Spindle Assembly Checkpoint Components Research Articles

Self-Assembly of the RZZ Complex into Filaments Drives Kinetochore Expansion in the Absence of Microtubule Attachment

SummaryThe kinetochore is a dynamic multi-protein assembly that forms on each sister chromatid and interacts with microtubules of the mitotic spindle to drive chromosome segregation. In animals, kinetochores without attached microtubules expand their outermost layer into crescent and ring shapes to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. Kinetochore expansion is an example of protein co-polymerization, but the mechanism is not understood. Here, we present evidence that kinetochore expansion is driven by oligomerization of the Rod-Zw10-Zwilch (RZZ) complex, an outer kinetochore component that recruits the motor dynein and the SAC proteins Mad1-Mad2. Depletion of ROD in human cells suppresses kinetochore expansion, as does depletion of Spindly, the adaptor that connects RZZ to dynein, although dynein itself is dispensable. Expansion is also suppressed by mutating ZWILCH residues implicated in Spindly binding. Conversely, supplying cells with excess ROD facilitates kinetochore expansion under otherwise prohibitive conditions. Using the C. elegans early embryo, we demonstrate that ROD-1 has a concentration-dependent propensity for oligomerizing into micrometer-scale filaments, and we identify the ROD-1 β-propeller as a key regulator of self-assembly. Finally, we show that a minimal ROD-1-Zw10 complex efficiently oligomerizes into filaments in vitro. Our results suggest that RZZ’s capacity for oligomerization is harnessed by kinetochores to assemble the expanded outermost domain, in which RZZ filaments serve as recruitment platforms for SAC components and microtubule-binding proteins. Thus, we propose that reversible RZZ self-assembly into filaments underlies the adaptive change in kinetochore size that contributes to chromosome segregation fidelity.

Abstract 1960: T790M-independent EGFR TKI resistance is associated with a broad multi-drug resistant phenotype but selective vulnerabilities to spindle assembly complex (SAC) and CDK inhibitors

Abstract While EGFR mutant NSCLC patients are initially responsive to EGFR targeted therapies, resistant disease inevitably emerges and in nearly half of resistance cases, tumors lack secondary EGFR mutations such as T790M and are refractory to 2nd and 3rd generation EGFR tyrosine kinase inhibitors (TKI). The identification of treatment regimens with efficacy against T790M-negative resistance remains a major clinical challenge. To address this unmet need, we derived a panel of NSCLC cell lines with acquired resistance to the EGFR TKI, erlotinib. A subset of EGFR TKI resistant variants were negative for secondary EGFR mutations, were resistant to 2nd and 3rd generation EGFR TKIs including osimertinb, afatinib, and dacomitinib, and had undergone epithelial to mesenchymal transition (EMT) as demonstrated by loss of E-cadherin, enhanced expression of N-cadherin and Axl and an increased invasive phenotype as determined by Boyden chamber assay. Proteomic profiling revealed that although EGFR TKI resistant cells displayed similar mesenchymal and invasive phenotypes, there was significant heterogeneity in protein expression and pathway activation among resistant variants derived from the same parental cell line. To identify therapeutic agents with activity against EMT-associated EGFR TKI resistance, we performed high-throughput drug screening to test the efficacy of 1,321 compounds. EMT-associated EGFR TKI resistance was accompanied by the acquisition of broad spectrum drug resistance. Compared to parental cells, mesenchymal EGFR TKI resistant cells were significantly more resistant to chemotherapeutic agents used to treat NSCLC including pemetrexed, irinotecan, vinblastine, and gemcitabine. EGFR TKI resistant cells displayed acquired resistance to 147 other tyrosine kinase and serine/threonine kinase inhibitors. In contrast, both parental cells and mesenchymal EGFR TKI resistant variants were highly sensitive to CDK inhibitors and agents targeting spindle assembly checkpoint (SAC) components including PLK1, Aurora, KSP, and survivin. These finding were validated by MTS and clonogenic assays. Treatment with SAC inhibitors induced the accumulation of cells in G2/M phase, enlarged nuclear size, and polyploidy. To clinically validate these findings, we established a cell line (MDA-011) from the pleural effusion of an EGFR mutant NSCLC patient with T790M-negative resistance to erlotinib. In vitro, MDA-011 cells were resistant to erlotinib and osimertinib. MDA-011 cells were highly sensitive to CDK inhibitors and SAC inhibitors as determined by MTS and clonogenic assays. These data indicate that EMT-associated resistance to EGFR TKIs is associated with broad spectrum drug resistance but vulnerabilities to CDK and SAC inhibition which can potentially be exploited to overcome resistant disease in NSCLC patients. Citation Format: Monique B. Nilsson, Jacqueline Robichaux, Huiying Sun, Pan Tong, Lixia Diao, Mia Hofstad, YouHong Fan, Jing Wang, John Heymach. T790M-independent EGFR TKI resistance is associated with a broad multi-drug resistant phenotype but selective vulnerabilities to spindle assembly complex (SAC) and CDK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1960.

