Cell Reports 2022 (In Press)

The cyclin-dependent kinase (Cdk1) oscillator is widely characterized in homogenized cytosolic extracts, leaving unclear the impact of nucleocytoplasmic compartmentalization. Here, by developing a Förster resonance energy transfer (FRET) biosensor, we track Cdk1 spatiotemporal dynamics in reconstituted nucleus-present and nucleus-absent cells side-by-side and find compartmentalization significantly modulates clock properties previously found in bulk studies. While nucleus-absent cells display highly tunable frequency, the nucleus-present cells maintain constant frequency against cyclin B1 variations. Despite high expression variability, cyclin degraded within the same duration, enabling a robust mitotic phase. Moreover, Cdk1 and cyclin B1 cycle rigorously out-of-phase, ensuring wide phase-plane orbits, essential for oscillation robustness. Although Cdk1 in homogeneous extracts is well-known for delayed switch-like activation, we find active cyclin B1-Cdk1 accumulates in nuclei, without delay, until the nuclear envelope breakdown (NEB) when another abrupt activation triggers anaphase. Cdk1 biphasic activation and spatial compartmentalization may together coordinate the accurate ordering of different downstream events.