Extended Data Fig. 1: Single-molecule tracking in living yeast.

a, ER and nucleolus shown by Elo3-GFP and Gar1-GFP. Scale bar, 1.0 μm. b, Coarse-grained representation of yeast interphase chromosomes, physically constrained by telomere (TEL)-nuclear envelope and centromere-spindle pole body (CEN-SPB) tethering. c, Overlay of RPB1^Rpo21-Halo^JF552 trajectories on the nucleus relative to ER and nucleolar GFP markers. Rainbow colours indicate the first appearance of each trajectory. Scale bar, 1 μm. d, Single-molecule trajectories of RPB1^Rpo21-Halo^JF552 (free or bound) with diffusion coefficients. Scale bar, 0.2 μm. e, Protein of interested (POI) fused with HaloTag, labeled with JF552-HaloTag ligand (JF552-HTL) (top). Initial laser exposure of JF552 resulted in strong ‘nuclear glow’, followed by shelving in dark state and stochastic reactivation, leading to single-molecule detection. Nucleus circled in red. Scale bar, 1.0 μm. (bottom). f, Strong 555 nm laser exposure for 5 min does not result in noticeable cell death or growth arrest. Scale bar, 4.0 μm. g, ER and nucleolar GFP markers for cell cycle stage identification. Scale bar, 1.0 μm. h, Diffusion coefficient histograms of various nuclear proteins (n: number of trajectories; mean value ± s.d.). i, Assessing the mean diffusion coefficients of the bound population reveals no substantial difference between histones (H2B, H3 and H2A.Z) and proteins that can move along DNA (for example RNAPII, RNAPIII, Paf1), while nucleolar RNAPI, and proteins that are physically tethered (Cse4 and Sir4) have notably lower values (n = 100 resamplings; mean value ± s.d.). Source numerical data are available in source data.

Source data