PLK1 has tumor-suppressive potential in APC-truncated colon cancer cells

The spindle assembly checkpoint (SAC) acts as a molecular safeguard in ensuring faithful chromosome transmission during mitosis, which is regulated by a complex interplay between phosphatases and kinases including PLK1. Adenomatous polyposis coli (APC) germline mutations cause aneuploidy and are responsible for familial adenomatous polyposis (FAP). Here we study the role of PLK1 in colon cancer cells with chromosomal instability promoted by APC truncation (APC-ΔC). The expression of APC-ΔC in colon cells reduces the accumulation of mitotic cells upon PLK1 inhibition, accelerates mitotic exit and increases the survival of cells with enhanced chromosomal abnormalities. The inhibition of PLK1 in mitotic, APC-∆C-expressing cells reduces the kinetochore levels of Aurora B and hampers the recruitment of SAC component suggesting a compromised mitotic checkpoint. Furthermore, Plk1 inhibition (RNAi, pharmacological compounds) promotes the development of adenomatous polyps in two independent ApcMin/+ mouse models. High PLK1 expression increases the survival of colon cancer patients expressing a truncated APC significantly.

Perturbing mitosis for anti-cancer therapy: is cell death the only answer?

Interfering with mitosis for cancer treatment is an old concept that has proven highly successful in the clinics. Microtubule poisons are used to treat patients with different types of blood or solid cancer since more than 20 years, but how these drugs achieve clinical response is still unclear. Arresting cells in mitosis can promote their demise, at least in a petri dish. Yet, at the molecular level, this type of cell death is poorly defined and cancer cells often find ways to escape. The signaling pathways activated can lead to mitotic slippage, cell death, or senescence. Therefore, any attempt to unravel the mechanistic action of microtubule poisons will have to investigate aspects of cell cycle control, cell death initiation in mitosis and after slippage, at single‐cell resolution. Here, we discuss possible mechanisms and signaling pathways controlling cell death in mitosis or after escape from mitotic arrest, as well as secondary consequences of mitotic errors, particularly sterile inflammation, and finally address the question how clinical efficacy of anti‐mitotic drugs may come about and could be improved.

The genome of Schmidtea mediterranea and the evolution of core cellular mechanisms

The planarian Schmidtea mediterranea is an important model for stem cell research and regeneration, but adequate genome resources for this species have been lacking. Here we report a highly contiguous genome assembly of S. mediterranea, using long-read sequencing and a de novo assembler (MARVEL) enhanced for low-complexity reads. The S. mediterranea genome is highly polymorphic and repetitive, and harbours a novel class of giant retroelements. Furthermore, the genome assembly lacks a number of highly conserved genes, including critical components of the mitotic spindle assembly checkpoint, but planarians maintain checkpoint function. Our genome assembly provides a key model system resource that will be useful for studying regeneration and the evolutionary plasticity of core cell biological mechanisms.

Whole chromosome loss and associated breakage-fusion-bridge cycles transform mouse tetraploid cells.

