The oncogene cyclin D1 promotes bipolar spindle integrity under compressive force.

The mitotic spindle is the bipolar, microtubule-based structure that segregates chromosomes at each cell division. Aberrant spindles are frequently observed in cancer cells, but how oncogenic transformation affects spindle mechanics and function, particularly in the mechanical context of solid tumors, remains poorly understood. Here, we constitutively overexpress the oncogene cyclin D1 in human MCF10A cells to probe its effects on spindle architecture and response to compressive force. We find that cyclin D1 overexpression increases the incidence of spindles with extra poles, centrioles, and chromosomes. However, it also protects spindle poles from fracturing under compressive force, a deleterious outcome linked to multipolar cell divisions. Our findings suggest that cyclin D1 overexpression may adapt cells to increased compressive stress, possibly contributing to its prevalence in cancers such as breast cancer by allowing continued proliferation in mechanically challenging environments.

Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling.

Accurate mitosis is coordinated by the spindle assembly checkpoint (SAC) through the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C). As an essential regulator, Cdc20 promotes mitotic exit through activating APC/C and monitors kinetochore-microtubule attachment through activating SAC. Cdc20 requires multiple interactions with APC/C and MCC subunits to elicit these functions. Functionally assessing these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNA interference (RNAi). Here we generated Cdc20 RNAi-sensitive cell lines which display a penetrant metaphase arrest by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron, was found critical for SAC by promoting MCC formation and its interaction with APC/C. These data reveal additional regulation within the SAC and establish a novel method to interrogate Cdc20.

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

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.

A missense SNP in the tumor suppressor SETD2 reduces H3K36me3 and mitotic spindle integrity in Drosophila.

Mutations in SETD2 are among the most prevalent drivers of renal cell carcinoma (RCC). We identified a novel single nucleotide polymorphism (SNP) in SETD2, E902Q, within a subset of RCC patients, which manifests as both an inherited or tumor-associated somatic mutation. To determine if the SNP is biologically functional, we used CRISPR-based genome editing to generate the orthologous mutation within the Drosophila melanogaster Set2 gene. In Drosophila, the homologous amino acid substitution, E741Q, reduces H3K36me3 levels comparable to Set2 knockdown, and this loss is rescued by reintroduction of a wild-type Set2 transgene. We similarly uncovered significant defects in spindle morphogenesis, consistent with the established role of SETD2 in methylating α-Tubulin during mitosis to regulate microtubule dynamics and maintain genome stability. These data indicate the Set2 E741Q SNP affects both histone methylation and spindle integrity. Moreover, this work further suggests the SETD2 E902Q SNP may hold clinical relevance.

LGN loss randomizes spindle orientation and accelerates tumorigenesis in PTEN-deficient epidermis.

Loss of cell polarity and disruption of tissue organization are key features of tumorigenesis that are intrinsically linked to spindle orientation. Epithelial tumors are often characterized by spindle orientation defects, but how these defects impact tumor formation driven by common oncogenic mutations is not fully understood. Here, we examine the role of spindle orientation in adult epidermis by deleting a key spindle regulator, LGN, in normal tissue and in a PTEN-deficient mouse model. We report that LGN deficiency in PTEN mutant epidermis leads to a threefold increase in the likelihood of developing tumors on the snout, and an over 10-fold increase in tumor burden. In this tissue, loss of LGN alone increases perpendicular and oblique divisions of epidermal basal cells, at the expense of a planar orientation of division. PTEN loss alone does not significantly affect spindle orientation in these cells, but the combined loss of PTEN and LGN fully randomizes basal spindle orientation. A subset of LGN- and PTEN-deficient animals have increased amounts of proliferative spinous cells, which may be associated with tumorigenesis. These results indicate that loss of LGN impacts spindle orientation and accelerates epidermal tumorigenesis in a PTEN-deficient mouse model.

Δ3-tubulin impairs mitotic spindle morphology and increases nuclear size in pancreatic cancer cells.

