Abstract Kinetochores (KTs) are large protein structures assembled upon centromeres during mitosis that bind to microtubules (MTs) of the mitotic spindle to orchestrate and power chromosome movements. We have recently discovered that a high proportion of human glioblastoma (GBM) isolates suffer from a lethal form of KT stress, which is triggered by oncogenic mitogen-activated protein kinase signaling [Mapk stressed KTs (MaSKs)]. MaSKs arise when the Ras-Raf-MEK-ERK cascade is inappropriately active in mitosis, resulting in hyperstimulation of a network of KT kinases that, in turn, hyperphosphorylate KTs, decreasing their MT binding capacity and causing excessive KT-MT turnover. MaSKs are highly relevant to cancer research. Our MaSK model provides a direct mechanism for chromosome instability induced by oncogenic Ras pathway signaling, which is a key knowledge gap. Moreover, we have only found MaSKs in cancer or transformed cells, where they trigger novel genetic and molecular dependencies not observed in normal cells. MaSK-containing cells differentially rely on two non-essential domains of the mitotic protein BubR1/BUB1B to facilitate recruitment of PP2A phosphatase to KTs to counteract MaSK-induced KT-MT instability. MaSKs themselves can serve as biomarkers for tumors and can be leveraged to discover new MaSK-targeted therapies and to define a patient responder class. At this meeting, we will provide an overview of the MaSK model, the data and associated assays leading to their discovery, and implications for novel therapeutic development.

The kinetochore, a megadalton structure composed of centromeric (CEN) DNA and protein complexes, is required for faithful chromosome segregation in eukaryotes. The evolutionarily conserved Dam1/DASH complex (Ska1 in metazoans) is one of the essential protein sub-complexes of the budding yeast kinetochore. Previous studies showed that methylation of lysine residue 233 in Dam1 by Set1 is important for haploid growth as mutation of lysine 233 to alanine results in lethality. In this study, we report that Set1-mediated cell cycle dependent Dam1 lysine methylation contributes to kinetochore assembly and chromosomal stability. Our results show that Dam1 methylation is cell cycle regulated with the highest levels of methylation in metaphase. Consistent with these results, co-immunoprecipitation experiments revealed an interaction between Dam1 with Set1 in metaphase cells. Set1 has been shown to colocalize with Jhd2, a histone lysine demethylase which demethylates Set1-methylated histones. Affinity purification-based mass spectroscopy of Jhd2 associated proteins identified seven of the ten subunits of the Dam1 complex; an association of Jhd2 with non-histone proteins, such as Dam1 has not been previously reported. We confirmed the interaction of Jhd2 with Dam1 and showed that cells overexpressing JHD2 exhibit reduced levels of methylated lysine in Dam1 in wild type and UBP8 deletion strains, growth defects in kinetochore mutants, reduced levels of kinetochore proteins at CEN chromatin, defects in kinetochore biorientation and chromosome missegregation. In summary, we have shown that cell cycle dependent methylation of Dam1 plays a crucial role in the maintenance of kinetochore assembly for faithful chromosome segregation.

BackgroundEsophageal carcinoma (ESCA) ranks among the most prevalent malignant tumors globally. Despite significant advancements in treatment options and improved patient outcomes, the 5-year survival rate remains unsatisfactory. The spindle and kinetochore associated complex subunit 1/2/3 (SKA1/2/3) attached to the kinetochore (KT) in the metaphase of mitosis are implicated in the occurrence and development of various tumors. However, the expression patterns, diagnostic significance and prognostic implications of SKA1/2/3 in ESCA have not been comprehensively determined.MethodsTCGA, UALCAN, Kaplan-Meier Plotter, and TIMER databases were leveraged to dissect the expression patterns, prognostic implications and diagnostic value of SKA1/2/3 in ESCA patients, as well as to investigate the potential regulatory mechanism of SKA1/2/3 in the onset and progression of ESCA.ResultsIn ESCA, SKA1/2/3 exhibited substantial expression, with higher levels relating significantly with clinicopathological features and patient prognosis. Enrichment analysis of genes co-expressed with SKA1/2/3 highlighted their involvement in the cell cycle, DNA replication and p53 signaling pathway. Protein-protein interaction (PPI) analysis identified ten hub genes that were not only markedly upregulated but also portended a poor prognosis in ESCA. Additionally, immune infiltration assays uncovered a significant link between SKA1/2/3 expression and the immune cell infiltration within ESCA. Silencing of SKA1/2/3 significantly suppresses cell proliferation and migration, while concurrently promoting apoptosis in ESCA cells.ConclusionsSKA1/2/3 may serve as promising biomarkers for the prognosis and diagnosis of ESCA, which holds promise as a novel therapeutic target for the disease.