Whole chromosome gains or losses (aneuploidy) are a hallmark of ~70% of human tumors. Modeling the consequences of aneuploidy has relied on perturbing spindle assembly checkpoint (SAC) components, but interpretations of these experiments are clouded by the multiple functions of these proteins. Here, we used a Cre recombinase-mediated chromosome loss strategy to individually delete mouse chromosomes 9, 10, 12, or 14 in tetraploid immortalized murine embryonic fibroblasts. This methodology also involves the generation of a dicentric chromosome intermediate, which subsequently undergoes a series of breakage-fusion-bridge (BFB) cycles. While the aneuploid cells generally display a growth disadvantage invitro, they grow significantly better in low adherence sphere-forming conditions and three of the four lines are transformed invivo, forming large and invasive tumors in immunocompromised mice. The aneuploid cells display increased chromosomal instability and DNA damage, a mutator phenotype associated with tumorigenesis invivo Thus, these studies demonstrate a causative role for whole chromosome loss and the associated BFB-mediated instability in tumorigenesis and may shed light on the early consequences of aneuploidy in mammalian cells.

Caenorhabditis elegans BUB-3 and SAN-1/MAD3 Spindle Assembly Checkpoint Components Are Required for Genome Stability in Response to Treatment with Ionizing Radiation.

Relatively little is known about the cross-talk between the spindle assembly checkpoint and the DNA damage response, especially in multicellular organisms. We performed a Caenorhabditis elegans forward genetic screen to uncover new genes involved in the repair of DNA damage induced by ionizing radiation. We isolated a mutation, gt2000, which confers hypersensitivity to ionizing radiation and showed that gt2000 introduces a premature stop in bub-3. BUB-3 is a key component of the spindle assembly checkpoint. We provide evidence that BUB-3 acts during development and in the germline; irradiated bub-3(gt2000) larvae are developmentally retarded and form abnormal vulvae. Moreover, bub-3(gt2000) embryos sired from irradiated worms show increased levels of lethality. Both bub-3 and san-1 (the C. elegans homolog of MAD3) deletion alleles confer hypersensitivity to ionizing radiation, consistent with the notion that the spindle assembly checkpoint pathway is required for the DNA damage response. bub-3(gt2000) is moderately sensitive to the cross-linking drug cisplatin but not to ultraviolet light or methyl methanesulfonate. This is consistent with a role in dealing with DNA double-strand breaks and not with base damage. Double mutant analysis revealed that bub-3 does not act within any of the three major pathways involved in the repair of double-strand breaks. Finally, the cdc-20 gain-of-function mutant cdc-20/fzy-1(av15), which is refractory to the cell cycle delay conferred by the spindle checkpoint, showed phenotypes similar to bub-3 and san-1 mutants. We speculate that BUB-3 is involved in the DNA damage response through regulation of cell cycle timing.

UBASH3B-mediated silencing of the mitotic checkpoint: Therapeutic perspectives in cancer

ABSTRACTDefects in mitosis can lead to aneuploidy, which is a common feature of human cancers. Spindle Assembly Checkpoint (SAC) controls fidelity of chromosome segregation in mitosis to prevent aneuploidy. The ubiquitin receptor protein Ubiquitin Associated and SH3 Domain Containing B (UBASH3B) was recently found to control SAC silencing and faithful chromosome segregation by relocalizing Aurora B kinase to the mitotic microtubules. Accordingly, loss and gain of function of UBASH3B have strong effects on mitotic progression. Downregulation of UBASH3B prevents SAC satisfaction leading to inhibition of chromosome segregation, mitotic arrest, and cell death. In contrast, increased cellular levels of UBASH3B trigger premature and uncontrolled chromosome segregation. Interestingly, elevated levels of UBASH3B were found in aggressive tumors. Therefore, we raised the question whether the oncogenic potential of UBASH3B is linked to its role in chromosome segregation. Here we show that in cancer cells expressing high levels of UBASH3B and SAC proteins, downregulation of UBASH3B, can further potentiate SAC response inducing mitotic arrest and cell death. Moreover, data mining approaches identified a correlation between mRNA levels of UBASH3B and SAC components in a set of primary patient tumors including kidney and liver carcinomas. Thus, inhibition of UBASH3B may offer an attractive therapeutic perspective for cancers.