Cancer cell proliferation is affected by post-translational modifications of tubulin. Especially, overexpression or depletion of enzymes for modifications on the tubulin C-terminal region perturbs dynamic instability of the spindle body. Those modifications include processing of C-terminal amino acids of α-tubulin; detyrosination, and a removal of penultimate glutamic acid (Δ2). We previously found a further removal of the third last glutamic acid, which generates so-called Δ3-tubulin. The effects of Δ3-tubulin on spindle integrities and cell proliferation remain to be elucidated. In this study, we investigated the impacts of forced expression of Δ3-tubulin on the structure of spindle bodies and cell division in a pancreatic cancer cell line, PANC-1. Overexpression of HA-tagged Δ3-tubulin impaired the morphology and orientation of spindle bodies during cell division in PANC-1 cells. In particular, spindle bending was most significantly increased. Expression of EGFP-tagged Δ3-tubulin driven by the endogenous promoter of human TUBA1B also deformed and misoriented spindle bodies. Spindle bending and condensation defects were significantly observed by EGFP-Δ3-tubulin expression. Furthermore, EGFP-Δ3-tubulin expression increased the nuclear size in a dose-dependent manner of EGFP-Δ3-tubulin expression. The expression of EGFP-Δ3-tubulin tended to slow down cell proliferation. Taken together, our results demonstrate that Δ3-tubulin affects the spindle integrity and cell division.

NuSAP regulates microtubule flux and Kif2A localization to ensure accurate chromosome congression.

Precise chromosome congression and segregation requires the proper assembly of a steady-state metaphase spindle, which is dynamic and maintained by continuous microtubule flux. NuSAP is a microtubule-stabilizing and -bundling protein that promotes chromosome-dependent spindle assembly. However, its function in spindle dynamics remains unclear. Here, we demonstrate that NuSAP regulates the metaphase spindle length control. Mechanistically, NuSAP facilitates kinetochore capture and spindle assembly by promoting Eg5 binding to microtubules. It also prevents excessive microtubule depolymerization through interaction with Kif2A, which reduces Kif2A spindle-pole localization. NuSAP is phosphorylated by Aurora A at Ser-240 during mitosis, and this phosphorylation promotes its interaction with Kif2A on the spindle body and reduces its localization with the spindle poles, thus maintaining proper spindle microtubule flux. NuSAP knockout resulted in the formation of shorter spindles with faster microtubule flux and chromosome misalignment. Taken together, we uncover that NuSAP participates in spindle assembly, dynamics, and metaphase spindle length control through the regulation of microtubule flux and Kif2A localization.

Pericentric major satellite transcription is essential for meiotic chromosome stability and spindle pole organization.

In somatic cells, mitotic transcription of major satellite non-coding RNAs is tightly regulated and essential for heterochromatin formation and the maintenance of genome integrity. We recently demonstrated that major satellite transcripts are expressed, and chromatin-bound during mouse oocyte meiosis. Pericentric satellite RNAs are also expressed in human oocytes. However, the specific biological function(s) during oocyte meiosis remain to be established. Here, we use validated locked nucleic acid gapmers for major satellite RNA depletion followed by live cell imaging, and superresolution analysis to determine the role of pericentric non-coding RNAs during female meiosis. Depletion of satellite RNA induces mesoscale changes in pericentric heterochromatin structure leading to chromosome instability, kinetochore attachment errors and abnormal chromosome alignment. Chromosome misalignment is associated with spindle defects, microtubule instability and, unexpectedly, loss of acentriolar microtubule organizing centre (aMTOC) tethering to spindle poles. Pericentrin fragmentation and failure to assemble ring-like aMTOCs with loss of associated polo-like kinase 1 provide critical insight into the mechanisms leading to impaired spindle pole integrity. Inhibition of transcription or RNA splicing phenocopies the chromosome alignment errors and spindle defects, suggesting that pericentric transcription during oocyte meiosis is required to regulate heterochromatin structure, chromosome segregation and maintenance of spindle organization.

Microtubules oppose cortical actomyosin-driven membrane ingression during C. elegans meiosis I polar body extrusion.