Proper chromosome segregation is required to ensure chromosomal stability. The centromere (CEN) is a unique chromatin domain defined by CENP-A and is responsible for recruiting the kinetochore (KT) during mitosis, ultimately regulating microtubule spindle attachment and mitotic checkpoint function. Upregulation of many CEN/KT genes is commonly observed in cancer. Here, we show that although FOXM1 occupies promoters of many CEN/KT genes with MYBL2, FOXM1 overexpression alone is insufficient to drive the FOXM1-correlated transcriptional program. CENP-F is canonically an outer kinetochore component; however, it functions with FOXM1 to coregulate G2/M transcription and proper chromosome segregation. Loss of CENP-F results in altered chromatin accessibility at G2/M genes and reduced FOXM1-MBB complex formation. We show that coordinated CENP-FFOXM1 transcriptional regulation is a cancer-specific function. We observe a small subset of CEN/KT genes including CENP-C, that are not regulated by FOXM1. Upregulation of CENP-C in the context of CENP-A overexpression leads to increased chromosome missegregation and cell death suggesting that escape of CENP-C from FOXM1 regulation is a cancer survival mechanism. Together, we show that FOXM1 and CENP-F coordinately regulate G2/M genes, and this coordination is specific to a subset of genes to allow for maintenance of chromosome instability levels and subsequent cell survival.

Proper chromosome segregation is required to ensure genomic and chromosomal stability. The centromere is a unique chromatin domain present throughout the cell cycle on each chromosome defined by the CENP-A nucleosome. Centromeres (CEN) are responsible for recruiting the kinetochore (KT) during mitosis, ultimately regulating spindle attachment and mitotic checkpoint function. Upregulation of many genes that encode the CEN/KT proteins is commonly observed in cancer. Here, we show although that FOXM1 occupies the promoters of many CEN/KT genes with MYBL2, occupancy is insufficient alone to drive the FOXM1 correlated transcriptional program. We show that CENP-F, a component of the outer kinetochore, functions with FOXM1 to coregulate G2/M transcription and proper chromosome segregation. Loss of CENP-F results in alteration of chromatin accessibility at G2/M genes, including CENP-A, and leads to reduced FOXM1-MBB complex formation. The FOXM1-CENP-F transcriptional coordination is a cancer-specific function. We observed that a few CEN/KT genes escape FOXM1 regulation such as CENP-C which when upregulated with CENP-A, leads to increased chromosome misegregation and cell death. Together, we show that the FOXM1 and CENP-F coordinately regulate G2/M gene expression, and this coordination is specific to a subset of genes to allow for proliferation and maintenance of chromosome stability for cancer cell survival.

Centromere (CEN) identity is specified epigenetically by specialized nucleosomes containing evolutionarily conserved CEN-specific histone H3 variant CENP-A (Cse4 in Saccharomyces cerevisiae, CENP-A in humans), which is essential for faithful chromosome segregation. However, the epigenetic mechanisms that regulate Cse4 function have not been fully defined. In this study, we show that cell cycle-dependent methylation of Cse4-R37 regulates kinetochore function and high-fidelity chromosome segregation. We generated a custom antibody that specifically recognizes methylated Cse4-R37 and showed that methylation of Cse4 is cell cycle regulated with maximum levels of methylated Cse4-R37 and its enrichment at the CEN chromatin occur in the mitotic cells. Methyl-mimic cse4-R37F mutant exhibits synthetic lethality with kinetochore mutants, reduced levels of CEN-associated kinetochore proteins and chromosome instability (CIN), suggesting that mimicking the methylation of Cse4-R37 throughout the cell cycle is detrimental to faithful chromosome segregation. Our results showed that SPOUT methyltransferase Upa1 contributes to methylation of Cse4-R37 and overexpression of UPA1 leads to CIN phenotype. In summary, our studies have defined a role for cell cycle-regulated methylation of Cse4 in high-fidelity chromosome segregation and highlight an important role of epigenetic modifications such as methylation of kinetochore proteins in preventing CIN, an important hallmark of human cancers.

