Spindle Architecture Research Articles

Optimized expansion microscopy reveals species-specific spindle microtubule organization in Xenopus egg extracts.

The spindle is a key structure in cell division as it orchestrates the accurate segregation of genetic material. While its assembly and function are well-studied, the mechanisms regulating spindle architecture remain elusive. In this study, we investigate the differences in spindle organization between Xenopus laevis and Xenopus tropicalis, leveraging expansion microscopy (ExM) to overcome the limitations of conventional imaging techniques. We optimized an ExM protocol tailored for Xenopus egg extract spindles, improving upon fixation, denaturation and gelation methods to achieve higher resolution imaging of spindles. Our protocol preserves spindle integrity and allows effective pre-expansion immunofluorescence. This method enabled detailed analysis of the differences in microtubule organization between the two species. X. laevis spindles overall exhibited a broader range of bundle sizes, while X. tropicalis spindles contained mostly smaller bundles. Moreover, while both species exhibited larger bundle sizes near and at the spindle center, X. tropicalis spindles otherwise consisted of very small bundles, and X. laevis spindles medium-sized bundles. By enhancing resolution and minimizing distortions and fixation artifacts, our optimized ExM approach offers new insights into spindle morphology and provides a robust tool for studying the structural intricacies of these large cellular assemblies. This work advances our understanding of spindle architecture and opens up new avenues for exploring underlying mechanisms.

Spindle architecture constrains karyotype evolution

The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell’s chromosomes in a carefully coordinated process. However, chromosome numbers vary dramatically between genomes, even on short evolutionary timescales. We sought to understand how the mitotic machinery senses and responds to karyotypic changes by using a series of budding yeast strains in which the native chromosomes have been successively fused. Using a combination of cell biological profiling, genetic engineering and experimental evolution, we show that chromosome fusions are well tolerated up until a critical point. Cells with fewer than five centromeres lack the necessary number of kinetochore-microtubule attachments needed to counter outward forces in the metaphase spindle, triggering the spindle assembly checkpoint and prolonging metaphase. Our findings demonstrate that spindle architecture is a constraining factor for karyotype evolution.

The cell cycle controls spindle architecture in Arabidopsis by activating the augmin pathway.

To ensure an even segregation of chromosomes during somatic cell division, eukaryotes rely on mitotic spindles. Here, we measured prime characteristics of the Arabidopsis mitotic spindle and built a three-dimensional dynamic model using Cytosim. We identified the cell-cycle regulator CYCLIN-DEPENDENT KINASE B1 (CDKB1) together with its cyclin partner CYCB3;1 as key regulators of spindle morphology in Arabidopsis. We found that the augmin component ENDOSPERM DEFECTIVE1 (EDE1) is a substrate of the CDKB1;1-CYCB3;1 complex. A non-phosphorylatable mutant rescue of ede1 resembled the spindle phenotypes of cycb3;1 and cdkb1 mutants and the protein associated less efficiently with spindle microtubules. Accordingly, reducing the level of augmin in simulations recapitulated the phenotypes observed in the mutants. Our findings emphasize the importance of cell-cycle-dependent phospho-control of the mitotic spindle in plant cells and support the validity of our model as a framework for the exploration of mechanisms controlling the organization of the eukaryotic spindle.

Spindle architecture constrains karyotype in budding yeast.

The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell's chromosomes in a carefully coordinated process. However, chromosome number varies dramatically between genomes, even on short evolutionary timescales. We sought to understand how the mitotic machinery senses and responds to karyotypic changes by using a series of budding yeast strains in which the native chromosomes have been successively fused. Using a combination of cell biological profiling, genetic engineering, and experimental evolution, we show that chromosome fusions are well tolerated up until a critical point. Cells with fewer than five centromeres lack the necessary number of kinetochore-microtubule attachments needed to counter outward forces in the metaphase spindle, triggering the spindle assembly checkpoint and prolonging metaphase. Our findings demonstrate that spindle architecture is a constraining factor for karyotype evolution.

