bims-micesi 2024-01-07 papers

Issue of 2024–01–07
eight papers selected by
Valentina Piano, Uniklinik Köln

Open Biol. 2024 Jan;14(1): 230379

Fission yeast Wee1 is required for stable kinetochore-microtubule attachment.

Masahiro Takado, Takaharu G Yamamoto, Yuji Chikashige, Tomohiro Matsumoto.

Wee1 is a cell cycle regulator that phosphorylates Cdk1/Cdc2 and inhibits G2/M transition. Loss of Wee1 in fission yeast results in an early onset of mitosis. Interestingly, we found that cells lacking Wee1 require the functional spindle checkpoint for their viability. Genetic analysis indicated that the requirement is not attributable to the early onset of mitosis. Live-cell imaging revealed that some kinetochores are not attached or bioriented in the wee1 mutant. Furthermore, Mad2, a component of the spindle checkpoint known to recognize unattached kinetochores, accumulates in the vicinity of the spindle, representing activation of the spindle checkpoint in the mutant. It appears that the wee1 mutant cannot maintain stable kinetochore-microtubule attachment, and relies on the delay imposed by the spindle checkpoint for establishing biorientation of kinetochores. This study revealed a role of Wee1 in ensuring accurate segregation of chromosomes during mitosis, and thus provided a basis for a new principle of cancer treatment with Wee1 inhibitors.

Keywords: Wee1; fission yeast; kinetochore; mitosis; spindle checkpoint

DOI: https://doi.org/10.1098/rsob.230379

Proc Natl Acad Sci U S A. 2024 Jan 09. 121(2): e2316583121

A coadapted KNL1 and spindle assembly checkpoint axis orchestrates precise mitosis in Arabidopsis.

Xingguang Deng, Ying He, Xiaoya Tang, Xianghong Liu, Yuh-Ru Julie Lee, Bo Liu, Honghui Lin.

The kinetochore scaffold 1 (KNL1) protein recruits spindle assembly checkpoint (SAC) proteins to ensure accurate chromosome segregation during mitosis. Despite such a conserved function among eukaryotic organisms, its molecular architectures have rapidly evolved so that the functional mode of plant KNL1 is largely unknown. To understand how SAC signaling is regulated at kinetochores, we characterized the function of the KNL1 gene in Arabidopsis thaliana. The KNL1 protein was detected at kinetochores throughout the mitotic cell cycle, and null knl1 mutants were viable and fertile but exhibited severe vegetative and reproductive defects. The mutant cells showed serious impairments of chromosome congression and segregation, that resulted in the formation of micronuclei. In the absence of KNL1, core SAC proteins were no longer detected at the kinetochores, and the SAC was not activated by unattached or misaligned chromosomes. Arabidopsis KNL1 interacted with SAC essential proteins BUB3.3 and BMF3 through specific regions that were not found in known KNL1 proteins of other species, and recruited them independently to kinetochores. Furthermore, we demonstrated that upon ectopic expression, the KNL1 homolog from the dicot tomato was able to functionally substitute KNL1 in A. thaliana, while others from the monocot rice or moss associated with kinetochores but were not functional, as reflected by sequence variations of the kinetochore proteins in different plant lineages. Our results brought insights into understanding the rapid evolution and lineage-specific connection between KNL1 and the SAC signaling molecules.

Keywords: Arabidopsis; KNL1; SAC; kinetochore

DOI: https://doi.org/10.1073/pnas.2316583121

J Cell Biol. 2024 Mar 04. pii: e202211122. [Epub ahead of print]223(3):

Multimerization of a disordered kinetochore protein promotes accurate chromosome segregation by localizing a core dynein module.

Jessica M McGory, Vikash Verma, Dylan M Barcelos, Thomas J Maresca.

Kinetochores connect chromosomes and spindle microtubules to maintain genomic integrity through cell division. Crosstalk between the minus-end directed motor dynein and kinetochore-microtubule attachment factors promotes accurate chromosome segregation by a poorly understood pathway. Here, we identify a linkage between the intrinsically disordered protein Spc105 (KNL1 orthologue) and dynein using an optogenetic oligomerization assay. Core pools of the checkpoint protein BubR1 and the adaptor complex RZZ contribute to the linkage. Furthermore, a minimal segment of Spc105 with a propensity to multimerize and which contains protein binding motifs is sufficient to link Spc105 to RZZ/dynein. Deletion of the minimal region from Spc105 compromises the recruitment of its binding partners to kinetochores and elevates chromosome missegregation due to merotelic attachments. Restoration of normal chromosome segregation and localization of BubR1 and RZZ requires both protein binding motifs and oligomerization of Spc105. Together, our results reveal that higher-order multimerization of Spc105 contributes to localizing a core pool of RZZ that promotes accurate chromosome segregation.

DOI: https://doi.org/10.1083/jcb.202211122

Nat Cell Biol. 2024 Jan 02.

Higher-order protein assembly controls kinetochore formation.

Gunter B Sissoko, Ekaterina V Tarasovetc, Océane Marescal, Ekaterina L Grishchuk, Iain M Cheeseman.

