Metaphase-anaphase Transition Research Articles

MEIKIN expression and its C-terminal phosphorylation by PLK1 is closely related the metaphase-anaphase transition by affecting cyclinB1 and Securin stabilization in meiotic oocyte.

Oocyte meiotic maturation failure and chromosome abnormality is one of the main causes of infertility, abortion, and diseases. The mono-orientation of sister chromatids during the first meiosis is important for ensuring accurate chromosome segregation in oocytes. MEIKIN is a germ cell-specific protein that can regulate the mono-orientation of sister chromatids and the protection of the centromeric cohesin complex during meiosisI. Here we found that MEIKIN is a maternal protein that was highly expressed in mouse oocytes before the metaphaseI (MI) stage, but became degraded by the MII stage and dramatically reduced after fertilization. Strikingly, MEIKIN underwent phosphorylation modification after germinal vesicle breakdown (GVBD), indicating its possible function in subsequent cellular event regulation. We further showed that MEIKIN phosphorylation was mediated by PLK1 at its carboxyl terminal region and its C-terminus was its key functional domain. To clarify the biological significance of meikin degradation during later stages of oocyte maturation, exogenous expression of MEIKIN was employed, which showed that suppression of MEIKIN degradation resulted in chromosome misalignment, cyclinB1 and Securin degradation failure, and MI arrest through a spindle assembly checkpoint (SAC)-independent mechanism. Exogenous expression of MEIKIN also inhibited metaphaseII (MII) exit and early embryo development. These results indicate that proper MEIKIN expression level and its C-terminal phosphorylation by PLK1 are critical for regulating the metaphase-anaphase transition in meiotic oocyte. The findings of this study are important for understanding the regulation of chromosome segregation and the prevention meiotic abnormality.

Propylparaben exposure impairs G2/M and metaphase-anaphase transition during mouse oocyte maturation

Propylparaben (PrPB) is a known endocrine disrupting chemicals that is widely applied as preservative in pharmaceuticals, food and cosmetics. PrPB has been detected in human urine samples and human serum and has been proven to cause functional decline in reproduction. However, the direct effects of PrPB on mammalian oocyte are still unknown. Here, we demonstrationed that exposure to PrPB disturbed mouse oocyte maturation in vitro, causing meiotic resumption arrest and first polar body extrusion failure. Our results indicated that 600 μM PrPB reduced the rate of oocyte germinal vesicle breakdown (GVBD). Further research revealed that PrPB caused mitochondrial dysfunction and oxidative stress, which led to oocyte DNA damage. This damage further disturbed the activity of the maturation promoting factor (MPF) complex Cyclin B1/ Cyclin-dependent kinase 1 (CDK1) and induced G2/M arrest. Subsequent experiments revealed that PrPB exposure can lead to spindle morphology disorder and chromosome misalignment due to unstable microtubules. In addition, PrPB adversely affected the attachment between microtubules and kinetochore, resulting in persistent activation of BUB3 amd BubR1, which are two spindle-assembly checkpoint (SAC) protein. Taken together, our studies indicated that PrPB damaged mouse oocyte maturation via disrupting MPF related G2/M transition and SAC depended metaphase-anaphase transition.

A force-sensitive mutation reveals a non-canonical role for dynein in anaphase progression.

The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties.

Live Cell Monitoring of Separase Activity, a Key Enzymatic Reaction for Chromosome Segregation, with Chimeric FRET-Based Molecular Sensor upon Cell Cycle Progression.

Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of the metaphase-anaphase transition in eukaryotes. Its activity is highly regulated by binding with securin and cyclinB-CDK1 complex. These bindings prevent the proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties in biochemical examinations due to the instability of separase in vitro and the fact that few spatiotemporal resolution approaches exist for monitoring live separase activity throughout mitotic processes. Here, we have developed FRET-based molecular sensors, including GFP variants, with separase-cleavable sequences as donors and covalently attached fluorescent dyes as acceptor molecules. These are applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated the performance of equivalent molecular sensors, either localized or not localized inside the nucleus under cell cycle control, using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems that can be used specifically or statistically, which could lead to the elucidation of separase properties in detail.