During C. elegans oocyte meiosis I cytokinesis and polar body extrusion, cortical actomyosin is locally remodeled to assemble a contractile ring that forms within and remains part of a much larger and actively contractile cortical actomyosin network. This network both mediates contractile ring dynamics and generates shallow ingressions throughout the oocyte cortex during polar body extrusion. Based on our analysis of requirements for CLS-2, a member of the CLASP family of proteins that stabilize microtubules, we recently proposed that a balance of actomyosin-mediated tension and microtubule-mediated stiffness limits membrane ingression throughout the oocyte during meiosis I polar body extrusion. Here, using live cell imaging and fluorescent protein fusions, we show that CLS-2 is part of a group of kinetochore proteins, including the scaffold KNL-1 and the kinase BUB-1, that also co-localize during meiosis I to structures called linear elements, which are present within the assembling oocyte spindle and also are distributed throughout the oocyte in proximity to, but appearing to underlie, the actomyosin cortex. We further show that KNL-1 and BUB-1, like CLS-2, promote the proper organization of sub-cortical microtubules and also limit membrane ingression throughout the oocyte. Moreover, nocodazole or taxol treatment to destabilize or stabilize oocyte microtubules leads to, respectively, excess or decreased membrane ingression throughout the oocyte. Furthermore, taxol treatment, and genetic backgrounds that elevate the levels of cortically associated microtubules, both suppress excess membrane ingression in cls-2 mutant oocytes. We propose that linear elements influence the organization of sub-cortical microtubules to generate a stiffness that limits cortical actomyosin-driven membrane ingression throughout the oocyte during meiosis I polar body extrusion. We discuss the possibility that this regulation of sub-cortical microtubule dynamics facilitates actomyosin contractile ring dynamics during C. elegans oocyte meiosis I cell division.

Spindle assembly checkpoint insensitivity allows meiosis-II despite chromosomal defects in aged eggs.

Chromosome segregation errors in mammalian oocyte meiosis lead to developmentally compromised aneuploid embryos and become more common with advancing maternal age. Known contributors include age-related chromosome cohesion loss and spindle assembly checkpoint (SAC) fallibility in meiosis-I. But how effective the SAC is in meiosis-II and how this might contribute to age-related aneuploidy is unknown. Here, we developed genetic and pharmacological approaches to directly address the function of the SAC in meiosis-II. We show that the SAC is insensitive in meiosis-II oocytes and that as a result misaligned chromosomes are randomly segregated. Whilst SAC ineffectiveness in meiosis-II is not age-related, it becomes most prejudicial in oocytes from older females because chromosomes that prematurely separate by age-related cohesion loss become misaligned in meiosis-II. We show that in the absence of a robust SAC in meiosis-II these age-related misaligned chromatids are missegregated and lead to aneuploidy. Our data demonstrate that the SAC fails to prevent cell division in the presence of misaligned chromosomes in oocyte meiosis-II, which explains how age-related cohesion loss can give rise to aneuploid embryos.

Distinct regions of the kinesin-5 C-terminal tail are essential for mitotic spindle midzone localization and sliding force.

Kinesin-5 motor proteins play essential roles during mitosis in most organisms. Their tetrameric structure and plus-end-directed motility allow them to bind to and move along antiparallel microtubules, thereby pushing spindle poles apart to assemble a bipolar spindle. Recent work has shown that the C-terminal tail is particularly important to kinesin-5 function: The tail affects motor domain structure, ATP hydrolysis, motility, clustering, and sliding force measured for purified motors, as well as motility, clustering, and spindle assembly in cells. Because previous work has focused on presence or absence of the entire tail, the functionally important regions of the tail remain to be identified. We have therefore characterized a series of kinesin-5/Cut7 tail truncation alleles in fission yeast. Partial truncation causes mitotic defects and temperature-sensitive growth, while further truncation that removes the conserved BimC motif is lethal. We compared the sliding force generated by cut7 mutants using a kinesin-14 mutant background in which some microtubules detach from the spindle poles and are pushed into the nuclear envelope. These Cut7-driven protrusions decreased as more of the tail was truncated, and the most severe truncations produced no observable protrusions. Our observations suggest that the C-terminal tail of Cut7p contributes to both sliding force and midzone localization. In the context of sequential tail truncation, the BimC motif and adjacent C-terminal amino acids are particularly important for sliding force. In addition, moderate tail truncation increases midzone localization, but further truncation of residues N-terminal to the BimC motif decreases midzone localization.

Characterization of a novel interaction of the Nup159 nucleoporin with asymmetrically localized spindle pole body proteins and its link with autophagy.

