chromosome congression
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2022 ◽  
Author(s):  
Stanislau Yatskevich ◽  
Kyle W Muir ◽  
Dom Bellini ◽  
Ziguo Zhang ◽  
Jing Yang ◽  
...  

Accurate chromosome segregation, controlled by kinetochore-mediated chromatid attachments to the mitotic spindle, ensures the faithful inheritance of genetic information. Kinetochores assemble onto specialized CENP-A nucleosomes (CENP-ANuc) of centromeric chromatin. In humans, this is mostly organized as thousands of copies of an ~171 bp α-satellite repeat. Here, we describe the cryo-EM structure of the human inner kinetochore CCAN (Constitutive Centromere Associated Network) complex bound to CENP-ANuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, while a linker DNA segment of the α-satellite repeat emerges from the fully-wrapped end of the nucleosome to thread through the central CENP-LN channel which tightly grips the DNA. The CENP-TWSX histone-fold module, together with CENP-HIKHead, further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the α-satellite repeat linker DNA by CCAN provides a robust mechanism by which the kinetochore withstands the pushing and pulling of centromeres associated with chromosome congression and segregation forces.


2021 ◽  
Author(s):  
Ines Berenguer ◽  
Pablo Lopez Jimenez ◽  
Irene Mena ◽  
Alberto Viera ◽  
Jesus Page ◽  
...  

Chromosome segregation requires that centromeres properly attach to spindle microtubules. This is an essential step towards the accuracy of cell division and therefore must be precisely regulated in both mitosis and meiosis. One of the main centromeric regulatory signaling pathways is the Haspin-H3T3ph-chromosomal passenger complex (CPC) cascade, which is responsible for the recruitment of the CPC to the centromeres. In mitosis, Haspin kinase phosphorylates H3 at threonine 3 (H3T3ph), the essential histone mark that recruits the CPC whose catalytic component is Aurora B kinase. To date, no data has yet been presented about the action of the centromeric Haspin-H3T3ph-CPC pathway in mammalian male meiosis. We have analyzed the consequences of Haspin chemical inhibition in cultured spermatocytes using LDN-192960. Our in vitro studies suggest that Haspin kinase activity is required for proper chromosome congression during both meiotic divisions and for the recruitment of phosphorylated Aurora B at meiotic centromeres. These results have been confirmed by the characterization of the meiotic phenotype of the genetic mouse model Haspin-/-, which displays similar defects. In addition, our work demonstrates that the absence of H3T3ph histone mark does not alter SGO2 localization to meiotic centromeres. These results add new and relevant information regarding the regulation of centromere function during meiosis.


2021 ◽  
Vol 220 (12) ◽  
Author(s):  
Amrita Kumari ◽  
Chandan Kumar ◽  
Rajaiah Pergu ◽  
Megha Kumar ◽  
Sagar P. Mahale ◽  
...  

The dynein motor performs multiple functions in mitosis by engaging with a wide cargo spectrum. One way to regulate dynein’s cargo-binding selectivity is through the C-terminal domain (CTD) of its light intermediate chain 1 subunit (LIC1), which binds directly with cargo adaptors. Here we show that mitotic phosphorylation of LIC1-CTD at its three cdk1 sites is required for proper mitotic progression, for dynein loading onto prometaphase kinetochores, and for spindle assembly checkpoint inactivation in human cells. Mitotic LIC1-CTD phosphorylation also engages the prolyl isomerase Pin1 predominantly to Hook2-dynein-Nde1-Lis1 complexes, but not to dynein-spindly-dynactin complexes. LIC1-CTD dephosphorylation abrogates dynein-Pin1 binding, promotes prophase centrosome–nuclear envelope detachment, and impairs metaphase chromosome congression and mitotic Golgi fragmentation, without affecting interphase membrane transport. Phosphomutation of a conserved LIC1-CTD SP site in zebrafish leads to early developmental defects. Our work reveals that LIC1-CTD phosphorylation differentially regulates distinct mitotic dynein pools and suggests the evolutionary conservation of this phosphoregulation.


2021 ◽  
Author(s):  
Alex F Thompson ◽  
Patrick R Blackburn ◽  
Dusica Babovic-Vuksanovic ◽  
Jane B Lian ◽  
Eric W Klee ◽  
...  

