Fig. 4.

mEos3.2–JF₆₄₆ allows tracking of single CENP-A protein complexes. a Schematic of labeling one protein with a donor PM fluorophore and a second protein with JF₆₄₆ such that FRET occurs when they are in close spatial proximity. b Tagging CENP-A with either mEos3.2 or the HaloTag protein in the same cell allows assembly of nucleosomes where FRET can be observed between mEos3.2 and JF₆₄₆ in the (CENP-A/Histone H4)₂ hetero-tetrameric core. c (top) Representative reconstructions of single mEos3.2-tagged CENP-A nucleosomes localized at centromeres (see inset) are shown in the absence or presence of JF₆₄₆-labeled CENP-A. Scale bar = 500 nm. (lower) Images of the JF₆₄₆ dye show that the HaloTag-tagged CENP-A has been successfully labeled. Scale bar = 1 µm. d Single mEos3.2-tagged CENP-A nucleosomes show decreased intensity and increased track length in the presence of JF₆₄₆-tagged CENP-A. (left) Montages of three representative 500 ms exposure single-molecule traces (one image for every four frames) are shown in the absence or presence of the JF₆₄₆-labeled CENP-A. (right) Histograms showing the percentage of molecules remaining with a particular on-state time (i.e., individual track length) after photo-conversion when performing sptPALM in mouse ES cells. Cells expressing mEos3.2-tagged CENP-A are compared with cells also expressing JF₆₄₆-tagged CENP-A