Both the spindle microtubule-organizing centers and the nuclear pore complexes (NPCs) are convoluted structures where many signaling pathways converge to coordinate key events during cell division. Interestingly, despite their distinct molecular conformation and overall functions, these structures share common components and collaborate in the regulation of essential processes. We have established a new link between microtubule-organizing centers and nuclear pores in budding yeast by unveiling an interaction between the Bfa1/Bub2 complex, a mitotic exit inhibitor that localizes on the spindle pole bodies, and the Nup159 nucleoporin. Bfa1/Bub2 association with Nup159 is reduced in metaphase to not interfere with proper spindle positioning. However, their interaction is stimulated in anaphase and assists the Nup159-dependent autophagy pathway. The asymmetric localization of Bfa1/Bub2 during mitosis raises the possibility that its interaction with Nup159 could differentially promote Nup159-mediated autophagic processes, which might be relevant for the maintenance of the replicative lifespan.

YAP co-localizes with the mitotic spindle and midbody to safeguard mitotic division in lung-cancer cells.

YES-associated protein (YAP) is a part of the Hippo pathway, with pivotal roles in several developmental processes and dual functionality as both a tumor suppressor and an oncogene. In the present study, we identified YAP activity as a microtubular scaffold protein that maintains the stability of the mitotic spindle and midbody by physically interacting with α-tubulin during mitotic progression. The interaction of YAP and α-tubulin was evident in co-immunoprecipitation assays, as well as observing their co-localization in the microtubular structure of the mitotic spindle and midbody in immunostainings. With YAP depletion, levels of ECT2, MKLP-1, and Aurora B are reduced, which is consistent with YAP functioning in midbody formation during cytokinesis. The concomitant decrease in α-tubulin and increase in acetyl-α-tubulin during YAP depletion occurred at the post-transcriptional level. This suggests that YAP maintains the stability of the mitotic spindle and midbody, which ensures appropriate chromosome segregation during mitotic division. The increase in acetyl-α-tubulin during YAP depletion may provide a lesion-halting mechanism in maintaining the microtubule structure. The depletion of YAP also results in multinuclearity and aneuploidy, which supports its role in stabilizing the mitotic spindle and midbody.

Microtubule nucleation for spindle assembly: one molecule at a time.

The cell orchestrates the dance of chromosome segregation with remarkable speed and fidelity. The mitotic spindle is built from scratch after interphase through microtubule (MT) nucleation, which is dependent on the γ-tubulin ring complex (γ-TuRC), the universal MT template. Although several MT nucleation pathways build the spindle framework, the question of when and how γ-TuRC is targeted to these nucleation sites in the spindle and subsequently activated remains an active area of investigation. Recent advances facilitated the discovery of new MT nucleation effectors and their mechanisms of action. In this review, we illuminate each spindle assembly pathway and subsequently consider how the pathways are merged to build a spindle.

Cell fragmentation in mouse preimplantation embryos induced by ectopic activation of the polar body extrusion pathway.

Cell fragmentation is commonly observed in human preimplantation embryos and is associated with poor prognosis during assisted reproductive technology (ART) procedures. However, the mechanisms leading to cell fragmentation remain largely unknown. Here, light sheet microscopy imaging of mouse embryos reveals that inefficient chromosome separation due to spindle defects, caused by dysfunctional molecular motors Myo1c or dynein, leads to fragmentation during mitosis. Extended exposure of the cell cortex to chromosomes locally triggers actomyosin contractility and pinches off cell fragments. This process is reminiscent of meiosis, during which small GTPase-mediated signals from chromosomes coordinate polar body extrusion (PBE) by actomyosin contraction. By interfering with the signals driving PBE, we find that this meiotic signaling pathway remains active during cleavage stages and is both required and sufficient to trigger fragmentation. Together, we find that fragmentation happens in mitosis after ectopic activation of actomyosin contractility by signals emanating from DNA, similar to those observed during meiosis. Our study uncovers the mechanisms underlying fragmentation in preimplantation embryos and, more generally, offers insight into the regulation of mitosis during the maternal-zygotic transition.

Centrosome linker diversity and its function in centrosome clustering and mitotic spindle formation.