Plk1 bound to Bub1 contributes to spindle assembly checkpoint activity during mitosis

For faithful chromosome segregation, the formation of stable kinetochore–microtubule attachment and its monitoring by the spindle assembly checkpoint (SAC) are coordinately regulated by mechanisms that are currently ill-defined. Here, we show that polo-like kinase 1 (Plk1), which is instrumental in forming stable kinetochore–microtubule attachments, is also involved in the maintenance of SAC activity by binding to Bub1, but not by binding to CLASP2 or CLIP-170. The effect of Plk1 on the SAC was found to be mediated through phosphorylation of Mps1, an essential kinase for the SAC, as well as through phosphorylation of the MELT repeats in Knl1. Bub1 acts as a platform for assembling other SAC components on the phosphorylated MELT repeats. We propose that Bub1-bound Plk1 is important for the maintenance of SAC activity by supporting Bub1 localization to kinetochores in prometaphase, a time when the kinetochore Mps1 level is reduced, until the formation of stable kinetochore-microtubule attachment is completed. Our study reveals an intricate mechanism for coordinating the formation of stable kinetochore–microtubule attachment and SAC activity.

Overexpression of the E2F target gene CENPI promotes chromosome instability and predicts poor prognosis in estrogen receptor-positive breast cancer.

During cell division, chromosome segregation is facilitated by the mitotic checkpoint, or spindle assembly checkpoint (SAC), which ensures correct kinetochore-microtubule attachments and prevents premature sister-chromatid separation. It is well established that misexpression of SAC components on the outer kinetochores promotes chromosome instability (CIN) and tumorigenesis. Here, we study the expression of CENP-I, a key component of the HIKM complex at the inner kinetochores, in breast cancer, including ductal, lobular, medullary and male breast carcinomas. CENPI mRNA and protein levels are significantly elevated in estrogen receptor-positive (ER+) but not in estrogen receptor-negative (ER-) breast carcinoma. Well-established prognostic tests indicate that CENPI overexpression constitutes a powerful independent marker for poor patient prognosis and survival in ER+ breast cancer. We further demonstrate that CENPI is an E2F target gene. Consistently, it is overexpressed in RB1-deficient breast cancers. However, CENP-I overexpression is not purely due to cell cycle-associated expression. In ER+ breast cancer cells, CENP-I overexpression promotes CIN, especially chromosome gains. In addition, in ER+ breast carcinomas the degree of CENPI overexpression is proportional to the level of aneuploidy and CENPI overexpression is one of the strongest markers for CIN identified to date. Our results indicate that overexpression of the inner kinetochore protein CENP-I promotes CIN and forecasts poor prognosis for ER+ breast cancer patients. These observations provide novel mechanistic insights and have important implications for breast cancer diagnostics and potentially therapeutic targeting.

Abstract 3460: TBK1 regulates mitotic progression by modulating spindle assembly checkpoint in cancer cells