The chromokinesin KIF22 uses plus end-directed motility and direct binding to chromosome arms to generate pushing forces that contribute to mitotic chromosome congression and alignment. Mutations in the motor domain of KIF22 have been identified in patients with abnormal skeletal development, and we report the identification of a patient with a novel mutation in the coiled-coil domain of the KIF22 tail. The mechanism by which these mutations affect development is unknown. We assessed whether pathogenic mutations affect the function of KIF22 in mitosis and demonstrate that mutations do not result in a loss of KIF22 function. Pathogenic mutations did not alter the localization or prometaphase function of KIF22. Instead, mutations disrupted chromosome segregation in anaphase, resulting in reduced proliferation, abnormal daughter cell nuclear morphology and, in a subset of cells, cytokinesis failure. This phenotype could be explained by a failure of KIF22 to inactivate in anaphase. Consistent with this model, a phosphomimetic mutation, which constitutively activates the motor, phenocopied the effects of pathogenic mutations. These findings offer insight into the mechanism by which mutations in KIF22 may affect human development, the balance between polar ejection forces and antiparallel microtubule sliding in anaphase, and potential mechanisms of KIF22 regulation.


2021 ◽  
Author(s):  
Morgan S Schrock ◽  
Luke Scarberry ◽  
Benjamin R Stromberg ◽  
Claire Sears ◽  
Adrian E Torres ◽  
...  

Mitotic kinesin-like protein 2 (MKLP2) is a motor protein with a well-established function in promoting cytokinesis. However, our results with siRNAs targeting MKLP2 and small molecule inhibitors of MKLP2 (MKLP2i) along with the observations of others suggested a function earlier in mitosis, prior to anaphase. In this study we provide direct evidence that MKLP2 facilitates chromosome congression in prometaphase. We employed live imaging to observe HeLa cells with fluorescently tagged histones treated with MKLP2i and discovered a pronounced chromosome congression defect. We show that MKLP2 inhibited cells had a significant increase in unstable kinetochore-microtubule attachments due to impaired error correction of syntelic attachments. We propose that MKLP2 mediates kinetochore microtubule attachment stability by regulating Aurora Kinase and a downstream target, pHEC1 (Ser 55). Lastly, we show that MKLP2 inhibition results in aneuploidy, confirming that MKLP2 safeguards cells against chromosomal instability.


2021 ◽  
Vol 118 (34) ◽  
pp. e2108145118
Author(s):  
Anja Bufe ◽  
Ana García del Arco ◽  
Magdalena Hennecke ◽  
Anchel de Jaime-Soguero ◽  
Matthias Ostermaier ◽  
...  

Canonical Wnt signaling plays critical roles in development and tissue renewal by regulating β-catenin target genes. Recent evidence showed that β-catenin–independent Wnt signaling is also required for faithful execution of mitosis. However, the targets and specific functions of mitotic Wnt signaling still remain uncharacterized. Using phosphoproteomics, we identified that Wnt signaling regulates the microtubule depolymerase KIF2A during mitosis. We found that Dishevelled recruits KIF2A via its N-terminal and motor domains, which is further promoted upon LRP6 signalosome formation during cell division. We show that Wnt signaling modulates KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle. Accordingly, inhibition of basal Wnt signaling leads to chromosome misalignment in somatic cells and pluripotent stem cells. We propose that Wnt signaling monitors KIF2A activity at the spindle poles during mitosis to ensure timely chromosome alignment. Our findings highlight a function of Wnt signaling during cell division, which could have important implications for genome maintenance, notably in stem cells.


2021 ◽  
Author(s):  
Alexander Julner ◽  
Marjan Abbasi ◽  
Victoria Menendez Benito

During mitosis, sister chromatids congress on either side of the spindle equator to facilitate the correct partitioning of the genomic material. Chromosome congression requires a finely tuned control of microtubule dynamics by the kinesin motor proteins. In Saccharomyces cerevisiae, the kinesin proteins Cin8, Kip1, and Kip3 have pivotal roles in chromosome congression. It has been hypothesized that additional proteins that modulate microtubule dynamics are also involved. Here, we show that the microtubule plus-end tracking protein Bik1 (the budding yeast ortholog of CLIP-170) is essential for chromosome congression. We find that nuclear Bik1 localizes to the kinetochores in a cell-cycle-dependent manner. Disrupting the nuclear pool of Bik1 with a nuclear export signal (Bik1-NES) leads to a slower cell cycle progression characterized by a delayed metaphase-anaphase transition. Bik1-NES cells have mispositioned kinetochores along the spindle in metaphase. Furthermore, using proximity-dependent methods, we identify Cin8 as an interaction partner of Bik1. Deleting CIN8 reduces the amount of Bik1 at the spindle. In contrast, Cin8 retains its typical bilobed distribution in Bik1-NES and does not localize to the unclustered kinetochores characteristic of Bik1-NES cells. Thus, we propose that Bik1 functions together with Cin8 to regulate kinetochore-microtubule dynamics for correct kinetochore positioning and chromosome congression.