The nucleolus is the most prominent membraneless compartment within the nucleus-dedicated to the metabolism of ribosomal RNA. Nucleoli are composed of hundreds of ribosomal DNA (rDNA) repeated genes that form large chromosomal clusters, whose high recombination rates can cause nucleolar dysfunction and promote genome instability. Intriguingly, the evolving architecture of eukaryotic genomes appears to have favored two strategic rDNA locations-where a single locus per chromosome is situated either near the centromere (CEN) or the telomere. Here, we deployed an innovative genome engineering approach to cut and paste to an ectopic chromosomal location-the ~1.5 mega-base rDNA locus in a single step using CRISPR technology. This "megablock" rDNA engineering was performed in a fused-karyotype strain of Saccharomyces cerevisiae. The strategic repositioning of this locus within the megachromosome allowed experimentally mimicking and monitoring the outcome of an rDNA migratory event, in which twin rDNA loci coexist on the same chromosomal arm. We showed that the twin-rDNA yeast readily adapts, exhibiting wild-type growth and maintaining rRNA homeostasis, and that the twin loci form a single nucleolus throughout the cell cycle. Unexpectedly, the size of each rDNA array appears to depend on its position relative to the CEN, in that the locus that is CEN-distal undergoes size reduction at a higher frequency compared to the CEN-proximal counterpart. Finally, we provided molecular evidence supporting a mechanism called paralogous cis-rDNA interference, which potentially explains why placing two identical repeated arrays on the same chromosome may negatively affect their function and structural stability.

The key to ensuring proper chromosome segregation during mitosis is the kinetochore (KT), a tightly regulated multiprotein complex that links the centromeric chromatin to the spindle microtubules and as such leads the segregation process. Understanding its architecture, function, and regulation is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in structural detail so far. In this study, we construct a nanometer-precise in situ map of the human-like regional KT of Schizosaccharomyces pombe using multi-color single-molecule localization microscopy. We measure each protein of interest (POI) in conjunction with two references, cnp1CENP-A at the centromere and sad1 at the spindle pole. This allows us to determine cell cycle and mitotic plane, and to visualize individual centromere regions separately. We determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI and, consequently, build an in situ KT model with unprecedented precision, providing new insights into the architecture.

Eukaryotic genomes are replicated in spatiotemporal patterns that are stereotypical for individual genomes and developmental profiles. In the model system Saccharomyces cerevisiae, two primary mechanisms determine the preferential activation of replication origins during early S phase, thereby largely defining the consequent replication profiles of these cells. Both mechanisms are thought to act through specific recruitment of a rate-limiting initiation factor, Dbf4-dependent kinase (DDK), to a subset of licensed replication origins. Fkh1/2 is responsible for stimulation of most early-firing origins, except for centromere (CEN)-proximal origins that recruit DDK via the kinetochore protein Ctf19, which is required for their early firing. The C-terminus of Dbf4 has been implicated in its recruitment to origins via both the Fkh1/2 and Ctf19 mechanisms. Here, we show that the Zn-finger motif within the C-terminus is specifically required for Dbf4 recruitment to CENs to stimulate CEN-proximal/Ctf19-dependent origins, whereas stimulation of origins via the Fkh1/2 pathway remains largely intact. These findings re-open the question of exactly how Fkh1/2 and DDK act together to stimulate replication origin initiation.

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Spindle and Kinetochore-Associated Complex Is Associated With Poor Prognosis in Adrenocortical Carcinoma.