Abstract TANK Binding Kinase 1 (TBK1) is a non-canonical IkB kinase that contributes to KRAS-driven lung cancer. It is activated by phosphorylation of Serine-172 by TLR and RIG1 signaling, and this circuit triggers phosphorylation of IRF3 and IRF7, activation of NFκB and the expression of proinflammatory genes and interferons. In addition to its role role in regulating innate immunity, TBK1 also promotes oncogenesis by phosphorylating Akt and enhancing cell survival and by promoting autophagy and mitophagy. TBK1 is also induced under hypoxic conditions and expressed at significant levels in many solid tumors. TBK1 also contributes to prostate cancer dormancy and drug resistance by inhibiting mTOR and to tamoxifen resistance of breast cancer cells by enhancing transcriptional activity of ERα. Recent studies from our lab revealed a novel role for TBK1 in regulating mitosis. It was found that levels of phospho-TBK1 increases and localizes to centrosomes and the mitotic spindles during mitosis. Depletion of TBK1 was shown to trigger defects in spindle apparatus and prevents mitotic progression (Pillai et al., Nature Communications, 2015). TBK1 physically interacts and phosphorylates centrosomal protein CEP170 and mitotic apparatus protein NuMA. At the same time, it is not clear whether TBK1 regulates mitosis my modulating the Spindle Assembly Checkpoint (SAC). Our results show that TBK1 colocalizes with Cdc20 on the centrosomes. Additionally, TBK1-inhibited cells showed an increase in the colocalization of BUBR1 and Cdc20, with enhanced recruitment of BUBR1 to kinetochores. To further study how TBK1 affects SAC, lung and breast cancer cells were depleted of TBK1 by CRISPR/Cas9 mediated genome editing. Cells depleted of TBK1 showed very few cells in the mitotic stage; those that entered mitosis had residual levels of TBK1 and showed multipolarity and unusually stable and bundled microtubules. TBK1 knockout cells not only showed aberrant mitotic structures and had elevated levels of SAC components including BUBR1 and Cdc20. Surprisingly, level of mitotic Cyclin B1 remained unchanged in spite of elevated levels of Cdc20 indicating a possible inactivation of Anaphase Promoting complex (APC/C). Also, percentage of Cyclin B1 positive cells was significantly high in mitotic cells enriched using double thymidine block in the presence of TBK1 inhibitor BX795 (R9+BX) and MRT67307 (R9+MRT) as compared to untreated mitotic cells (R9). Further, double thymidine blocked released cells displayed elevated levels of SAC components upon treatment with TBK1 inhibitors. All these findings suggest that TBK1 facilitates mitotic progression through satisfying SAC. Citation Format: Neha Jaiswal, Smitha Pillai, Namrata BoraSinghal, Srikumar Chellappan. TBK1 regulates mitotic progression by modulating spindle assembly checkpoint in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3460. doi:10.1158/1538-7445.AM2017-3460

Cell division cycle 20 overexpression predicts poor prognosis for patients with lung adenocarcinoma.

The cell division cycle 20, a key component of spindle assembly checkpoint, is an essential activator of the anaphase-promoting complex. Aberrant expression of cell division cycle 20 has been detected in various human cancers. However, its clinical significance has never been deeply investigated in non-small-cell lung cancer. By analyzing The Cancer Genome Atlas database and using some certain online databases, we validated overexpression of cell division cycle 20 in both messenger RNA and protein levels, explored its clinical significance, and evaluated the prognostic role of cell division cycle 20 in non-small-cell lung cancer. Cell division cycle 20 expression was significantly correlated with sex (p = 0.003), histological classification (p < 0.0001), and tumor size (p = 0.0116) in non-small-cell lung cancer patients. In lung adenocarcinoma patients, overexpression of cell division cycle 20 was significantly associated with bigger primary tumor size (p = 0.0023), higher MKI67 level (r = 0.7618, p < 0.0001), higher DNA ploidy level (p < 0.0001), and poor prognosis (hazard ratio = 2.39, confidence interval: 1.87-3.05, p < 0.0001). However, in lung squamous cell carcinoma patients, no significant association of cell division cycle 20 expression was observed with any clinical parameter or prognosis. Overexpression of cell division cycle 20 is associated with poor prognosis in lung adenocarcinoma patients, and its overexpression can also be used to identify high-risk groups. In conclusion, cell division cycle 20 might serve as a potential biomarker for lung adenocarcinoma patients.

Premature Silencing of the Spindle Assembly Checkpoint Is Prevented by the Bub1-H2A-Sgo1-PP2A Axis in Saccharomyces cerevisiae.