F-actin coordinates spindle morphology and function in Drosophila meiosis.

Meiosis is a highly conserved feature of sexual reproduction that ensures germ cells have the correct number of chromosomes prior to fertilization. A subset of microtubules, known as the spindle, are essential for accurate chromosome segregation during meiosis. Building evidence in mammalian systems has recently highlighted the unexpected requirement of the actin cytoskeleton in chromosome segregation; a network of spindle actin filaments appear to regulate many aspects of this process. Here we show that Drosophila oocytes also have a spindle population of actin that appears to regulate the formation of the microtubule spindle and chromosomal movements throughout meiosis. We demonstrate that genetic and pharmacological disruption of the actin cytoskeleton has a significant impact on spindle morphology, dynamics, and chromosome alignment and segregation during maturation and the metaphase-anaphase transition. We further reveal a role for calcium in maintaining the microtubule spindle and spindle actin. Together, our data highlights potential conservation of morphology and mechanism of the spindle actin during meiosis.

The Contribution of Oxidative Stress to NF1-Altered Tumors.

The neurofibromatosis-1 gene (NF1) was initially characterized because its germline mutation is responsible for an inherited syndromic disease predisposing tumor development, in particular neurofibromas but also various malignancies. Recently, large-scale tumor sequencing efforts have demonstrated NF1 as one of the most frequently mutated genes in human cancer, being mutated in approximately 5-10% of all tumors, especially in malignant peripheral nerve sheath tumors and different skin tumors. NF1 acts as a tumor suppressor gene that encodes neurofibromin, a large protein that controls neoplastic transformation through several molecular mechanisms. On the other hand, neurofibromin loss due to NF1 biallelic inactivation induces tumorigenic hyperactivation of Ras and mTOR signaling pathways. Moreover, neurofibromin controls actin cytoskeleton structure and the metaphase-anaphase transition. Consequently, neurofibromin deficiency favors cell mobility and proliferation as well as chromosomal instability and aneuploidy, respectively. Growing evidence supports the role of oxidative stress in NF1-related tumorigenesis. Neurofibromin loss induces oxidative stress both directly and through Ras and mTOR signaling activation. Notably, innovative therapeutic approaches explore drug combinations that further increase reactive oxygen species to boost the oxidative unbalance of NF1-altered cancer cells. In our paper, we review NF1-related tumors and their pathogenesis, highlighting the twofold contribution of oxidative stress, both tumorigenic and therapeutic.

MTA1 localizes to the mitotic spindle apparatus and interacts with TPR in spindle assembly checkpoint regulation

We previously identified a cell cycle-dependent periodic subcellular distribution of cancer metastasis-associated antigen 1 (MTA1) and unraveled a novel role of MTA1 in inhibiting spindle damage-induced spindle assembly checkpoint (SAC) activation in cancer cells. However, the more detailed subcellular localization of MTA1 in mitotic cells and its copartner in SAC regulation in cancer cells are still poorly understood. Here, through immunofluorescent colocalization analysis of MTA1 and alpha-tubulin in mitotic cancer cells, we reveal that MTA1 is dynamically localized to the spindle apparatus throughout the entire mitotic process. We also demonstrated a reversible upregulation of MTA1 expression upon spindle damage-induced SAC activation, and time-lapse imaging assays indicated that MTA1 silencing delayed the mitotic metaphase-anaphase transition in cancer cells. Further investigation revealed that MTA1 interacts and colocalizes with Translocated Promoter Region (TPR) on spindle microtubules in mitotic cells, and this interaction is attenuated on SAC activation. TPR is well-implicated in SAC regulation via binding the MAD1-MAD2 complex, however, no interactions between MTA1 and MAD1 or MAD2 were detected in our coimmunoprecipitation (co-IP) assays, suggesting that the MTA1-TPR may represent a distinct SAC-associated complex separate from the previously reported TPR-MAD1/MAD2 complex. Our data provide new insights into the subcellular localization and molecular function of MTA1 in SAC regulation in cancer, and indicate that intervention of the MTA1-TPR interaction may be effective to modulate SAC and hence chromosomal instability (CIN) in tumorigenesis.

