Fig. 5.

FRET-enhanced PA-JF₅₄₉ allows tracking of single H2B complexes. a Schematic showing one histone H2B molecule in a nucleosome labeled with a donor PA-JF₅₄₉, and a second labeled with an acceptor JF₆₄₆ such that FRET occurs either within a nucleosome or between nucleosomes when they are in close spatial proximity. The reduced inter-nucleosome distance in heterochromatin leads to higher FRET efficiency. b Representative images and intensity vs. time traces of single PA-JF₅₄₉-tagged H2B molecules are shown in the absence or presence of JF₆₄₆-labeled H2B (scale bar = 5 µm). c Single PA-JF₅₄₉-tagged H2B nucleosomes show increased track length in the presence of JF₆₄₆-tagged H2B. A 1% confidence interval was defined to show whether average trajectory lengths were below (yellow) or above 12 s (blue)—only 1% of PA-JF₅₄₉-tagged molecules have average track lengths longer than 12 s. Density maps were generated to show where the long trajectories are concentrated, and representative examples of long trajectories are shown in the upper panels in the same colors, where the one circled in red shows long periods of confined motion interrupted by a short period of rapid linear motion. d Inter-nuclear differences in track length were observed (left) for three different cells with less condensed, more condensed, or mitotic chromatin (top to bottom). Density maps show that regions where track lengths were on average above the 12-s cutoff (middle) also had a high number of localized PA-JF₅₄₉-tagged H2B molecules (right)