The spindle assembly checkpoint (SAC) monitors mistakes in kinetochore-microtubule interaction and its activation prevents anaphase entry. The SAC remains active until all chromosomes have achieved bipolar attachment which applies tension on kinetochores. Our previous data in budding yeast Saccharomyces cerevisiae show that Ipl1/Aurora B kinase and a centromere-associated protein, Sgo1, are required to prevent SAC silencing prior to tension generation, but we believe that this regulatory network is incomplete. Bub1 kinase is one of the SAC components, and Bub1-dependent H2A phosphorylation triggers centromere recruitment of Sgo1 by H2A in yeast and human cells. Although yeast cells lacking the kinase domain of Bub1 show competent SAC activation, we found that the mutant cells fail to maintain a prolonged checkpoint arrest in the presence of tensionless attachment. Mutation of the Bub1 phosphorylation site in H2A also results in premature SAC silencing in yeast cells. Previous data indicate that Sgo1 protein binds to PP2ARts1, and we found that rts1Δ mutants exhibited premature SAC silencing as well. We further revealed that sgo1 mutants with abolished binding to H2A or PP2ARts1 displayed premature SAC silencing. Together, our results suggest that, in budding yeast S. cerevisiae, the Bub1-H2A-Sgo1-PP2ARts1 axis prevents SAC silencing and helps prolonged checkpoint arrest prior to tension establishment at kinetochores.

Mitotic spindle disruption in human preimplantation embryos activates the spindle assembly checkpoint but not apoptosis until Day 5 of development

Is the spindle assembly checkpoint(SAC) active during human preimplantation development? Mitotic spindle disruption during mitosis activates the SAC from at least Day 3 of human preimplantation development, but this does not lead to apoptosis until Day 5. Human preimplantation embryos frequently acquire chromosomal abnormalities, but the mechanisms behind this are poorly understood. It has been speculated that a dysfunctional SAC could be responsible. Although research has shown that the SAC components are present during early human development, functional studies are lacking. In vitro study using human preimplantation embryos in a university research laboratory. We studied a total of 38 Day-3, 38 Day-4, 29 Day-5 and 21 Day-6 human preimplantation embryos, donated for research, during 16 h of incubation. We cultured human preimplantation embryos overnight in a time-lapse imaging system, in control or in a nocodazole-containing medium that prevents the formation of a proper mitotic spindle. The embryos were subsequently fixed and analysed by immunocytochemistry for tubulin or mitotic and apoptotic markers, or by FISH. All embryos showed an increase in M-phase cells from 4.1-8.8% to 21.4-53.5% when exposed to nocodazole (P < 0.05; two-way ANOVA for all groups except Day-4 embryos, P = 0.128) suggesting SAC functionality. Apoptosis, which was rarely detected between Day 3 and Day 6 in good-quality control embryos, increased from Day 5 onwards in nocodazole-treated embryos and became statistically different from Day 6 (P < 0.01; two-way ANOVA). The FISH data suggest that in compacted Day-4 embryos, approximately one in six cells started a polyploid new cell cycle rather than to go in apoptosis after the failure to maintain the SAC-mediated M-phase arrest. These results suggest that during early embryo development, blastomeres with unresolved chromosome misalignments during M-phase can escape SAC-mediated apoptosis, continue cell division which can then result in aneuploid daughter cells. Not applicable. This study used nocodazole to inhibit microtubule polymerization, a drug that is regularly used to induce metaphase arrest and SAC activation. Results should be extrapolated to naturally occurring chromosome misalignments with care. Our results provide functional data that can help explain the high aneuploidy rates seen in human cleavage-stage embryos and suggest that this is due to their unusual cell cycle control. This work was supported by the Fund for Scientific Research Flanders (Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen) and the Methusalem grant to Karen Sermon of the Research Council of the Vrije Universiteit Brussel. The authors declare no competing financial interests.

Dynamic location changes of Bub1-phosphorylated-H2AThr133 with CENH3 nucleosome in maize centromeric regions.

