Pim J. Huis in 't Veld

Aurora B and Cdk1 mediate Wapl activation and release of acetylated cohesin from chromosomes by phosphorylating Sororin

Sister chromatid cohesion depends on Sororin, a protein that stabilizes acetylated cohesin complexes on DNA by antagonizing the cohesin release factor Wings-apart like protein (Wapl). Cohesion is essential for chromosome biorientation but has to be dissolved to enable sister chromatid separation. To achieve this, the majority of cohesin is removed from chromosome arms in prophase and prometaphase in a manner that depends on Wapl and phosphorylation of cohesin’s subunit stromal antigen 2 (SA2), whereas centromeric cohesin is cleaved in metaphase by the protease separase. Here we show that the mitotic kinases Aurora B and Cyclin-dependent kinase 1 (Cdk1) destabilize interactions between Sororin and the cohesin subunit precocious dissociation of sisters protein 5 (Pds5) by phosphorylating Sororin, leading to release of acetylated cohesin from chromosome arms and loss of cohesion. At centromeres, the cohesin protector shugoshin (Sgo1)-protein phosphatase 2A (PP2A) antagonizes Aurora B and Cdk1 partly by dephosphorylating Sororin and thus maintains cohesion until metaphase. We propose that the stepwise loss of cohesion between chromosome arms and centromeres is caused by local regulation of Wapl activity, which is controlled by the phosphorylation state of Sororin.

Rapid movement and transcriptional re-localization of human cohesin on DNA

The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring‐shaped protein complexes that are thought to function by entrapping DNA. It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three‐dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single‐molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA‐bound proteins and nucleosomes but is constrained in its movement by transcription and DNA‐bound CCCTC‐binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion.

Cohesin’s ATPase Activity Couples Cohesin Loading onto DNA with Smc3 Acetylation

Summary Background Cohesin mediates sister chromatid cohesion by topologically entrapping sister DNA molecules inside its ring structure. Cohesin is loaded onto DNA by the Scc2/NIPBL-Scc4/MAU2-loading complex in a manner that depends on the adenosine triphosphatase (ATPase) activity of cohesin’s Smc1 and Smc3 subunits. Subsequent cohesion establishment during DNA replication depends on Smc3 acetylation by Esco1 and Esco2 and on recruitment of sororin, which “locks” cohesin on DNA by inactivating the cohesin release factor Wapl. Results Human cohesin ATPase mutants associate transiently with DNA in a manner that depends on the loading complex but cannot be stabilized on chromatin by depletion of Wapl. These mutants cannot be acetylated, fail to interact with sororin, and do not mediate cohesion. The absence of Smc3 acetylation in the ATPase mutants is not a consequence of their transient association with DNA but is directly caused by their inability to hydrolyze ATP because acetylation of wild-type cohesin also depends on ATP hydrolysis. Conclusions Our data indicate that cohesion establishment involves the following steps. First, cohesin transiently associates with DNA in a manner that depends on the loading complex. Subsequently, ATP hydrolysis by cohesin leads to entrapment of DNA and converts Smc3 into a state that can be acetylated. Finally, Smc3 acetylation leads to recruitment of sororin, inhibition of Wapl, and stabilization of cohesin on DNA. Our finding that cohesin’s ATPase activity is required for both cohesin loading and Smc3 acetylation raises the possibility that cohesion establishment is directly coupled to the reaction in which cohesin entraps DNA.

Cohesin acetylation and Wapl‐Pds5 oppositely regulate translocation of cohesin along DNA

Cohesin is a ring‐shaped protein complex that plays a crucial role in sister chromatid cohesion and gene expression. The dynamic association of cohesin with chromatin is essential for these functions. However, the exact nature of cohesin dynamics, particularly cohesin translocation, remains unclear. We evaluated the dynamics of individual cohesin molecules on DNA and found that the cohesin core complex possesses an intrinsic ability to traverse DNA in an adenosine triphosphatase (ATPase)‐dependent manner. Translocation ability is suppressed in the presence of Wapl‐Pds5 and Sororin; this suppression is alleviated by the acetylation of cohesin and the action of mitotic kinases. In Xenopus laevis egg extracts, cohesin is translocated on unreplicated DNA in an ATPase‐ and Smc3 acetylation‐dependent manner. Cohesin movement changes from bidirectional to unidirectional when cohesin faces DNA replication; otherwise, it is incorporated into replicating DNA without being translocated or is dissociated from replicating DNA. This study provides insight into the nature of individual cohesin dynamics and the mechanisms by which cohesin achieves cohesion in different chromatin contexts.

biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes.

Significance At the molecular level, most processes in living systems are mediated by multisubunit protein complexes. Recombinant forms of these complexes are essential for analyzing their structure and function. Multigene expression constructs greatly improve recombinant protein complex preparations, but the generation of such constructs can be a rate-limiting step. To overcome this limitation, we have adapted Gibson assembly reactions for the rapid, efficient, and fast generation of numerous expression constructs in parallel and used the resulting biGBac method for expression of different cell-cycle complexes, composed of up to 17 different subunits. The biGBac technique enables the analyses of large protein complexes by systematic mutagenesis approaches that were not feasible before. Analyses of protein complexes are facilitated by methods that enable the generation of recombinant complexes via coexpression of their subunits from multigene DNA constructs. However, low experimental throughput limits the generation of such constructs in parallel. Here we describe a method that allows up to 25 cDNAs to be assembled into a single baculoviral expression vector in only two steps. This method, called biGBac, uses computationally optimized DNA linker sequences that enable the efficient assembly of linear DNA fragments, using reactions developed by Gibson for the generation of synthetic genomes. The biGBac method uses a flexible and modular “mix and match” approach and enables the generation of baculoviruses from DNA constructs at any assembly stage. Importantly, it is simple, efficient, and fast enough to allow the manual generation of many multigene expression constructs in parallel. We have used this method to generate and characterize recombinant forms of the anaphase-promoting complex/cyclosome, cohesin, and kinetochore complexes.

