Nuclear-cytoplasmic compartmentalization promotes robust timing of mitotic events by cyclin B1-Cdk1

Abstract

Studies applying well-mixed cytosolic extracts found the mitotic network centered on cyclin-dependent kinase (Cdk1) performs robust relaxation oscillations with tunable frequency [1-6]. However, recent work also highlighted the importance of cyclin B1-Cdk1 nuclear translocation in mitotic timing [7, 8]. How nuclear compartmentalization affects the oscillator properties and the accurate ordering of mitotic events, especially in embryos lacking checkpoints, remains elusive. Here we developed a Forster resonance energy transfer (FRET) biosensor for analyzing Cdk1 spatiotemporal dynamics in synthetic cells containing nuclei compared to those without. We found cellular compartmentalization significantly impacts clock behaviors. While the amplitude-frequency dependency measured in the homogeneous cytoplasm showed highly tunable frequency for a fixed amplitude, confirming predictions by non-spatial models [4], the frequency remains constant against cyclin variations when nuclei are present, suggesting a possible buffering mechanism of nuclear compartments to ensure robust timing. We also found all cyclin degrades within similar mitotic durations despite variable interphase cyclin expression. This scalable degradation of cyclin may further promote the precise mitotic duration. Simultaneous measurements revealed Cdk1 and cyclin B1 cycle rigorously out of phase, producing a wide orbit on their phase plane, essential for robust oscillations. We further mapped mitotic events on the phase-plane orbits. Unlike cytoplasmic-only cells showing delayed Cdk1 activation, nucleus-containing cells exhibit steady cyclin B1-Cdk1 nuclear accumulation until nuclear envelope breakdown (NEB) followed by an abrupt cyclin-independent activation to trigger anaphase. Thus, both biphasic activation and subcellular localization of Cdk1 ensure accurate ordering of substrates.