The genomic stability of all organisms requires precise cell division with proper chromosome orientation. The Bub1-H2Aph-Sgo1 pathway and spindle assembly checkpoint (SAC) components have been identified in yeast and mammals that are important for sister centromere orientation and chromosome segregation. However, their roles in plants are not clear. Maize meiotic mutants and minichromosomes were used to study the role of H2AThr133 phosphorylation and SAC components in sister centromere orientation and chromosome segregation. Unlike previously reported, SAC protein Bub1-Sgo1 recruitment was independent of Rec8 in maize and did not play a role in centromere protection in meiosis I. Chromatin immunoprecipitation sequencing analysis with immnolocalization results indicate most CENH3 nucleosomes contain phosphorylated H2AThr133 in centromeric regions. H2AThr133ph spreads to encompass centromeric regions including the inner centromeric and pericentromeric regions during (pro)metaphase. The presence and localization of SAC components and H2AThr133ph on maize lines containing sister chromatids separate precociously in anaphase I revealed no direct role of these proteins on centromere orientation in meiosis I . This work sheds light on the relationship between H2AThr133ph and CENH3 nucleosome in plants, and the phosphorylation with dynamic location changes in centomeric regions suggests temporal and spatial regulation roles for H2A phosphorylation in chromosome segregation.

Molecular Mechanisms of Spindle Assembly Checkpoint Activation and Silencing.

In eukaryotic cell division, the Spindle Assembly Checkpoint (SAC) plays a key regulatory role by monitoring the status of chromosome-microtubule attachments and allowing chromosome segregation only after all chromosomes are properly attached to spindle microtubules. While the identities of SAC components have been known, in some cases, for over two decades, the molecular mechanisms of the SAC have remained mostly mysterious until very recently. In the past few years, advances in biochemical reconstitution, structural biology, and bioinformatics have fueled an explosion in the molecular understanding of the SAC. This chapter seeks to synthesize these recent advances and place them in a biological context, in order to explain the mechanisms of SAC activation and silencing at a molecular level.

Whole Chromosome Instability induces senescence and promotes SASP.

Age-related accumulation of ploidy changes is associated with decreased expression of genes controlling chromosome segregation and cohesin functions. To determine the consequences of whole chromosome instability (W-CIN) we down-regulated the spindle assembly checkpoint component BUB1 and the mitotic cohesin SMC1A, and used four-color-interphase-FISH coupled with BrdU incorporation and analyses of senescence features to reveal the fate of W-CIN cells. We observed significant correlations between levels of not-diploid cells and senescence-associated features (SAFs). W-CIN induced DNA double strand breaks and elevated oxidative stress, but caused low apoptosis. SAFs of W-CIN cells were remarkably similar to those induced by replicative senescence but occurred in only 13 days versus 4 months. Cultures enriched with not-diploid cells acquired a senescence-associated secretory phenotype (SASP) characterized by IL1B, CXCL8, CCL2, TNF, CCL27 and other pro-inflammatory factors including a novel SASP component CLEC11A. These findings suggest that W-CIN triggers premature senescence, presumably to prevent the propagation of cells with an abnormal DNA content. Cells deviating from diploidy have the ability to communicate with their microenvironment by secretion of an array of signaling factors. Our results suggest that aneuploid cells that accumulate during aging in some mammalian tissues potentially contribute to age-related pathologies and inflammation through SASP secretion.

Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint.

The multi-functional molecular motor cytoplasmic dynein performs diverse essential roles during mitosis. The mechanistic importance of the dynein Light Intermediate Chain homologs, LIC1 and LIC2 is unappreciated, especially in the context of mitosis. LIC1 and LIC2 are believed to exist in distinct cytoplasmic dynein complexes as obligate subunits. LIC1 had earlier been reported to be required for metaphase to anaphase progression by inactivating the kinetochore-microtubule attachment-sensing arm of the spindle assembly checkpoint (SAC). However, the functional importance of LIC2 during mitosis remains elusive. Here we report prominent novel roles for the LIC2 subunit of cytoplasmic dynein in regulating the spindle assembly checkpoint. LIC2 depletion in mammalian cells led to prolonged metaphase arrest in the presence of an active SAC and also to stretched kinetochores, thus implicating it in SAC inactivation. Quantitative fluorescence microscopy of SAC components revealed accumulation of both attachment- and tension-sensing checkpoint proteins at metaphase kinetochores upon LIC2 depletion. These observations support a stronger and more diverse role in checkpoint inactivation for LIC2 in comparison to its close homolog LIC1. Our study uncovers a novel functional hierarchy during mitotic checkpoint inactivation between the closely related but homologous LIC subunits of cytoplasmic dynein. These subtle functional distinctions between dynein subpopulations could be exploited to study specific aspects of the spindle assembly checkpoint, which is a key mediator of fidelity in eukaryotic cell division.