Abstract Background: BAL0891 is a dual inhibitor of threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1). These kinases collaborate in activating the mitotic spindle assembly checkpoint (SAC) at the kinetochore (KT) to regulate chromosome alignment and segregation prior to mitotic exit. In this work, kinase inhibition by BAL0891 was linked to effects on SAC integrity and aberrant mitotic progression in tumor cells. Comparison with a TTK-specific inhibitor (CFI-402257, CFI) allowed further evaluation of the contribution of dual TTK/PLK1 inhibition to anti-cancer activity, associated with a promising anti-proliferative profile across diverse tumor cell lines. Methods: Kinase assays used a radiometric assay. Target residency was measured using surface plasmon resonance with recombinant kinase. Anti-proliferative activity was assessed with crystal violet or YO-PRO assay (5 days incubation), in-cell target inhibition by immunoblotting for phospho-TTK following drug wash-out. Effects on SAC integrity were followed by immunoprecipitation (IP) mitotic progression by flow cytometry/mitotic marker expression. Cells were blocked in mitosis using the microtubule-targeting agent nocodazole or the PLK1 inhibitor onvansertib. SAC KT accumulation was evaluated by immunofluorescence (IF) for co-localization of BubR1 with CENPC. Comparative studies with CFI used anti-proliferative IC50 concentrations. Results: In vitro kinase profiling showed that BAL0891 has low nM IC50s against TTK and PLK1, with prolonged TTK (>12 h) and transient PLK1 (4 min) target residency. Prolonged TTK inhibition (≥38 h) was also observed in HT29 tumor cells. Consistent with a dominant TTK-targeting activity, BAL0891 treatment of HT29 cells blocked in mitosis with nocodazole or the PLK1 inhibitor onvansertib led to aberrant mitotic release and accumulation of polyploid cells. This was preceded by SAC disruption as visualized by IP assays. Effects on the SAC and mitotic exit were evaluated in comparative studies with CFI; BAL0891 exhibited faster kinetics for both parameters suggesting a contribution of PLK1 inhibition. This was confirmed by directly evaluating acute effects on SAC integrity at the KT by IF. Specifically, 1 h BAL0891 treatment of mitotic HT29 cells resulted in a highly reproducible and significant reduction in KT-associated SAC (p<0.0001) which was not observed with CFI in the same conditions. An extensive in vitro BAL0891 anti-proliferative screen indicated a broad anti-cancer potential, with low nM GI50s observed for most tumor lines and minimal activity on non-immortalized cells (GI50s >5 uM). Conclusion: BAL0891 is a novel dual TTK/PLK1 mitotic checkpoint inhibitor. In tumor cells, prolonged effects on TTK and transient effects on PLK1 contribute to rapid SAC disruption and aberrant mitotic exit. This is associated with potent anti-proliferative activity in diverse tumor lines. Citation Format: Elisa Zanini, Nicole Forster-Gross, Felix Bachmann, Nicole Willemsen-Seegers, Jos de Man, Guido J. Zaman, Rogier C. Buijsman, Anna Groner, Mila Roceri, Karin Burger, Paul McSheehy, Laurenz Kellenberger, Heidi A. Lane. BAL0891: A novel, small molecule, dual TTK/PLK1 mitotic checkpoint inhibitor (MCI) that drives aberrant tumor cell division [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5646.

Abstract Background: BAL0891 is a dual inhibitor of threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1). These kinases collaborate in activating the mitotic spindle assembly checkpoint (SAC) at the kinetochore (KT) to regulate chromosome alignment and segregation prior to mitotic exit. In vitro, BAL0891 has a combined prolonged effect on TTK and a transient effect on PLK1, leading to rapid disruption of the SAC that potentiates aberrant mitotic progression of tumor cells. In this work, efficacy of BAL0891 was investigated in mouse models of human triple negative breast cancer (TNBC) including evaluation of dose-dependency, drug exposure, target occupancy and a screen of activity across a panel of PDX models. Methods: The MDA-MB-231 cell line was grown sc in nude mice and treated with BAL0891, administered IV weekly (QW) or twice-weekly (2QW). Thirteen sc TNBC PDX models were screened for BAL0891 response using 2QW administration. Efficacy was quantified as deltaT/C (treated/control tumors). Plasma and tumor were analyzed for drug levels or TTK target occupancy by LC-MS/MS. The latter used a biotinylated TTK-specific probe and streptavidin-mediated isolation of unoccupied TTK, trypsin digestion and quantification of TTK-representative peptides. Results: BAL0891 efficacy was tested in the TNBC xenograft model MDA-MB-231 with QW or 2QW IV dosing schedules. All treatments were well tolerated, with no drug-related animal deaths. With MTD dosing, tumor regressions were observed, while different MTD fractions for both QW and 2QW schedules showed dose-dependent anti-tumor activity. The weekly MTD group was followed for an additional 20 days after treatment cessation on day 100. Strikingly, 3 of 8 tumors continued to shrink resulting in 2 (25%) pathologically confirmed cures. Consistent with the potent efficacy of intermittent MTD dosing, and prolonged tumor drug exposure, tumor TTK was fully drug-occupied for ≥ 6 days after the last administration; target occupancy was also dose-and drug exposure-dependent. To further evaluate BAL0891 anti-cancer activity in TNBC, a screen in 13 TNBC PDX models was conducted. Seven models exhibited deltaT/C < 50%, with regressions observed in 3. Of these, 2 models showed persistent regressions ≥ 70% vs. baseline. Interestingly, evaluation of TTK target occupancy in selected models showed high target occupancy independent of tumor response, indicating target dependency rather than drug availability is important for anti-cancer activity. Conclusion: BAL0891 is a novel dual TTK/PLK1 mitotic checkpoint inhibitor with potent anti-cancer activity in TNBC models. Intermittent IV administration is well tolerated and associated with prolonged tumor drug exposure, prolonged TTK inhibition and notable anti-tumor efficacy. These data support further investigation of BAL0891 for the treatment of cancer patients (incl. TNBC). Citation Format: Heidi A. Lane, Felix Bachmann, Elisa Zanini, Paul McSheehy, Karine Litherland, Nicole Forster-Gross, Luc Bury, Diep Vu-Pham, Jos de Man, Wilhelmina E. van Riel, Guido JR Zaman, Rogier C. Buijsman, Laurenz Kellenberger. BAL0891: A novel dual TTK/PLK1 mitotic checkpoint inhibitor (MCI) that drives aberrant tumor cell division resulting in potent anti-cancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5645.