The centrosome linker joins the two interphase centrosomes of a cell into one microtubule organizing center. Despite increasing knowledge on linker components, linker diversity in different cell types and their role in cells with supernumerary centrosomes remained unexplored. Here, we identified Ninein as a C-Nap1-anchored centrosome linker component that provides linker function in RPE1 cells while in HCT116 and U2OS cells, Ninein and Rootletin link centrosomes together. In interphase, overamplified centrosomes use the linker for centrosome clustering, where Rootletin gains centrosome linker function in RPE1 cells. Surprisingly, in cells with centrosome overamplification, C-Nap1 loss prolongs metaphase through persistent activation of the spindle assembly checkpoint indicated by BUB1 and MAD1 accumulation at kinetochores. In cells lacking C-Nap1, the reduction of microtubule nucleation at centrosomes and the delay in nuclear envelop rupture in prophase probably cause mitotic defects like multipolar spindle formation and chromosome mis-segregation. These defects are enhanced when the kinesin HSET, which normally clusters multiple centrosomes in mitosis, is partially inhibited indicating a functional interplay between C-Nap1 and centrosome clustering in mitosis.

PARP12 regulates mouse oocyte meiotic maturation.

Poly(ADP-ribosyl)ation (PARylation) is an important post-translational modification of proteins that involves the transfer of ADP-ribose moieties, and plays important roles in many biological processes including DNA repair, gene expression, RNA processing, ribosome biogenesis, and protein translation. Though it is accepted that PARylation is crucial for oocyte maturation, little is known about how Mono(ADP-ribosyl)ation (MARylation) regulates this process. Here, we report that Parp12, a mon(ADP-ribosyl) transferase of poly(ADP-ribosyl) polymerase (PARP) family, was highly expressed at all stages of oocytes during meiotic maturation. At germinal vesicle (GV) stage, PARP12 was mainly distributed in cytoplasm. Interestingly, PARP12 formed granular aggregation near to spindle poles during metaphase I (MI) and metaphase II (MII). PARP12 depletion results in abnormal spindle organization and chromosome misalignment in mouse oocytes. Chromosome aneuploidy frequency in PARP12 knockdown oocytes was significantly increased. Importantly, PARP12 knockdown triggers activation of spindle assembly checkpoint as shown by active BUBR1 in PARP12-KD MI oocytes. Besides, F actin was significantly attenuated in PARP12-KD MI oocytes which may affect the asymmetric division process. Transcriptomic analysis demonstrated that PARP12 depletion disrupts transcriptome homeostasis. Collectively, our results showed that the maternally expressed mono(ADPribosyl) transferases PARP12 was essential for oocyte meiotic maturation in mouse.

Caspase cleaves Drosophila BubR1 to modulate spindle assembly checkpoint function and lifespan of the organism.

Caspases cleave over 1500 substrates in the human proteome in both lethal and non-lethal scenarios. However, reports of the physiological consequences of substrate cleavage are limited. Additionally, the manner in which caspase cleaves only a subset of substrates in the non-lethal scenario remains to be elucidated. BubR1, a spindle assembly checkpoint component, is a caspase substrate in humans, the physiological function of which remains unclear. Here, we found that caspases, especially Drice, cleave Drosophila BubR1 between the N-terminal KEN box motif and C-terminal kinase domain. By using proximity labelling, we found that Drice, but not Dcp-1, is in proximity to BubR1, suggesting that protein proximity facilitates substrate preference. The cleaved fragments displayed altered subcellular localization and protein-protein interactions. Flies that harboured cleavage-resistant BubR1 showed longer duration of BubR1 localization to the kinetochore upon colchicine treatment. Furthermore, these flies showed extended lifespan. Thus, we propose that the caspase-mediated cleavage of BubR1 limits spindle assembly checkpoint and organismal lifespan. Our results highlight the importance of the individual analysis of substrates in vivo to determine the biological significance of caspase-dependent non-lethal cellular processes.

[Progress in the Study of Spindle Assembly Checkpoint in Lung Cancer].

Spindle assembly checkpoint (SAC) is a protective mechanism for cells to undergo accurate mitosis. SAC prevented chromosome segregation when kinetochores were not, or incorrectly attached to microtubules in the anaphase of mitosis, thus avoiding aneuploid chromosomes in daughter cells. Aneuploidy and altered expression of SAC component proteins are common in different cancers, including lung cancer. Therefore, SAC is a potential new target for lung cancer therapy. Five small molecule inhibitors of monopolar spindle 1 (MPS1), an upstream component protein of SAC, have entered clinical trials. This article introduces the biological functions of SAC, summarizes the abnormal expression of SAC component proteins in various cancers and the research progress of MPS1 inhibitors, and expects to provide a reference for the future development of lung cancer therapeutic strategies targeting SAC components.
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A kinesin-based approach for inducing chromosome-specific mis-segregation in human cells.

Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve.