Sue Biggins

Tension Directly Stabilizes Reconstituted Kinetochore-Microtubule Attachments

Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore–microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore–microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore–microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

The Ipl1-Aurora protein kinase activates the spindle checkpoint by creating unattached kinetochores

The spindle checkpoint ensures accurate chromosome segregation by delaying cell-cycle progression until all sister kinetochores capture microtubules from opposite poles and come under tension (for reviews, see refs 1, 2). Although the checkpoint is activated by either the lack of kinetochore-microtubule attachments or defects in the tension exerted by microtubule-generated forces, it is not clear whether these signals are linked. We investigated the connection between tension and attachment by studying the conserved budding yeast Ipl1Aurora protein kinase that is required for checkpoint activation in the absence of tension but not attachment. Here, we show that spindle-checkpoint activation in kinetochore mutants that seem to have unattached kinetochores depends on Ipl1 activity. When Ipl1 function was impaired in these kinetochore mutants, the attachments were restored and the checkpoint was turned off. These data indicate that Ipl1 activates the checkpoint in response to tension defects by creating unattached kinetochores. Moreover, although the Dam1 kinetochore complex has been implicated as a key downstream target, we found the existence of unidentified Ipl1 sites on Dam1 or additional important substrates that regulate both microtuble detachment and the checkpoint.

The NoCut Pathway Links Completion of Cytokinesis to Spindle Midzone Function to Prevent Chromosome Breakage

During anaphase, spindle elongation pulls sister chromatids apart until each pair is fully separated. In turn, cytokinesis cleaves the cell between the separated chromosomes. What ensures that cytokinesis proceeds only after that all chromosome arms are pulled out of the cleavage plane was unknown. Here, we show that a signaling pathway, which we call NoCut, delays the completion of cytokinesis in cells with spindle-midzone defects. NoCut depends on the Aurora kinase Ipl1 and the anillin-related proteins Boi1 and Boi2, which localize to the site of cleavage in an Ipl1-dependent manner and act as abscission inhibitors. Inactivation of NoCut leads to premature abscission and chromosome breakage by the cytokinetic machinery and is lethal in cells with spindle-elongation defects. We propose that NoCut monitors clearance of chromatin from the midzone to ensure that cytokinesis completes only after all chromosomes have migrated to the poles.

Phosphoregulation of Spc105 by Mps1 and PP1 Regulates Bub1 Localization to Kinetochores

Kinetochores are the macromolecular complexes that interact with microtubules to mediate chromosome segregation. Accurate segregation requires that kinetochores make bioriented attachments to microtubules from opposite poles. Attachments between kinetochores and microtubules are monitored by the spindle checkpoint, a surveillance system that prevents anaphase until every pair of chromosomes makes proper bioriented attachments. Checkpoint activity is correlated with the recruitment of checkpoint proteins to the kinetochore. Mps1 is a conserved protein kinase that regulates segregation and the spindle checkpoint, but few of the targets that mediate its functions have been identified. Here, we show that Mps1 is the major kinase activity that copurifies with budding yeast kinetochore particles and identify the conserved Spc105/KNL-1/blinkin kinetochore protein as a substrate. Phosphorylation of conserved MELT motifs within Spc105 recruits the Bub1 protein to kinetochores, and this is reversed by protein phosphatase I (PP1). Spc105 mutants lacking Mps1 phosphorylation sites are defective in the spindle checkpoint and exhibit growth defects. Together, these data identify Spc105 as a key target of the Mps1 kinase and show that the opposing activities of Mps1 and PP1 regulate the kinetochore localization of the Bub1 protein.

Proteolysis Contributes to the Exclusive Centromere Localization of the Yeast Cse4/CENP-A Histone H3 Variant

Kinetochores are the specialized protein structures that form on centromeric DNA and direct chromosome segregation. It is critical that all chromosomes assemble a single kinetochore every cell cycle. One hallmark of all eukaryotic kinetochores is CENP-A, an essential centromeric histone H3 (CenH3) variant. Overexpression of CENP-A causes mislocalization to euchromatin, which could lead to deleterious consequences because CENP-A overexpression is associated with colorectal cancer . Although CENP-A protein levels are important for genomic stability, little is known about the mechanisms of CenH3 regulation. Here, we show that the levels of the budding yeast CenH3, Cse4, are regulated by ubiquitin-proteasome-mediated proteolysis. Because mutation of all Cse4 lysine residues did not completely stabilize the protein, we isolated a dominant lethal mutant, CSE4-351, that was stable. The Cse4-351 protein localized to euchromatin, suggesting that proteolysis prevents CenH3 euchromatic localization. When wild-type Cse4 was fused to a degron signal, the soluble Cse4 protein was rapidly degraded, but the centromere bound Cse4 was stable, indicating that centromere localization protects Cse4 from degradation. Taken together, these data identify proteolysis as one mechanism that contributes to the restricted centromere localization of the yeast CenH3.