A bioinformatics analysis of the clinicopathological and prognostic significance of FAM64A mRNA expression in gynecological cancers

FAM64A is a mitotic regulator which promotes cell metaphase-anaphase transition and is highly expressed in a cell-cycle-dependent manner. In this study, we examined the clinicopathological and prognostic significance of FAM64A mRNA expression in gynecological cancers. We conducted a bioinformatics analysis of FAM64A mRNA expression using Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), xiantao, The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN), and Kaplan-Meier (KM) plotter databases. FAM64A expression was elevated in breast, cervical, endometrial, and ovarian cancers when compared with normal tissue. Expression was positively correlated with white race, low T stages, infiltrating ductal carcinoma, or favourable PAM50 classification in breast cancer patients, and with clinical stage, histological grade and TP53 mutation, and endometrial cancer serous subtype. FAM64A expression was negatively associated with overall and/or recurrence-free survival rates in breast and endometrial cancer patients, while the opposite was observed in cervical and ovarian cancer patients. FAM64A functioned as an independent predictor of overall and disease-specific survival in breast cancer patients. FAM64A-correlated genes were involved in ligand-receptor interactions, and chromosomal, cell cycle, and DNA replication processes in breast, cervical, endometrial and ovarian cancers. Top hub genes primarily included cell cycle-related proteins in breast cancer, mucins and acetylgalactosaminyl transferases in cervical cancer, kinesin family members in endometrial cancer, and synovial sarcoma X and the cancer/testis antigen in ovarian cancer. FAM64A mRNA expression was positively related to Th2 cell infiltration, but negatively associated with neutrophil and Th17 cell infiltration in breast, cervical, endometrial, and ovarian cancers. FAM64A expression may be considered a potential biomarker reflecting carcinogenesis, histogenesis, aggressive behaviour, and prognosis in gynecological cancers. Impact statement What is already known on this subject? FAM64A is located in cell nucleolar and nucleoplasmic regions, and during mitosis it putatively controls metaphase-to-anaphase transition. FAM64A appears to regulate different physiological processes, including apoptosis, tumorigenesis, neural differentiation, stress responses, and the cell cycle. What the results of this study add? FAM64A expression was up-regulated in breast, cervical, endometrial, and ovarian cancers, and positively correlated with white race, low T stages, infiltrating ductal carcinoma, or favourable PAM50 classification in breast cancer patients, and with clinical stage, histological grade, and TP53 mutation, and a serous subtype in endometrial cancer. FAM64A expression was negatively associated with overall and/or recurrence-free survival rates in breast and endometrial cancer patients, while the opposite was observed in cervical and ovarian cancer patients. FAM64A functioned as an independent predictor of overall and disease-specific survival in breast cancer. FAM64A-correlated genes were involved in ligand-receptor interactions, chromosomal, cell cycle, and DNA replication processes, while FAM64A mRNA expression was positively related to Th2 cell infiltration but negatively correlated with neutrophil and Th17 cell infiltration in four gynecological cancers. What the implications of these findings for clinical practice and/or further research? In the future, abnormal FAM64A mRNA expression may serve as a biomarker of carcinogenesis, histogenesis, aggressiveness, and prognosis in gynecological malignancies.

Abstract 6231: Activation of GCN2 by HC-7366 results in significant antitumor efficacy as monotherapy and in combination with multiple standard of care agents in various solid cancer models