PLoS Genetics ◽  
2021 ◽  
Vol 17 (5) ◽  
pp. e1009567
Author(s):  
Nikita S. Divekar ◽  
Amanda C. Davis-Roca ◽  
Liangyu Zhang ◽  
Abby F. Dernburg ◽  
Sarah M. Wignall

The widely conserved kinase Aurora B regulates important events during cell division. Surprisingly, recent work has uncovered a few functions of Aurora-family kinases that do not require kinase activity. Thus, understanding this important class of cell cycle regulators will require strategies to distinguish kinase-dependent from independent functions. Here, we address this need in C. elegans by combining germline-specific, auxin-induced Aurora B (AIR-2) degradation with the transgenic expression of kinase-inactive AIR-2. Through this approach, we find that kinase activity is essential for AIR-2’s major meiotic functions and also for mitotic chromosome segregation. Moreover, our analysis revealed insight into the assembly of the ring complex (RC), a structure that is essential for chromosome congression in C. elegans oocytes. AIR-2 localizes to chromosomes and recruits other components to form the RC. However, we found that while kinase-dead AIR-2 could load onto chromosomes, other components were not recruited. This failure in RC assembly appeared to be due to a loss of RC SUMOylation, suggesting that there is crosstalk between SUMOylation and phosphorylation in building the RC and implicating AIR-2 in regulating the SUMO pathway in oocytes. Similar conditional depletion approaches may reveal new insights into other cell cycle regulators.


Cell Cycle ◽  
2021 ◽  
pp. 1-19
Author(s):  
Divya Subramonian ◽  
Te-An Chen ◽  
Nicholas Paolini ◽  
Xiang-Dong “David” Zhang

Author(s):  
Sara Abdelfatah ◽  
Janine Naß ◽  
Caroline Knorz ◽  
Sabine M. Klauck ◽  
Jan-Heiner Küpper ◽  
...  

AbstractPyrrolizidine alkaloids (PAs) are a large group of highly toxic chemical compounds, which are found as cross-contaminants in numerous food products (e.g., honey), dietary supplements, herbal teas, and pharmaceutical herbal medicines. PA contaminations are responsible for serious hepatotoxicity and hepatocarcinogenesis. Health authorities have to set legal limit values to guarantee the safe consumption of plant-based nutritional and medical products without harmful health. Toxicological and chemical analytical methods are conventionally applied to determine legally permitted limit values for PAs. In the present investigation, we applied a highly sensitive transcriptomic approach to investigate the effect of low concentrations of five PAs (lasiocarpine, riddelliine, lycopsamine, echimidine, and monocrotaline) on human cytochrome P450 3A4-overexpressing HepG2 clone 9 hepatocytes. The transcriptomic profiling of deregulated gene expression indicated that the PAs disrupted important signaling pathways related to cell cycle regulation and DNA damage repair in the transfected hepatocytes, which may explain the carcinogenic PA effects. As PAs affected the expression of genes that involved in cell cycle regulation, we applied flow cytometric cell cycle analyses to verify the transcriptomic data. Interestingly, PA treatment led to an arrest in the S phase of the cell cycle, and this effect was more pronounced with more toxic PAs (i.e., lasiocarpine and riddelliine) than with the less toxic monocrotaline. Using immunofluorescence, high fractions of cells were detected with chromosome congression defects upon PA treatment, indicating mitotic failure. In conclusion, the tested PAs revealed threshold concentrations, above which crucial signaling pathways were deregulated resulting in cell damage and carcinogenesis. Cell cycle arrest and DNA damage repair point to the mutagenicity of PAs. The disturbance of chromosome congression is a novel mechanism of Pas, which may also contribute to PA-mediated carcinogenesis. Transcriptomic, cell cycle, and immunofluorescence analyses should supplement the standard techniques in toxicology to unravel the biological effects of PA exposure in liver cells as the primary target during metabolization of PAs. Graphical abstract


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