SlimVar: rapid in vivo single-molecule tracking of chromatin regulators in plants

Epigenetic regulation maintains gene expression patterns over many rounds of cell division in higher organisms. However, visualization of factors regulating epigenetic switches in vivo is limited by the challenge of imaging cells deep in living tissue, with molecular sensitivity and rapid sampling. We report an easy-to-implement method called Variable-angle Slimfield microscopy (SlimVar), which by simple modification of an inverted optical microscope, enables single-molecule tracking of fluorescent reporters in Arabidopsis thaliana. Using SlimVar, we imaged stepwise photobleaching of chromatin-protein assemblies in individual nuclei, 30 microns deep in root tips through multiple cell layers. We find that two homologous proteins key to the epigenetic switch at FLOWERING LOCUS C (FLC) - cold-induced VERNALIZATION INSENSITIVE3 (VIN3) and constitutively expressed VERNALIZATION 5 (VRN5) - exhibit dynamic nuclear assemblies during FLC silencing. Upon cold exposure, these assemblies increase in stoichiometry by up to 100% to a median of ~20 molecules. Larger VRN5 assemblies preferentially co-localize with an FLC lacO transgenic reporter during prolonged cold, persisting after return to warm conditions. Our findings support a hybrid model of epigenetic memory in which nucleation of histone trimethylation is assisted by dynamic protein assemblies over extended durations. SlimVar therefore has potential to offer molecular insights into proteins expressed at physiological levels in a range of tissues. ### Competing Interest Statement The authors have declared no competing interest.