Centromere identity is specified by a single centromeric nucleosome in budding yeast.

Chromosome segregation ensures that DNA is equally divided between daughter cells during each round of cell division. The centromere (CEN) is the specific locus on each chromosome that directs formation of the kinetochore, the multiprotein complex that interacts with the spindle microtubules to promote proper chromosomal alignment and segregation during mitosis. CENs are organized into a specialized chromatin structure due to the incorporation of an essential CEN-specific histone H3 variant (CenH3) in the centromeric nucleosomes of all eukaryotes. Consistent with its essential role at the CEN, the loss or up-regulation of CenH3 results in mitotic defects. Despite the requirement for CenH3 in CEN function, it is unclear how CenH3 nucleosomes structurally organize centromeric DNA to promote formation of the kinetochore. To address this issue, we developed a modified chromatin immunoprecipitation approach to analyze the number and position of CenH3 nucleosomes at the budding yeast CEN. Using this technique, we show that yeast CENs have a single CenH3 nucleosome positioned over the CEN-determining elements. Therefore, a single CenH3 nucleosome forms the minimal unit of centromeric chromatin necessary for kinetochore assembly and proper chromosome segregation.

Signalling dynamics in the spindle checkpoint response

The spindle checkpoint ensures proper chromosome segregation during cell division. Unravelling checkpoint signalling has been a long-standing challenge owing to the complexity of the structures and forces that regulate chromosome segregation. New reports have now substantially advanced our understanding of checkpoint signalling mechanisms at the kinetochore, the structure that connects microtubules and chromatin. In contrast to the traditional view of a binary checkpoint response — either completely on or off — new findings indicate that the checkpoint response strength is variable. This revised perspective provides insight into how checkpoint bypass can lead to aneuploidy and informs strategies to exploit these errors for cancer treatments.

The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly.

Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1–GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

An E3 Ubiquitin Ligase Prevents Ectopic Localization of the Centromeric Histone H3 Variant via the Centromere Targeting Domain

Proper centromere function is critical to maintain genomic stability and to prevent aneuploidy, a hallmark of tumors and birth defects. A conserved feature of all eukaryotic centromeres is an essential histone H3 variant called CENP-A that requires a centromere targeting domain (CATD) for its localization. Although proteolysis prevents CENP-A from mislocalizing to euchromatin, regulatory factors have not been identified. Here, we identify an E3 ubiquitin ligase called Psh1 that leads to the degradation of Cse4, the budding yeast CENP-A homolog. Cse4 overexpression is toxic to psh1Δ cells and results in euchromatic localization. Strikingly, the Cse4 CATD is a key regulator of its stability and helps Psh1 discriminate Cse4 from histone H3. Taken together, we propose that the CATD has a previously unknown role in maintaining the exclusive localization of Cse4 by preventing its mislocalization to euchromatin via Psh1-mediated degradation.

Protein Phosphatase 1 Regulates Exit from the Spindle Checkpoint in Budding Yeast

Accurate chromosome segregation depends on sister kinetochores coming under tension when they make bioriented attachments to microtubules from opposite poles. The spindle checkpoint halts the cell cycle in response to defects in generating proper attachments or tension on kinetochores, although the precise signal that triggers the checkpoint is unclear because tension and attachment are coupled. The target of the checkpoint is the Cdc20 protein, which initiates the anaphase-promoting complex (APC)-dependent degradation of the anaphase inhibitor Pds1/securin. Although the molecular details of spindle checkpoint activation are still being elucidated, phosphorylation by at least four kinases is a crucial requirement. However, less is known about the mechanisms that silence the checkpoint after kinetochores biorient. Here, we show that the catalytic subunit of the budding yeast protein phosphatase 1 (PP1) homolog, Glc7, regulates exit from the checkpoint. Glc7 overexpression prevents spindle checkpoint activation in response to both tension and attachment defects. Although glc7 mutant cells are able to efficiently release from a non-checkpoint-mediated metaphase arrest, they are uniquely sensitive to transient spindle checkpoint activation as a result of a failure in spindle checkpoint exit. We therefore propose that PP1 activity silences the checkpoint by reversing key phosphorylation events.