Abstract The integrated stress response (ISR) is an adaptive signaling pathway that cells utilize to respond to a wide range of extrinsic and intrinsic stresses, which are important for tumorigenesis. Activation of ISR is suggested to play a dual role in cell fate decisions. While the ISR promotes survival, prolonged activation of ISR induces apoptosis. We are developing HC-7366, a first-in-class, first-in-human GCN2 activator, and are currently evaluating it in a phase 1 clinical trial in solid tumors (NCT05121948). In this study, we present the characterization of the antitumor effects of HC-7366 in solid tumors. In vivo efficacy studies using HC-7366 montherapy showed significant tumor growth inhibition (TGI%) in preclinical cancer models of colorectal (78-95%), head and neck (33% regression), sarcoma (80%) and prostate (65%). HC-7366 activated the ISR in tumors from treated mice as evidenced by induction of the ATF4 target genes ASNS and PSAT1. Additionally, HC-7366 induced the proapoptotic protein PUMA and reduced HIF1⍺ and HIF2⍺ levels. Furthermore, HC-7366 showed significant benefit in colorectal models when combined with DC101 (anti-VEGFR2 antibody), 5-fluorouracil (chemotherapy), alpelisib (PI3Kα inhibitor), or trametinib (MEK1/2 inhibitor). Using GCN2 CRISPR-knockout cells, we confirmed that the HC-7366 mediated reduction of cell growth and induction of ISR markers was dependent on GCN2. We performed multi-omics analyses to further understand the mechanism of action. Metabolomics analysis of tumors treated with HC-7366 revealed that HC-7366 altered several metabolites involved in amino acid metabolism, oxidative stress, the urea cycle, and pyrimidine biosynthesis. Additionally, proteomics analysis showed that HC-7366 significantly reduced proteins involved in oxidative phosphorylation. Analysis of the transcriptome in tumors from treated mice demonstrated that HC-7366 reduced the activity of HIF and E2F1-driven transcription, including expression of metaphase-anaphase transition genes, consistent with decreased Ki67 staining in tumors. ATF4 and JUN transcriptional activity was enhanced with HC-7366 treatment consistent with activation of ISR. Collectively, our in vitro and in vivo results demonstrate that HC-7366 is a potent GCN2 activator with strong antitumor activity across multiple solid tumor models as a monotherapy or in combination with standard of care agents. Citation Format: Feven Tameire, Paulina Wojnarowicz, Crissy Dudgeon, Sho Fujisawa, Sharon Huang, Owen B. Reilly, Nicholas Collette, Jeremy Drees, Kathryn Bieging-Rolett, Takashi O. Kangas, Weiyu Zhang, Maria Fumagalli, Iman Dewji, Yunfang Li, Anissa SH Chan, Xiaohong Qiu, Ben Harrison, Ashley LaCayo, Ricardo A. Cordova, Kirk A. Staschke, Alan C. Rigby, Savithri Ramurthy, Eric S. Lightcap, David Surguladze, Nandita Bose. Activation of GCN2 by HC-7366 results in significant antitumor efficacy as monotherapy and in combination with multiple standard of care agents in various solid cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6231.

Introduction of a Geminin mScarlet Reporter into H2B-mTurq2 hiPSCs for Live-cell Imaging of Proliferation and Cell Cycling

We previously generated a doxycycline-inducible H2B-mTurq2 reporter in hiPSCs to track cells and study cell division and apoptosis. To improve visualization of cycling cells, we introduced a ubiquitously transcribed mScarletI-Geminin (GMMN) (1–110) into the previously untargeted second AAVS1 allele. Fusion to the N-terminal part of GMNN provided tightly controlled mScarletI expression during the cell cycle. mScarletI fluorescence increased gradually from the S-phase through the M-phase of the cell cycle and was lost at the metaphase-anaphase transition. The resulting hiPSC reporter line generated, which we named ProLiving, is a valuable tool to study cell division and cell cycle characteristics in living hiPSC-derived cells.

Space microgravity improves proliferation of human iPSC-derived cardiomyocytes.

SummaryIn microgravity, cells undergo profound changes in their properties. However, how human cardiac progenitors respond to space microgravity is unknown. In this study, we evaluated the effect of space microgravity on differentiation of human induced pluripotent stem cell (hiPSC)-derived cardiac progenitors compared with 1G cultures on the International Space Station (ISS). Cryopreserved 3D cardiac progenitors were cultured for 3 weeks on the ISS. Compared with 1G cultures, the microgravity cultures had 3-fold larger sphere sizes, 20-fold higher counts of nuclei, and increased expression of proliferation markers. Highly enriched cardiomyocytes generated in space microgravity showed improved Ca2+ handling and increased expression of contraction-associated genes. Short-term exposure (3 days) of cardiac progenitors to space microgravity upregulated genes involved in cell proliferation, survival, cardiac differentiation, and contraction, consistent with improved microgravity cultures at the late stage. These results indicate that space microgravity increased proliferation of hiPSC-cardiomyocytes, which had appropriate structure and function.