42P BAL0891: A novel, small molecule, dual TTK/PLK1 mitotic checkpoint inhibitor (MCI) with potent single agent activity

During mitosis, the interaction of kinetochores (KTs) with microtubules (MTs) drives chromosome congression to the spindle equator and supports the segregation of sister chromatids. Faithful genome partition critically relies on the ability of chromosomes to establish and maintain proper amphitelic end-on attachments, a configuration in which sister KTs are connected to robust MT fibers emanating from opposite spindle poles. Because the capture of spindle MTs by KTs is error prone, cells use mechanisms that sense and correct inaccurate KT-MT interactions before committing to segregate sister chromatids in anaphase. If left unresolved, these errors can result in the unequal distribution of chromosomes and lead to aneuploidy, a hallmark of cancer. In this review, we provide an overview of the molecular strategies that monitor the formation and fine-tuning of KT-MT attachments. We describe the complex network of proteins that operates at the KT-MT interface and discuss how AURORA B and PLK1 coordinate several concurrent events so that the stability of KT-MT attachments is precisely modulated throughout mitotic progression. We also outline updated knowledge on how the RZZ complex is regulated to ensure the formation of end-on attachments and the fidelity of mitosis.

The spindle and kinetochore-associated complex is composed of three members: SKA1, SKA2, and SKA3. It is necessary for stabilizing spindle microtubules attaching to kinetochore (KT) in the middle stage of mitosis. The SKA complex is associated with poor prognosis in several human cancers. However, the role of SKA complex in rare malignant diseases, such as gliomas, has not been fully investigated. We investigated several databases, including Oncomine, UALCAN, and cBioPortal to explore the expression profile and prognostic significance of SKA complex in patients with gliomas. Gene ontology and Kyoto Encyclopedia of Genes and Genome pathways were used to analyze the potential enriched pathways. The genes co-expressed with SKA complex were identified and used for developing a protein-protein interaction (PPI) network using the STRING database. We found a significant overexpression of the mRNA levels of SKA1, SKA2, and SKA3 in patients with glioma patients. Higher expression of SKA1 and SKA3, but not SKA2, was significantly correlated with shorter overall survival of patients with glioma. In glioma, SKA complex was found to be involved in nuclear division, chromosome segregation, and DNA replication. The results of PPI network identified 10 hub genes (CCNB2, UBE2C, BUB1B, TPX2, CCNA2, CCNB1, MELK, TOP2A, PBK, and KIF11), all of which were overexpressed and negatively associated with prognosis of patients with glioma. In conclusion, our study sheds new insights into the biological role and prognostic significance of SKA complex in glioma.

