Our central effort: the molecular machines that drive and police cell division — separase, the CDK/cyclin complexes that set its timing, and the APC/C ubiquitin ligase that controls mitotic exit.
"Cell division is studied both for its beauty and for the danger that it represents. When all goes well, new healthy cells are born." — Silke Hauf, Nature 2021
Aberrant cell division transforms normal growing cells into cancer cells. To maintain genome stability, each daughter cell must receive an identical set of sister chromatids — demanding precision during DNA replication in S phase and during chromosome segregation in mitosis.
In early mitosis, duplicated chromosomes are held together by the ring-shaped cohesin complex. Separation at anaphase is triggered by separase, a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (RAD21). Separase is activated by degradation of its inhibitors, securin and cyclin B. We showed that both securin and the Cdk1–cyclin B1–Cks1 complex inhibit separase through pseudosubstrate motifs that block substrate binding at the catalytic site and nearby docking sites.
As in C. elegans and yeast, human securin carries its own pseudosubstrate motifs. CDK1–cyclin B1 instead deploys pseudosubstrate motifs from intrinsically disordered loops within separase itself: one autoinhibitory loop blocks the catalytic sites of both separase and CDK1, another blocks substrate docking, and a third contains a phosphoserine that anchors the complex by binding a conserved phosphate pocket in cyclin B1. Together this reveals how chromosome segregation is so robustly controlled.
Cohesin encircles the sister chromatids in early mitosis; at anaphase onset, separation is triggered by separase cleaving the cohesin subunit SCC1/RAD21. SCC1 has two cleavage sites, and cleavage is stimulated by SCC1 phosphorylation — but the mechanisms of recognition were only partly understood.
We determined a series of cryo-EM structures of human separase in apo and substrate-bound forms. Combined with biochemistry, these verify the first SCC1 cleavage site and reassign the second. Multiple substrates — separase autocleavage sites and both SCC1 sites — engage several docking sites, including four phosphate-binding sites that explain why phosphorylation enhances cleavage. We also describe how the cohesin subunit SA2/STAG2 interacts with separase to promote cleavage at the second SCC1 site, and propose a model for how cohesin is targeted overall.
Phosphorylation of substrates by cyclin-dependent kinases (CDKs) drives cell-cycle progression, yet how cyclins contribute to substrate specificity is poorly understood. With the Thomas Mayer group (University of Konstanz), we discovered a positively charged pocket in cyclin B1 — conserved only within B-type cyclins — that binds phosphorylated serine/threonine residues and is essential for correct execution of mitosis.
HeLa cells expressing pocket-mutant cyclin B1 are strongly delayed in anaphase onset, with defects in spindle function and in timely APC/C activation. Pocket integrity is required for APC/C phosphorylation, particularly at non-consensus CDK1 sites. The pocket appears to act as a specificity-site factor for sequential phosphorylations, where priming events facilitate subsequent pocket-dependent phosphorylation.
The multi-subunit anaphase-promoting complex/cyclosome (APC/C) is a master regulator of cell division, marking mitotic cyclins, securin and other regulators for degradation via the ubiquitin–proteasome pathway. It is itself regulated by its coactivators CDC20 and CDH1, by post-translational modification, and by inhibitors such as EMI1 and the mitotic checkpoint complex.
We determined structures of human APC/C bound to CDH1 and EMI1, and of apo-APC/C, at 2.9 Å and 3.2 Å. The high-resolution maps allowed unambiguous assignment of an N-terminal CDH1 α-helix at the APC1–APC8 interface, revealed a novel zinc-binding module in APC2 (confirmed experimentally), and — aided by AlphaFold — let us build several intrinsically disordered regions important for complex assembly.