Role of phosphorylation of Cdc20 in the regulation of the action of APC/C in mitosis

The ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) is essential for the control of mitosis, and its activity is subject to tight regulation. In early mitosis, APC/C is inhibited by the mitotic checkpoint system, but subsequently it regains activity and promotes metaphase-anaphase transition by targeting cyclin B and securin for degradation. The phosphorylation of APC/C by the mitotic protein kinase Cdk1-cyclin B facilitates its interaction with its coactivator Cdc20, while the phosphorylation of Cdc20 inhibits its binding to APC/C. This raises the question of how Cdc20 binds to APC/C under conditions of high Cdk1 activity. It seemed possible that the opposing action of protein phosphatases produces a fraction of unphosphorylated Cdc20 that binds to APC/C. We found, however, that while inhibitors of protein phosphatases PP2A and PP1 increased the overall phosphorylation of Cdc20 in anaphase extracts from Xenopus eggs, they did not decrease the levels of Cdc20 bound to APC/C. Searching for alternative mechanisms, we found that following the binding of Cdc20 to APC/C, it became significantly protected against phosphorylation by Cdk1. Protection was mainly at threonine sites at the N-terminal region of Cdc20, known to affect its interaction with APC/C. A model is proposed according to which a pool of unphosphorylated Cdc20, originating from initial stages of mitosis or from phosphatase action, combines with phosphorylated APC/C and thus becomes stabilized against further phosphorylation, possibly by steric hindrance of Cdk1 action. This pool of APCCdc20 appears to be required for the regulation of APC/C activity at different stages of mitosis.

The Role of the APC/C and Its Coactivators Cdh1 and Cdc20 in Cancer Development and Therapy.

To sustain genomic stability by correct DNA replication and mitosis, cell cycle progression is tightly controlled by the cyclic activity of cyclin-dependent kinases, their binding to cyclins in the respective phase and the regulation of cyclin levels by ubiquitin-dependent proteolysis. The spindle assembly checkpoint plays an important role at the metaphase-anaphase transition to ensure a correct separation of sister chromatids before cytokinesis and to initiate mitotic exit, as an incorrect chromosome distribution may lead to genetically unstable cells and tumorigenesis. The ubiquitin ligase anaphase-promoting complex or cyclosome (APC/C) is essential for these processes by mediating the proteasomal destruction of cyclins and other important cell cycle regulators. To this end, it interacts with the two regulatory subunits Cdh1 and Cdc20. Both play a role in tumorigenesis with Cdh1 being a tumor suppressor and Cdc20 an oncogene. In this review, we summarize the current knowledge about the APC/C-regulators Cdh1 and Cdc20 in tumorigenesis and potential targeted therapeutic approaches.

Single-cell monitoring of dry mass and dry mass density reveals exocytosis of cellular dry contents in mitosis.

Cell mass and composition change with cell cycle progression. Our previous work characterized buoyant mass dynamics in mitosis (Miettinen et al., 2019), but how dry mass and cell composition change in mitosis has remained unclear. To better understand mitotic cell growth and compositional changes, we develop a single-cell approach for monitoring dry mass and the density of that dry mass every ~75 s with 1.3% and 0.3% measurement precision, respectively. We find that suspension grown mammalian cells lose dry mass and increase dry mass density following mitotic entry. These changes display large, non-genetic cell-to-cell variability, and the changes are reversed at metaphase-anaphase transition, after which dry mass continues accumulating. The change in dry mass density causes buoyant and dry mass to differ specifically in early mitosis, thus reconciling existing literature on mitotic cell growth. Mechanistically, cells in early mitosis increase lysosomal exocytosis, and inhibition of lysosomal exocytosis decreases the dry mass loss and dry mass density increase in mitosis. Overall, our work provides a new approach for monitoring single-cell dry mass and dry mass density, and reveals that mitosis is coupled to extensive exocytosis-mediated secretion of cellular contents.

