Figure 5.

Phase separation propensity and MT binding of Borealin subunit of the CPC are important for MT nucleation and bundling.A, scheme of the CPC; CEN: centromere-targeting domain composed of full-length Survivin and Borealin, N-terminal fragment of INCENP. Mutations introduced to Borealin amino acid sequence are presented as blue (MTBM) and orange (LLPS) lines. The regions of the CPC that are not part of the CEN domain are faded out. B, DIC and fluorescence images of rhodamine-labeled MTs generated by CEN-Borealin^wt, CEN-Borealin^LLPS, and CEN-Borealin^MTBM. Experiment was repeated three times. Plot shows the number of MT bundles per μm²; total number of MT bundles for CEN-Borealin^wt (n = 612), for CEN-Borealin^LLPS (n = 61); p^wt-LLPS = 5.5 × 10⁻⁵, CEN-Borealin^MTBM—not detected (N/D); scale bar represents 5 μm. C, GMpCpp-stabilized rhodamine-labeled MT bundled by CEN-Borealin^wt, CEN-Borealin^LLPS, and CEN-Borealin^MTBM imaged in TIRF mode with excitation at 555 nm (rhodamine tubulin). Plot shows quantification of the width of single bundles of MTs. Experiment was repeated twice; CEN-Borealin^wt (n = 49), CEN-Borealin^LLPS (n = 43), CEN-Borealin^MTBM (n = 49), MTs (n = 39); p^wt-LLPS = 1.05 × 10⁻⁹; p^wt-MTBM = 1.68 ×10⁻⁹. Statistical analysis was performed by applying Kolmogorov–Smirnov test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; scale bar represents 5 μm. CPC, chromosomal passenger complex; DIC, differential interference contrast; LLPS, liquid–liquid phase separation; MT, microtubule; TIRF, total internal reflection fluorescence.