To faithfully segregate chromosomes during vertebrate mitosis, kinetochore-microtubule interactions must be restricted to a single site on each chromosome. Prior work on pair-wise kinetochore protein interactions has been unable to identify the mechanisms that prevent outer kinetochore formation in regions with a low density of CENP-A nucleosomes. To investigate the impact of higher-order assembly on kinetochore formation, we generated oligomers of the inner kinetochore protein CENP-T using two distinct, genetically engineered systems in human cells. Although individual CENP-T molecules interact poorly with outer kinetochore proteins, oligomers that mimic centromeric CENP-T density trigger the robust formation of functional, cytoplasmic kinetochore-like particles. Both in cells and in vitro, each molecule of oligomerized CENP-T recruits substantially higher levels of outer kinetochore components than monomeric CENP-T molecules. Our work suggests that the density dependence of CENP-T restricts outer kinetochore recruitment to centromeres, where densely packed CENP-A recruits a high local concentration of inner kinetochore proteins.

DOI: https://doi.org/10.1038/s41556-023-01313-7

Cell Death Dis. 2024 Jan 03. 15(1): 2

BCL-XL regulates the timing of mitotic apoptosis independently of BCL2 and MCL1 compensation.

Chun Yin Yu, Tsz Kwan Yeung, Wai Kuen Fu, Randy Y C Poon.

Mitotic catastrophe induced by prolonged mitotic arrest is a major anticancer strategy. Although antiapoptotic BCL2-like proteins, including BCL-XL, are known to regulate apoptosis during mitotic arrest, adaptive changes in their expression can complicate loss-of-function studies. Our studies revealed compensatory alterations in the expression of BCL2 and MCL1 when BCL-XL is either downregulated or overexpressed. To circumvent their reciprocal regulation, we utilized a degron-mediated system to acutely silence BCL-XL just before mitosis. Our results show that in epithelial cell lines including HeLa and RPE1, BCL-XL and BCL2 acted collaboratively to suppress apoptosis during both unperturbed cell cycle and mitotic arrest. By tagging BCL-XL and BCL2 with a common epitope, we estimated that BCL-XL was less abundant than BCL2 in the cell. Nonetheless, BCL-XL played a more prominent antiapoptotic function than BCL2 during interphase and mitotic arrest. Loss of BCL-XL led to mitotic cell death primarily through a BAX-dependent process. Furthermore, silencing of BCL-XL led to the stabilization of MCL1, which played a significant role in buffering apoptosis during mitotic arrest. Nevertheless, even in a MCL1-deficient background, depletion of BCL-XL accelerated mitotic apoptosis. These findings underscore the pivotal involvement of BCL-XL in controlling timely apoptosis during mitotic arrest, despite adaptive changes in the expression of other BCL2-like proteins.

DOI: https://doi.org/10.1038/s41419-023-06404-9

bioRxiv. 2023 Dec 19. pii: 2023.12.12.571338. [Epub ahead of print]

Securin Regulates the Spatiotemporal Dynamics of Separase.

Christopher G Sorensen Turpin, Dillon Sloan, Marian LaForest, Lindsey Uehlein Klebanow, Diana Mitchell, Aaron F Severson, Joshua N Bembenek.

Separase is a key regulator of the metaphase to anaphase transition with multiple functions. Separase cleaves cohesin to allow chromosome segregation and localizes to vesicles to promote exocytosis in mid-anaphase. The anaphase promoting complex/cyclosome (APC/C) activates separase by ubiquitinating its inhibitory chaperone, securin, triggering its degradation. How this pathway controls the exocytic function of separase has not been investigated. During meiosis I, securin is degraded over several minutes, while separase rapidly relocalizes from kinetochore structures at the spindle and cortex to sites of action on chromosomes and vesicles at anaphase onset. The loss of cohesin coincides with the relocalization of separase to the chromosome midbivalent at anaphase onset. APC/C depletion prevents separase relocalization, while securin depletion causes precocious separase relocalization. Expression of non-degradable securin inhibits chromosome segregation, exocytosis, and separase localization to vesicles but not to the anaphase spindle. We conclude that APC/C mediated securin degradation controls separase localization. This spatiotemporal regulation will impact the effective local concentration of separase for more precise targeting of substrates in anaphase.

DOI: https://doi.org/10.1101/2023.12.12.571338

Nat Struct Mol Biol. 2024 Jan 04.

Aurora kinase A-mediated phosphorylation triggers structural alteration of Rab1A to enhance ER complexity during mitosis.

Morphological rearrangement of the endoplasmic reticulum (ER) is critical for metazoan mitosis. Yet, how the ER is remodeled by the mitotic signaling remains unclear. Here, we report that mitotic Aurora kinase A (AURKA) employs a small GTPase, Rab1A, to direct ER remodeling. During mitosis, AURKA phosphorylates Rab1A at Thr75. Structural analysis demonstrates that Thr75 phosphorylation renders Rab1A in a constantly active state by preventing interaction with GDP-dissociation inhibitor (GDI). Activated Rab1A is retained on the ER and induces the oligomerization of ER-shaping protein RTNs and REEPs, eventually triggering an increase of ER complexity. In various models, from Caenorhabditis elegans and Drosophila to mammals, inhibition of Rab1AThr75 phosphorylation by genetic modifications disrupts ER remodeling. Thus, our study reveals an evolutionarily conserved mechanism explaining how mitotic kinase controls ER remodeling and uncovers a critical function of Rab GTPases in metaphase.