PLEKHA5 Regulates Mitotic Progression by Promoting APC/C Localization to Microtubules

Progression through major cell cycle checkpoints involves spatiotemporal coordination of cytoskeletal dynamics with changes in protein activities, chromosomal dynamics, and signaling events. A major role of the ubiquitin–proteasome system is to orchestrate cell cycle progression through temporally controlled protein degradation programs. The anaphase‐promoting complex/cyclosome (APC/C) is the primary E3 ligase controlling all aspects of mitosis, including G2/M transition, early mitosis, the metaphase–anaphase transition, and mitotic exit, via polyubiquitination of important mitotic effector proteins. The temporal control of APC/C‐mediated ubiquitination in these processes is well established, including identities of ubiquitination substrates and accessory factors that modulate E3 ligase activity. Much less is known, however, about the spatial organization of APC/C function, including its potential interplay with the microtubule cytoskeleton, which serves as a signaling platform and whose dynamic behavior is central to advancement through mitosis. Here, we investigate and describe pleckstrin homology domain‐containing family A, member 5 (PLEKHA5) as a new regulator of APC/C function and the ubiquitination pathway in mitosis. We found that PLEKHA5 localized to the microtubule network and interacted with APC/C. PLEKHA5 knockdown antagonized mitotic entry and progression, causing a buildup of APC/C substrates implicated in both the G2/M and metaphase–anaphase transitions, in a manner dependent upon the PLEKHA5 interaction with APC/C. In vitro ubiquitination assays showed that APC/C isolated from mitotic cells lacking PLEKHA5 had lower catalytic activity and a decreased association with its key mitotic co‐activator CDC20. We investigated the dynamic localizations of the APC/C and the potential role of PLEKHA5 in its regulation by developing a TurboID proximity biotinylation tool to assess microtubule localization of endogenous proteins. Microtubule‐targeted TurboIDs revealed that PLEKHA5, APC/C subunits, and the co‐activator CDC20 all localized to microtubules both in interphase and in M phase. Importantly, upon PLEKHA5 knockdown, a pool of APC/C, but not CDC20, lost its microtubule localization, which could explain the decreased APC/C‐CDC20 association. We propose that PLEKHA5 functions as a novel APC/C adaptor to promote its subcellular localization to microtubules and enable APC/C to search for its co‐activator CDC20 more efficiently in one dimension along the microtubule network. In this model PLEKHA5 acts to coordinate the spatial regulation of APC/CCDC20 and facilitate its efficient polyubiquitination of key mitotic effector proteins to ensure proper progression of mitosis.

The Cell Biology of Heterochromatin.

A conserved feature of virtually all higher eukaryotes is that the centromeres are embedded in heterochromatin. Here we provide evidence that this tight association between pericentric heterochromatin and the centromere is essential for proper metaphase exit and progression into telophase. Analysis of chromosome rearrangements that separate pericentric heterochromatin and centromeres indicates that they must remain associated in order to balance Cohesin/DNA catenation-based binding forces and centromere-based pulling forces during the metaphase–anaphase transition. In addition, a centromere embedded in heterochromatin facilitates nuclear envelope assembly around the entire complement of segregating chromosomes. Because the nuclear envelope initially forms on pericentric heterochromatin, nuclear envelope formation proceeds from the pole, thus providing time for incorporation of lagging and trailing chromosome arms into the newly formed nucleus. Additional analysis of noncanonical mitoses provides further insights into the functional significance of the tight association between heterochromatin and centromeres.

Kinetochore scaffold 1 regulates SAC function during mouse oocyte meiotic maturation.

Precise regulation of chromosome separation through spindle assembly checkpoint (SAC) during oocyte meiosis is critical for mammalian reproduction. The kinetochore plays an important role in the regulation of SAC through sensing microtubule tension imbalance or missing microtubule connections. Here, we report that kinetochore scaffold 1 (KNL1, also known as CASC5), an outer kinetochore protein, plays a critical role in the SAC function of mouse oocytes. KNL1localized at kinetochores from GVBD to the MII stage, and microinjection of KNL1-siRNA caused accelerated metaphase-anaphase transition and premature first meiosis completion, producing aneuploid eggs. The SAC was prematurely silenced in the presence of unstable kinetochore-microtubule attachments and misaligned chromosomes in KNL1-depleted oocytes. Additionally, KNL1 and MPS1had a synergistic effect on the activation and maintenance of SAC. Taken together, our results suggest that KNL1, as a kinetochore platform protein, stabilizes SAC to ensure timely anaphase entry and accurate chromosome segregation during oocyte meiotic maturation.