Kinetochore (KTs) are macromolecular protein assemblies that attach sister chromatids to spindle microtubules (MTs) and mediate accurate chromosome segregation during mitosis. The outer KT consists of the KMN network, a protein super-complex comprising Knl1 (yeast Spc105), Mis12 (yeast Mtw1), and Ndc80 (yeast Ndc80), which harbours sites for MT binding. Within the KMN network, Spc105 acts as an interaction hub of components involved in spindle assembly checkpoint (SAC) signalling. It is known that Spc105 forms a complex with KT component Kre28. However, where Kre28 physically localizes in the budding yeast KT is not clear. The exact function of Kre28 at the KT is also unknown. Here, we investigate how Spc105 and Kre28 interact and how they are organized within bioriented yeast KTs using genetics and cell biological experiments. Our microscopy data show that Spc105 and Kre28 localize at the KT with a 1 : 1 stoichiometry. We also show that the Kre28-Spc105 interaction is important for Spc105 protein turn-over and essential for their mutual recruitment at the KTs. We created several truncation mutants of kre28 that affect Spc105 loading at the KTs. When over-expressed, these mutants sustain the cell viability, but SAC signalling and KT biorientation are impaired. Therefore, we conclude that Kre28 contributes to chromosome biorientation and high-fidelity segregation at least indirectly by regulating Spc105 localization at the KTs.

SummaryProtection of peri-centromeric (periCEN) REC8 cohesin from Separase and sister kinetochore (KT) attachment to microtubules emanating from the same spindle pole (co-orientation) ensures that sister chromatids remain associated after meiosis I. Both features are lost during meiosis II, resulting in sister chromatid disjunction and the production of haploid gametes. By transferring spindle-chromosome complexes (SCCs) between meiosis I and II in mouse oocytes, we discovered that both sister KT co-orientation and periCEN cohesin protection depend on the SCC, and not the cytoplasm. Moreover, the catalytic activity of Separase at meiosis I is necessary not only for converting KTs from a co- to a bi-oriented state but also for deprotection of periCEN cohesion, and cleavage of REC8 may be the key event. Crucially, selective cleavage of REC8 in the vicinity of KTs is sufficient to destroy co-orientation in univalent chromosomes, albeit not in bivalents where resolution of chiasmata may also be required

KNL1 is a large intrinsically disordered kinetochore (KT) protein that recruits spindle assembly checkpoint (SAC) components to mediate SAC signaling. The N-terminal region (NTR) of KNL1 possesses two activities that have been implicated in SAC silencing: microtubule (MT) binding and protein phosphatase 1 (PP1) recruitment. The NTR of Drosophila melanogaster KNL1 (Spc105) has never been shown to bind MTs or to recruit PP1. Furthermore, the phosphoregulatory mechanisms known to control SAC protein binding to KNL1 orthologues is absent in D. melanogaster. Here, these apparent discrepancies are resolved using in vitro and cell-based assays. A phosphoregulatory circuit that utilizes Aurora B kinase promotes SAC protein binding to the central disordered region of Spc105 while the NTR binds directly to MTs in vitro and recruits PP1-87B to KTs in vivo. Live-cell assays employing an optogenetic oligomerization tag and deletion/chimera mutants are used to define the interplay of MT and PP1 binding by Spc105 and the relative contributions of both activities to the kinetics of SAC satisfaction.

The dynamic process of mitotic spindle assembly depends on multitudes of inter-dependent interactions involving kinetochores (KTs), microtubules (MTs), spindle pole bodies (SPBs), and molecular motors. Before forming the mitotic spindle, multiple visible microtubule organizing centers (MTOCs) coalesce into a single focus to serve as an SPB in the pathogenic budding yeast, Cryptococcus neoformans. To explain this unusual phenomenon in the fungal kingdom, we propose a "search and capture" model, in which cytoplasmic MTs (cMTs) nucleated by MTOCs grow and capture each other to promote MTOC clustering. Our quantitative modeling identifies multiple redundant mechanisms mediated by a combination of cMT-cell cortex interactions and inter-cMT coupling to facilitate MTOC clustering within the physiological time limit as determined by time-lapse live-cell microscopy. Besides, we screen various possible mechanisms by computational modeling and propose optimal conditions that favor proper spindle positioning-a critical determinant for timely chromosome segregation. These analyses also reveal that a combined effect of MT buckling, dynein pull, and cortical push maintains spatiotemporal spindle localization.