Molecular basis of outer kinetochore assembly on CENP-T.

Stable kinetochore-microtubule attachment is essential for cell division. It requires recruitment of outer kinetochore microtubule binders by centromere proteins C and T (CENP-C and CENP-T). To study the molecular requirements of kinetochore formation, we reconstituted the binding of the MIS12 and NDC80 outer kinetochore subcomplexes to CENP-C and CENP-T. Whereas CENP-C recruits a single MIS12:NDC80 complex, we show here that CENP-T binds one MIS12:NDC80 and two NDC80 complexes upon phosphorylation by the mitotic CDK1:Cyclin B complex at three distinct CENP-T sites. Visualization of reconstituted complexes by electron microscopy supports this model. Binding of CENP-C and CENP-T to MIS12 is competitive, and therefore CENP-C and CENP-T act in parallel to recruit two MIS12 and up to four NDC80 complexes. Our observations provide a molecular explanation for the stoichiometry of kinetochore components and its cell cycle regulation, and highlight how outer kinetochore modules bridge distances of well over 100 nm. DOI: http://dx.doi.org/10.7554/eLife.21007.001

Molecular requirements for the inter-subunit interaction and kinetochore recruitment of SKAP and Astrin

Accurate chromosome segregation during cell division is crucial for propagating life and protects from cellular transformation. The SKAP:Astrin heterodimer localizes to spindle microtubules and to mature microtubule–kinetochore attachments during mitosis. Depletion of either subunit disrupts spindle structure and destabilizes kinetochore–microtubule attachments. Here, we identify molecular requirements for the inter-subunit interaction of SKAP and Astrin, and discuss requirements for their kinetochore recruitment. We also identify and characterize a microtubule-binding domain in SKAP, distinct from the SXIP motif that mediates end binding (EB) protein binding and plus end tracking, and show that it stimulates the growth-rate of microtubules, possibly through a direct interaction with tubulin. Mutations targeting this microtubule-binding domain impair microtubule plus-end tracking but not kinetochore targeting, and recapitulate many effects observed during depletion of SKAP. Collectively, our studies represent the first thorough mechanistic analysis of SKAP and Astrin, and significantly advance our functional understanding of these important mitotic proteins.

Hyperosmosis enhances radiation and hydroxyurea resistance of Schizosaccharomyces pombe checkpoint mutants through the spindle checkpoint and delayed cytokinesis

The DNA damage and stress response pathways interact to regulate cellular responses to genotoxins and environmental stresses. How these pathways interact in Schizosaccharomyces pombe is not well understood. We demonstrate that osmotic stress suppresses the DNA damage sensitivity of checkpoint mutants, and that this occurs through three distinct cell cycle delays. A delay in G2/M is dependent on Srk1. Progression through mitosis is halted by the Mad2‐dependent spindle checkpoint. Finally, cytokinesis is impaired by modulating Cdc25 expression. These three delays, imposed by osmotic stress, together compensate for the loss of checkpoint signalling.

Multivalency of NDC80 in the outer kinetochore is essential to track shortening microtubules and generate forces

Presence of multiple copies of the microtubule-binding NDC80 complex is an evolutionary conserved feature of kinetochores, points of attachment of chromosomes to spindle microtubules. This may enable multivalent attachments to microtubules, with implications that remain unexplored. Using recombinant human kinetochore components, we show that while single NDC80 complexes do not track depolymerizing microtubules, reconstituted particles containing the NDC80 receptor CENP-T bound to three or more NDC80 complexes do so effectively, as expected for a kinetochore force coupler. To study multivalency systematically, we engineered modules allowing incremental addition of NDC80 complexes. The modules’ residence time on microtubules increased exponentially with the number of NDC80 complexes. Modules with two or more complexes tracked depolymerizing microtubules with increasing efficiencies, and stalled and rescued microtubule depolymerization in a force-dependent manner when conjugated to cargo. Our observations indicate that NDC80, rather than through biased diffusion, tracks depolymerizing microtubules by harnessing force generated during microtubule disassembly.

The kinetochore proteins CENP-E and CENP-F directly and specifically interact with distinct BUB mitotic checkpoint Ser/Thr kinases

The segregation of chromosomes during cell division relies on the function of the kinetochores, protein complexes that physically connect chromosomes with microtubules of the spindle. The metazoan proteins, centromere protein E (CENP-E) and CENP-F, are components of a fibrous layer of mitotic kinetochores named the corona. Several of their features suggest that CENP-E and CENP-F are paralogs: they are very large (comprising ∼2700 and 3200 residues, respectively), contain abundant predicted coiled-coil structures, are C-terminally prenylated, and are endowed with microtubule-binding sites at their termini. Moreover, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus end–directed motion. Here, we show that both CENP-E and CENP-F are recruited to mitotic kinetochores independently of the main corona constituent, the Rod/Zwilch/ZW10 (RZZ) complex. We identified specific interactions of CENP-F and CENP-E with budding uninhibited by benzimidazole 1 (BUB1) and BUB1-related (BUBR1) mitotic checkpoint Ser/Thr kinases, respectively, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. Whereas BUBR1 was dispensable for kinetochore localization of CENP-E, BUB1 was stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrated that the CENP-E/BUBR1 and CENP-F/BUB1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BUBR1 or BUB1. Our findings are consistent with the existence of structurally similar BUB1/CENP-F and BUBR1/CENP-E complexes, supporting the notion that CENP-E and CENP-F are evolutionarily related.