Phosphorylation of Bub1 by Mph1 promotes Bub1 signaling at the kinetochore to ensure accurate chromosome segregation.

Bub1 is a conserved mitotic kinase involved in signaling of the spindle assembly checkpoint. Multiple phosphorylation sites on Bub1 have been characterized, yet it is challenging to understand the interplay between the multiple phosphorylation sites due to the limited availability of phosphospecific antibodies. In addition, phosphoregulation of Bub1 in Schizosaccharomyces pombe is poorly understood. Here we report the identification of a new Mph1/Mps1-mediated phosphorylation site, i.e., Ser532, of Bub1 in Schizosaccharomyces pombe. A phosphospecific antibody against phosphorylated Bub1-Ser532 was developed. Using the phosphospecific antibody, we demonstrated that phosphorylation of Bub1-Ser352 was mediated specifically by Mph1/Mps1 and took place during early mitosis. Moreover, live-cell microscopy showed that inhibition of the phosphorylation of Bub1 at Ser532 impaired the localization of Bub1, Mad1, and Mad2 to the kinetochore. In addition, inhibition of the phosphorylation of Bub1 at Ser532 caused anaphase B lagging chromosomes. Hence, our study constitutes a model in which Mph1/Mps1-mediated phosphorylation of fission yeast Bub1 promotes proper kinetochore localization of Bub1 and faithful chromosome segregation.

SpindlesTracker: An Automatic and Low-Cost Labeled Workflow for Spindle Analysis.

Quantitative analysis of spindle dynamics in mitosis through fluorescence microscopy requires tracking spindle elongation in noisy image sequences. Deterministic methods, which use typical microtubule detection and tracking methods, perform poorly in the sophisticated background of spindles. In addition, the expensive data labeling cost also limits the application of machine learning in this field. Here we present a fully automatic and low-cost labeled workflow that efficiently analyzes the dynamic spindle mechanism of time-lapse images, called SpindlesTracker. In this workflow, we design a network named YOLOX-SP which can accurately detect the location and endpoint of each spindle under box-level data supervision. We then optimize the algorithm SORT and MCP for spindle's tracking and skeletonization. As there was no publicly available dataset, we annotated a S.pombe dataset that was entirely acquired from the real world for both training and evaluation. Extensive experiments demonstrate that SpindlesTracker achieves excellent performance in all aspects, while reducing label costs by 60%. Specifically, it achieves 84.1% mAP in spindle detection and over 90% accuracy in endpoint detection. Furthermore, the improved algorithm enhances tracking accuracy by 1.3% and tracking precision by 6.5%. Statistical results also indicate that the mean error of spindle length is within 1 µm. In summary, SpindlesTracker holds significant implications for the study of mitotic dynamic mechanisms and can be readily extended to the analysis of other filamentous objects. The code and the dataset are both released on GitHub.

The fission yeast kinetochore complex Mhf1-Mhf2 regulates the spindle assembly checkpoint and faithful chromosome segregation.

The outer kinetochore serves as a platform for the initiation of the spindle assembly checkpoint (SAC) and for mediating kinetochore-microtubule attachments. How the inner kinetochore subcomplex CENP-S-CENP-X is involved in regulating the SAC and kinetochore-microtubule attachments has not been well characterized. Using live-cell microscopy and yeast genetics, we found that Mhf1-Mhf2, the CENP-S-CENP-X counterpart in the fission yeast Schizosaccharomyces pombe, plays crucial roles in promoting the SAC and regulating chromosome segregation. The absence of Mhf2 attenuates the SAC, impairs the kinetochore localization of most of the components in the constitutive centromere-associated network (CCAN), and alters the localization of the kinase Ark1 (yeast homolog of Aurora B) to the kinetochore. Hence, our findings constitute a model in which Mhf1-Mhf2 ensures faithful chromosome segregation by regulating the accurate organization of the CCAN complex, which is required for promoting SAC signaling and for regulating kinetochore-microtubule attachments. This article has an associated First Person interview with the first author of the paper.

Structural insights reveal the specific recognition of meiRNA by the Mei2 protein.

In the fission yeast Schizosaccharomyces pombe, Mei2, an RNA-binding protein essential for entry into meiosis, regulates meiosis initiation. Mei2 binds to a specific non-coding RNA species, meiRNA, and accumulates at the sme2 gene locus, which encodes meiRNA. Previous research has shown that the Mei2 C-terminal RNA recognition motif (RRM3) physically interacts with the meiRNA 5' region in vitro and stimulates meiosis in vivo. However, the underlying mechanisms still remain elusive. We first employed an in vitro crosslinking and immunoprecipitation sequencing (CLIP-seq) assay and demonstrated a preference for U-rich motifs of meiRNA by Mei2 RRM3. We then solved the crystal structures of Mei2 RRM3 in the apo form and complex with an 8mer RNA fragment, derived from meiRNA, as detected by in vitro CLIP-seq. These results provide structural insights into the Mei2 RRM3-meiRNA complex and reveal that Mei2 RRM3 binds specifically to the UUC(U) sequence. Furthermore, a structure-based Mei2 mutation, Mei2F644A causes defective karyogamy, suggesting an essential role of the RNA-binding ability of Mei2 in regulating meiosis.

Klp2 and Ase1 synergize to maintain meiotic spindle stability during metaphase I.

The spindle apparatus segregates bi-oriented sister chromatids during mitosis but mono-oriented homologous chromosomes during meiosis I. It has remained unclear if similar molecular mechanisms operate to regulate spindle dynamics during mitosis and meiosis I. Here, we employed live-cell microscopy to compare the spindle dynamics of mitosis and meiosis I in fission yeast cells and demonstrated that the conserved kinesin-14 motor Klp2 plays a specific role in maintaining metaphase spindle length during meiosis I but not during mitosis. Moreover, the maintenance of metaphase spindle stability during meiosis I requires the synergism between Klp2 and the conserved microtubule cross-linker Ase1, as the absence of both proteins causes exacerbated defects in metaphase spindle stability. The synergism is not necessary for regulating mitotic spindle dynamics. Hence, our work reveals a new molecular mechanism underlying meiotic spindle dynamics and provides insights into understanding differential regulation of meiotic and mitotic events.