Fig. 7: Modulating Pit’s interactions with the nucleolus or HP1a alters its subnucleolar localization and PCH–nucleolar associations.
a, Attraction matrix showing the pairwise interaction strengths (blue gradient) between Fib (F), rDNA (rD), PCH (H) and Pit (P) (left). In this refined model, an interaction between Pit and rDNA is included (εrD–P = 2.1), which results in the simulated outcome recapitulating the WT in vivo organization. Additional simulations at εH–P = 0 represent the condition where Pit–HP1a interactions are disrupted, whereas εrD–P = 0 simulates conditions where the Pit–rDNA association is lost. Snapshots of the three corresponding simulation outcomes are shown (right). b, Radial distribution of Pit calculated from the centre of mass of the Fib beads (nucleolar centre) under the three interaction conditions. The x axis shows the radial distance (σ, bead size), where zero corresponds to the nucleolar centre. c, Representative live-cell fluorescence images of S2R+ cells transfected with Pit–mYFP and mCherry–HP1a, and treated with vehicle (dimethylsulfoxide) or 0.08 µg ml−1 ActD, imaged between 10 and 60 min of drug treatment. Pit is enriched at the edge of the nucleolus and HP1a localization around the nucleolus increases after ActD treatment. The dashed lines indicate the nuclear boundaries. d, Levels of HP1a occupancy around the nucleolus in S2R+ cells treated with vehicle (n = 10 nucleoli, with one nucleolus per cell) or 0.08 µg ml−1 ActD (n = 12 nucleoli). Data are the mean ± s.d. The P value was calculated using an unpaired two-tailed Student’s t-test. e, Line scans show Pit distribution in the nucleolus of S2R+ cells treated with vehicle (n = 18 nucleoli, with one nucleolus per cell) or 0.08 µg ml−1 ActD (n = 19 nucleoli). Intensities were normalized to the average value for each profile and lengths were normalized to the diameter of the corresponding nucleolus. The mean is indicated by the solid line and the shaded region indicates the s.d. f, Model summarizing how the dual-affinity linker Pit mediates the spatial organization of PCH and nucleolar condensates in vivo. Modulating nucleation sites, Pit concentrations and interaction motifs alter the affinity hierarchies leading to specific rearrangements in condensate architecture and Pit distribution. Scenarios relating to (i)–(v) are described in Discussion.
