The CyclinB1-Cdk1 kinase is the catalytic activity at the heart of mitosis-promoting factor (MPF), yet fundamental questions concerning its role in mitosis remained unresolved. It is not known when and how rapidly CyclinB1-Cdk1 is activated in mammalian cells, nor how its activation coordinates the substantial changes in the cell at mitosis. Here, we have developed a FRET biosensor specific for CyclinB1-Cdk1 that enables us to assay its activity with very high temporal precision in living human cells. We show that CyclinB1-Cdk1 is inactive in G2 phase and activated at a set time before nuclear envelope breakdown, thereby initiating the events of prophase. CyclinB1-Cdk1 levels rise to their maximum extent over the course of approximately 30 min, and we demonstrate that different levels of CyclinB1-Cdk1 kinase activity trigger different mitotic events, thus revealing how the remarkable reorganization of the cell is coordinated at mitotic entry.

A biosensor for cyclin B–Cdk1 activity shows that it uses an unconventional yet simple mechanism for nuclear accumulation.

Stathmin is a highly conserved ubiquitous cytoplasmic protein, phosphorylated in response to extracellular signals and during the cell cycle. Stathmin has recently been shown to destabilize microtubules, but the molecular mechanisms of this function remained unclear. We show here that stathmin directly interacts with tubulin. We assessed the conditions of this interaction and determined some its quantitative parameters using plasmon resonance, gel filtration chromatography, and analytical ultracentrifugation. The stathmin/ tubulin interaction leads to the formation of a 7.7 S complex with a 60-Å Stokes radius, associating one stathmin with two tubulin heterodimer molecules as determined by direct quantification by Western blotting. This interaction is sensitive to pH and ionic environment. Its equilibrium dissociation constant, determined by plasmon resonance measurement of kinetic constants, has an optimum value of 0.5 M at pH 6.5. The affinity was lowered with a fully "pseudophosphorylated" 4-Glu mutant form of stathmin, suggesting that it is modulated in vivo by stathmin phosphorylation. Finally, analysis of microtubule dynamics by video microscopy shows that, in our conditions, stathmin reduces the growth rate of microtubules with no effect on the catastrophe frequency. Overall, our results suggest that the stathmin destabilizing activity on microtubules is related to tubulin sequestration by stathmin.Stathmin (1, 2), also designated Op18, p18, p19, prosolin, and metablastin (3-6), is a ubiquitous cytosolic phosphoprotein highly conserved in vertebrates (7,8) and specifically abundant in neurons (9 -11). Expression and phosphorylation of stathmin are modulated in various situations related to the control of cellular activities, and it has been proposed that it may act as a relay integrating various intracellular signaling pathways (1). Expression of stathmin was shown to be regulated in vivo during development (7,(12)(13)(14), during tissue regeneration (15, 16), and in cell culture by cell/cell interactions (17). Stathmin is also up-regulated in many malignant cell types and tumors (5,18,19). Phosphorylation of stathmin is observed in response to hormones (20), cytokines (21), neurotransmitters (22), and growth and differentiation factors (23). Moreover, progression through the cell cycle appears to require multisite phosphorylation of stathmin (24). Actually, overexpression of a nonphosphorylatable mutant of stathmin resulted in a large population of cells blocked in G 2 /M with a high DNA content (24,25). Finally, stathmin is the generic element of a protein family whose other members most probably play distinct roles related to the control of neuronal differentiation or to the expression of neuron-specific traits (8, 26).The molecular mechanism(s) by which stathmin acts in these processes remain largely unknown. Two domains can be distinguished in the primary structure of stathmin, an N-terminal "regulatory" domain that contains the four phosphorylation sites that account for all of the electrophoretic f...