DOI: https://doi.org/10.1038/s41594-023-01165-7

Sichuan Da Xue Xue Bao Yi Xue Ban. 2023 Nov 20. 54(6): 1167-1175

[Prognostic Value of PCMT1 Expression in Gastric Cancer and Its Regulatory Effect on Spindle Assembly Checkpoints].

Yueyue Wang, Min Zhang, Zhen Zhang, Bohan Li, Ju Huang, Jing Li, Zhijun Geng, Xiaofeng Zhang, Xue Song, Lian Wang, Lugen Zuo, Jianguo Hu.

Objective: The study was conducted to investigate the expression of protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) in gastric cancer and its effect on the prognosis, and to analyze its potential mechanism.
Methods: UALCAN, a cancer data analysis platform, was used to conduct online analysis of the expression of PCMT1 in gastric cancer tissues. Through the Database for Annotation, Visualization and Integrated Discovery (DAVID), Gene Ontology (GO) annotation and signaling pathway enrichment by Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to analyze the possible functions and signaling pathways. A total of 120 patients who underwent radical gastrectomy for gastric cancer between January 2014 and December 2017 in our hospital were enrolled for the study. Immunohistochemical staining was performed to determine the expression of PCMT1 and Ki67 in gastric cancer tissues. Cox regression, Kaplan-Meier curve, and receiver operating characteristic (ROC) curves were used for prognostic analysis of 5-year survival in gastric cancer patients after surgery. Lentivirus was used to construct PCMT1-interfering or PCMT1-overexpressing vectors, which were then used to transfect human gastric cancer cell lines of MGC-803 and HGC-27 cells. The interfering empty vector (sh-NC) group, the interfering PCMT1 vector (sh-PCMT1) group, the overexpressing empty vector (LV-Vec) group, and the overexpressing PCMT1 vector (LV-PCMT1) group were set up. Western blot was performed to determine the protein expression levels of PCMT1, CyclinB1, and CDC20. CCK-8 assay was performed to measure the proliferation of gastric cancer cells. Flow cytometry was performed to determine the cell cycle. MGC-803 cells were injected in four groups of nude mice to construct a subcutaneous xenograft tumor model, with three nude mice in each group. The body mass of the nude mice was measured. The nude mice were sacrificed after 14 days and the tumor volume was monitored. The expression levels of CyclinB1 and CDC20 proteins in the tumor tissues were determined by Western blot assay.
Results: Analysis with UALCAN showed that PCMT1 was highly expressed in gastric cancer tissues. Moreover, elevated expression was found in gastric tumor tissues of different pathological stages and grades and those with lymph node metastasis (P<0.05). GO and KEGG enrichment analyses showed that PCMT1 was mainly involved in the signal regulation of mitosis, spindle assembly checkpoints, and cell cycle. The immunohistochemical results showed that PCMT1 and Ki67 were highly expressed in gastric cancer tissues and that they were positively correlated with each other (P<0.05). Cox multivariate analysis showed that high PCMT1 expression (hazard ratio [HR]=2.921, 95% confidence interval [CI]:1.628-5.239) was one of the independent risk factors affecting the 5-year survival rate of gastric cancer patients after surgery. Kaplan-Meier curve showed that patients with high PCMT1 expression had a lower 5-year survival after surgery (16.7%, HR=4.651, 95% CI: 2.846-7.601) than patients with low PCMT1 expression (70.0%, HR=0.215, 95% CI: 0.132-0.351) did. The ROC curve showed that PCMT1 had an area under the curve (AUC) of 0.764 (95% CI: 0.674-0.854) for predicting 5-year patient survival after surgery. Western blot results showed that lentiviral interference or overexpression of PCMT1 cell lines was successfully constructed. The results of CCK-8 showed that the proliferative ability of MGC-803 and HGC-27 cells was weakened with the downregulation of PCMT1, and the overexpression of PCMT1 promoted cell proliferation (P<0.05). With the interference of PCMT1, the expression of CDC20 protein was decreased, the expression of CyclinB1 protein was increased, and the cell cycle was arrested in the G2/M phase. In contrast, the overexpression of PCMT1 led to the opposite trends (P<0.05). In the sh-PCMT1 group, the tumor volume and mass were decreased and the expression of CDC20 protein was decreased and the expression of CyclinB1 protein was increased in the tumor tissues of the nude mice (P<0.05, compared with those of the sh-NC group. In contrast, the LV-PCMT1 group showed the opposite trends (P<0.05, compared with those of the LV-Vec group).
Conclusion: The high expression of PCMT1 in gastric cancer tissues is associated with poor prognosis in patients and may affect tumor cell malignant proliferation via regulating spindle checkpoints in the process of mitosis.

Keywords: Clinical prognosis; Gastric cancer; PCMT1; Spindle assembly checkpoint

DOI: https://doi.org/10.12182/20231160211