it to liquid-liquid phase-separated condensates formed from an EGFP fluorescent reporter of flowering control locus A (FCA), a protein that associates with chromosomal DNA in plants during epigenetic regulation of the flowering process. First, we use home-built single-molecule Slimfield microscopy to establish a fluorescent protein standard. This sample comprises live yeast cells expressing Mig1 protein, a metabolic regulator which localizes to the nucleus under conditions of high glucose, fused to the same type of EGFP label as for the FCA fusion construct. Then we employ commercial confocal AiryScan microscopy to study the same standard. Finally, we demonstrate how to quantify FCA-EGFP nuclear condensates in intact root tips at rapid timescales and apply this calibration. This method is a valuable approach to obtaining single-molecule precise stoichiometry and copy number estimates of protein condensates that are integrated into the chromosome architecture of plants, using confocal instrumentation that lacks de facto single-molecule detection sensitivity.