Absence of a conventional spindle mitotic checkpoint in the binucleated single-celled parasite Giardia intestinalis

The spindle assembly checkpoint (SAC) joins the machinery of chromosome-to-spindle microtubule attachment with that of the cell cycle to prevent missegregation of chromosomes during mitosis. Although a functioning SAC has been verified in a limited number of organisms, it is regarded as an evolutionarily conserved safeguard mechanism. In this report, we focus on the existence of the SAC in a single-celled parasitic eukaryote, Giardia intestinalis. Giardia belongs to Excavata, a large and diverse supergroup of unicellular eukaryotes in which SAC control has been nearly unexplored. We show that Giardia cells with absent or defective mitotic spindles due to the inhibitory effects of microtubule poisons do not arrest in mitosis; instead, they divide without any delay, enter the subsequent cell cycle and even reduplicate DNA before dying. We identified a limited repertoire of kinetochore and SAC components in the Giardia genome, indicating that this parasite is ill equipped to halt mitosis before the onset of anaphase via SAC control of chromosome-spindle microtubule attachment. Finally, based on overexpression, we show that Giardia Mad2, a core SAC protein in other eukaryotes, localizes along intracytoplasmic portions of caudal flagellar axonemes, but never within nuclei, even in mitotic cells with blocked spindles, where the SAC should be active. These findings are consistent with the absence of a conventional SAC, known from yeast and metazoans, in the parasitic protist Giardia.

Abstract 2728: In silico identification of a MAD2-interacting motif in MAD2 spliced isoforms suggest a functional interaction with the spindle assemble checkpoint in cancer

Abstract Abnormal chromosome segregation plays a key role in cancer development. MAD2 is a component of the spindle assembly checkpoint (SAC), a cell cycle control mechanism that ensures an accurate segregation of chromosomes during mitosis. Changes in MAD2 expression have been associated with chemo-resistance both to spindle inhibitors and to DNA damaging agents. Also, a previous study has shown that the exogenous expression of MAD2β, a splicing variant of MAD2, was associated with resistance to Adriamycin and Vincristine in gastric cell lines. Additionally, we have previously identified that exogenous overexpression of MAD2γ upon paclitaxel-induced SAC activation in the colorectal cancer cell HCT116, reduces drug-induced mitotic arrest. These findings suggested a possible structural interaction of MAD2 isoforms with SAC components. To determine possible structural interactions between MAD2 isoforms and key SAC components (i.e. MAD1 and CDC20), we performed an in silico analysis of MAD2 isoforms, Interestingly, we found that alternative splicing of MAD2 generates a premature stop codon and a frameshift in exon 4 in MAD2γ and MAD2β. This change generates a new C-terminal region in MAD2γ and MAD2β isoforms that comprise 16 amino acids, which are not present in the major isoform (MAD2α). We aligned this region with the amino acid sequence of CDC20 from various species and identified a MAD2-interacting motif (MIM). This finding suggests that MAD2 isoforms may interact with the active conformation of MAD2 (C-MAD2). Since MAD2 isoforms and CDC20 may compete for the same region in MAD2, we propose a new model whereby MAD2 isoforms inhibits SAC by interfering with C-MAD2/CDC20 formation. This model helps to explain previous results where MAD2 isoforms over expression seem to have an opposite role in SAC signaling. Citation Format: Miguel Ramirez-Otero, Alejandro Lopez-Saavedra, Marco Andonegui, Jose Diaz-Chavez, Luis Alonso Herrera. In silico identification of a MAD2-interacting motif in MAD2 spliced isoforms suggest a functional interaction with the spindle assemble checkpoint in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2728.