Cohesin pairs sister chromatids by forming a tripartite Scc1-Smc1-Smc3 ring around them. In mitosis, cohesin is removed from chromosome arms by the phosphorylation-dependent prophase pathway. Centromeric cohesin is protected by shugoshin 1 and protein phosphatase 2A (Sgo1-PP2A) and opened only in anaphase by separase-dependent cleavage of Scc1 (refs 4-6). Following chromosome segregation, centrioles loosen their tight orthogonal arrangement, which licenses later centrosome duplication in S phase. Although a role of separase in centriole disengagement has been reported, the molecular details of this process remain enigmatic. Here, we identify cohesin as a centriole-engagement factor. Both premature sister-chromatid separation and centriole disengagement are induced by ectopic activation of separase or depletion of Sgo1. These unscheduled events are suppressed by expression of non-cleavable Scc1 or inhibition of the prophase pathway. When endogenous Scc1 is replaced by artificially cleavable Scc1, the corresponding site-specific protease triggers centriole disengagement. Separation of centrioles can alternatively be induced by ectopic cleavage of an engineered Smc3. Thus, the chromosome and centrosome cycles exhibit extensive parallels and are coordinated with each other by dual use of the cohesin ring complex.

SignificanceKinesins are a superfamily of ATP-dependent motors important for many microtubule-based functions, including multiple roles in mitosis. Small-molecule inhibitors of mitotic kinesins disrupt cell division and are being developed as antimitotic therapies. We investigated the molecular mechanism of the multitasking human mitotic kinesin Kif18A and its inhibition by the small molecule BTB-1. We used cryo-electron microscopy to visualize nucleotide-dependent conformational changes in microtubule-bound Kif18A, and the conformation of microtubule-bound, BTB-1-bound Kif18A. We calculated a putative BTB-1–binding site and validated this site experimentally to reveal the BTB-1 inhibition mechanism. Our work points to a general mechanism of kinesin inhibition, with wide implications for a targeted blockade of these motors in both dividing and interphase cells.

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The free energy provided by adenosine triphosphate (ATP) hydrolysis is centra l to many cellular processes and, therefore, the number of enzymes utilizing ATP as a substrate is almost innumerable. Modified ana logues of ATP are a valuable means to understand the biological function of ATPases. Although these enzymes have evo lved towards binding to ATP, large differences in active site architectures were found. In order to systematica lly access the specific active site constraints of different ATPases su itable tools are required. Here, we present the synthesis of six new ATP-based ATPase probes modified at three different positions of the nucleobase and the ribose, respectively. Subsequently, we studied the ATPases focal adhesion kinase FAK, the ubiquitin-activating protein UBA 1 and the kinesin Eg5 as exa mples for ATPases that process ATP by different mechanisms. We find that for each of these enzymes at least one position in ATP can be modified without loss of acceptance by the enzyme. However, the positions at which modifications are tolerated significantly differ between the studied enzymes allowing fingerprints to be drawn for reactivity. The introduced ATP ana logues may form the basis for the design of tailored probes with increased affinity and specificity for a specific ATPase of interest.

The mitotic spindle, a highly dynamic structure composed of microtubules, mediates the segregation of the previously duplicated genome into the two nascent daughter cells. Errors in this process contribute to pathology including tumor formation. Key for the shape and function of the mitotic spindle are kinesins, molecular motor proteins that convert chemical energy into mechanical work. Due to their fast mode of action, small molecules are valuable tools to dissect the dynamic functions of kinesins during mitosis. In this study, we report the identification of optimized small molecule inhibitors of the mitotic kinesin Kif18A. Using BTB-1, the first identified Kif18A inhibitor, as a lead compound, we synthesized a collection of derivatives. We demonstrate that some of the synthesized derivatives potently inhibited the ATPase activity of Kif18A with a half maximal inhibitory concentration (IC50) value in the low micromolar range. In vitro analysis of a panel of Kif18A-related kinesins revealed that the two most potent compounds show improved selectivity compared to BTB-1. Structure-activity relationship studies identified substituents mediating undesired inhibitory effects on microtubule polymerization. In summary, our study provides key insights into the mechanism of action of BTB-1 and its analogs, which will have a great impact on the further development of highly selective and bioactive Kif18A inhibitors. Since Kif18A is frequently overexpressed in solid tumors, such compounds are not only of great interest for basic research but also have the potential to open up new strategies for the treatment of human diseases.