Polarity protein Par3 sensitizes breast cancer to paclitaxel by promoting cell cycle arrest.

Paclitaxel, belongs to tubulin-binding agents (TBAs), shows a great efficacy against breast cancer via stabilizing microtubules. Drug resistance limits its clinical application. Here we aimed to explore a role of Polarity protein Par3 in improving paclitaxel effectiveness. Breast cancer specimens from 45 patients were collected to study the relationship between Par3 expression and paclitaxel efficacy. The Kaplan-Meier method was used for survival analysis. Cell viability was measured in breast cancer cells (SK-BR-3 and T-47D) with Par3 over-expression or knockdown. The flow cytometry assays were performed to measure cell apoptosis and cell cycle. BrdU incorporation assay and Hoechst 33,258 staining were performed to measure cell proliferation and cell apoptosis, respectively. Immunofluorescence was used to detect microtubule structures. Par3 expression was associated with good response of paclitaxel in breast cancer patients. Consistently, Par3 over-expression significantly sensitized breast cancer cells to paclitaxel by promoting cell apoptosis and reducing cell proliferation. In Par3 overexpressing cells upon paclitaxel treatment, we observed intensified cell cycle arrests at metaphase. Further exploration showed that Par3 over-expression stabilized microtubules of breast cancer cells in response to paclitaxel and resists to microtubules instability induced by nocodazole, a microtubule-depolymerizing agent. Par3 facilitates polymeric forms of tubulin and stabilizes microtubule structure, which aggravates paclitaxel-induced delay at the metaphase-anaphase transition, leading to proliferation inhibition and apoptosis of breast cancer cells. Par3 has a potential role in sensitizing breast cancer cells to paclitaxel, which may provide a more precise assessment of individual treatment and novel therapeutic targets.

TORC1 inactivation promotes APC/C-dependent mitotic slippage in yeast and human cells

SummaryUnsatisfied kinetochore-microtubule attachment activates the spindle assembly checkpoint to inhibit the metaphase-anaphase transition. However, some cells eventually override mitotic arrest by mitotic slippage. Here, we show that inactivation of TORC1 kinase elicits mitotic slippage in budding yeast and human cells. Yeast mitotic slippage was accompanied with aberrant aspects, such as degradation of the nucleolar protein Net1, release of phosphatase Cdc14, and anaphase-promoting complex/cyclosome (APC/C)-Cdh1-dependent degradation of securin and cyclin B in metaphase. This mitotic slippage caused chromosome instability. In human cells, mammalian TORC1 (mTORC1) inactivation also invoked mitotic slippage, indicating that TORC1 inactivation-induced mitotic slippage is conserved from yeast to mammalian cells. However, the invoked mitotic slippage in human cells was not dependent on APC/C-Cdh1. This study revealed an unexpected involvement of TORC1 in mitosis and provides information on undesirable side effects of the use of TORC1 inhibitors as immunosuppressants and anti-tumor drugs.

Dynamic crotonylation of EB1 by TIP60 ensures accurate spindle positioning in mitosis.

Spindle position control is essential for cell fate determination and organogenesis. Early studies indicate the essential role of the evolutionarily conserved Gαi/LGN/NuMA network in spindle positioning. However, the regulatory mechanisms that couple astral microtubules dynamics to the spindle orientation remain elusive. Here we delineated a new mitosis-specific crotonylation-regulated astral microtubule-EB1-NuMA interaction in mitosis. EB1 is a substrate of TIP60, and TIP60-dependent crotonylation of EB1 tunes accurate spindle positioning in mitosis. Mechanistically, TIP60 crotonylation of EB1 at Lys66 forms a dynamic link between accurate attachment of astral microtubules to the lateral cell cortex defined by NuMA-LGN and fine tune of spindle positioning. Real-time imaging of chromosome movements in HeLa cells expressing genetically encoded crotonylated EB1 revealed the importance of crotonylation dynamics for accurate control of spindle orientation during metaphase-anaphase transition. These findings delineate a general signaling cascade that integrates protein crotonylation with accurate spindle positioning for chromosome stability in mitosis.