Condensation-dependent multivalent interactions of EB1 and CENP-R regulate chromosome oscillations in mitosis

Mitotic chromosomes oscillate between the spindle poles upon the establishment of bi-orientation, which is essential for chromosome alignment and subsequent synchronous segregation. However, the molecular mechanisms underlying the oscillatory movement remain unclear. Recent studies revealed that phase separation of the end-binding protein 1 (EB1) is essential for eukaryotic cell division. Here, we show that EB1 interacts with CENP-R and that the phase separation-defective EB1 mutant fails to power the chromosome oscillations. Biochemical analyses reveal a co-condensation of EB1 and CENP-R, a subunit of the constitutive centromere-associated network. Nuclear magnetic resonance assays reveal that the interaction and co-condensation are largely mediated by the structured end-binding homology domain of EB1 and the non-structured N-terminal intrinsic disorder region of CENP-R. Chromosome oscillation is perturbed in cells expressing the EB1-binding-defective CENP-R mutant. Thus, phase-separated EB1 binding to CENP-R forms a physical link between inner kinetochore and dynamic spindle microtubule plus-ends to guide accurate chromosome oscillations.