(A) The sequences encoding the five Cbx proteins were fused with HaloTag to generate the HaloTag-Cbx fusions that were stably expressed in wild-type (PGK12.1) mES cells. The expression level of HaloTag-Cbx fusions was controlled by Tet-responsive element (TRE). HaloTag is shown in yellow, CD (chromodomain) in red, AT (AT-hook) motif in light blue; ATL (AT-hook-like) motif in cyan, and Cbox (chromobox) in emerald. (B) Schematic representation of highly inclined and laminated optical sheet (HILO) microscopy. (C) Live-cell single-molecule visualization of HaloTag-Cbx7 molecules in mES cells during a 30-ms exposure. Oval white dash circle outlines the nucleus of the cell. The individual white points represent single HaloTag-Cbx7 molecules. Scale bar, 2 µm. (D) Normalized histograms of the log maximum likelihood diffusion coefficient for H2A-HaloTag (N = 19 cells, n = 2675 trajectories) and HaloTag-NLS (N = 69 cells, n = 2087 trajectories) in wild-type mES cells. The H2A-HaloTag histogram was fitted with a three-component Gaussian and the HaloTag-NLS histogram a two-component Gaussian. The color bars indicate that the fraction of proteins in the chromatin-bound (CB, red), intermediate (ID, cyan), and fast diffusion (FD, green) population. NLS, nuclear localization sequence. (E) Normalized histograms of the log maximum likelihood diffusion coefficient for HaloTag-Cbx2 (N = 44 cells, n = 2833 trajectories), HaloTag-Cbx4 (N = 34 cells, n = 11,343 trajectories), HaloTag-Cbx6 (N = 33 cells, n = 7457 trajectories), HaloTag-Cbx7 (N = 51 cells, n = 3097 trajectories), and HaloTag-Cbx8 (N = 36 cells, n = 3351 trajectories) in wild-type mES cells. The histograms were fitted with a three-component Gaussian. (F) Fraction of the CB (red), ID (cyan), and FD (green) population for H2A-HaloTag, HaloTag-NLS, HaloTag-Cbx2, HaloTag-Cbx4, HaloTag-Cbx6, HaloTag-Cbx7, and HaloTag-Cbx8. The data were obtained from Figure 1D and E fitted with a Gaussian. Results are means ± SD.