Microtubule length correlates with spindle length in C. elegans meiosis.

The accurate segregation of chromosomes during female meiosis relies on the precise assembly and function of the meiotic spindle, a dynamic structure primarily composed of microtubules. Despite the crucial role of microtubule dynamics in this process, the relationship between microtubule length and spindle size remains elusive. Leveraging Caenorhabditis elegans as a model system, we combined electron tomography and live imaging to investigate this correlation. Our analysis revealed significant changes in spindle length throughout meiosis, coupled with alterations in microtubule length. Surprisingly, while spindle size decreases during the initial stages of anaphase, the size of antiparallel microtubule overlap decreased as well. Detailed electron tomography shows a positive correlation between microtubule length and spindle size, indicating a role of microtubule length in determining spindle dimensions. Notably, microtubule numbers displayed no significant association with spindle length, highlighting the dominance of microtubule length regulation in spindle size determination. Depletion of the microtubule depolymerase KLP-7 led to elongated metaphase spindles with increased microtubule length, supporting the link between microtubule length and spindle size. These findings underscore the pivotal role of regulating microtubule dynamics, and thus microtubule length, in governing spindle rearrangements during meiotic division, shedding light on fundamental mechanisms dictating spindle architecture.

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

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

Comparative Spray Performance of a Solid Set Canopy Delivery System and an Airblast Sprayer in Modern Apple Orchards

Highlights Airblast sprayer applications in modern apple orchards can produce high off-target drift. A pneumatic spray delivery based solid set canopy delivery system (PSD-SSCDS) can be an alternative solution. PSD-SSCDS had comparable spray deposition, but lower spray coverage compared to airblast sprayer. Airblast sprayer had significantly higher losses to the ground and aerial drift. PSD-SSCDS confined ground drift within 4.1 m with minimal spray losses to air. Abstract. A pneumatic spray delivery-based solid set canopy delivery system (PSD-SSCDS) has been successfully configured for efficient spray applications in modern apple orchards typical of the state of Washington state. This study investigates the spray performance of optimized PSD-SSCDS against an axial-fan airblast sprayer in terms of spray deposition, coverage, and off-target drift in a tall spindle architecture of a commercial apple orchard. For field evaluation, mylar cards and water sensitive paper samplers were placed in different canopy zones on either side of the leaf surface, and 500 ppm of biodegradable fluorescent tracer solution was sprayed with PSD-SSCDS and an airblast sprayer. Additionally, aerial and downwind samplers were installed to evaluate ground runoff and drift, as well as aerial drift. The spray deposited on the mylar cards was analyzed using fluorometry, and spray coverage on WSPs was evaluated using image processing techniques. PSD-SSCDS (645.0 ± 94.7 ng cm-2, mean ± standard error) and an airblast sprayer treatment (661.6 ± 26.9 ng cm-2) had comparable mean spray deposition with no significant difference in different canopy zones for both treatments. However, the spray coverage was significantly higher for the airblast sprayer (32.7 ± 2.4%) compared to PSD-SSCDS (22.5 ± 3.1%). These differences are likely due to air-assist in airblast spraying, which is absent in PSD-SSCDS. Evidently, the airblast sprayer had significantly higher losses to the ground and aerial drift compared to PSD-SSCDS, as the ground drift for the latter was mostly confined within 4.1 m downwind of the sprayed row and spray losses to air were minimal. Keywords: Crop protection, Fixed spray delivery, Off-target drift, Pneumatic spray delivery, Spray coverage, Spray deposition.

Sleep spindle architecture associated with distinct clinical phenotypes in older adults at risk for dementia

Sleep spindles are a hallmark of non-REM sleep and play a fundamental role in memory consolidation. Alterations in these spindles are emerging as sensitive biomarkers for neurodegenerative diseases of ageing. Understanding the clinical presentations associated with spindle alterations may help to elucidate the functional role of these distinct electroencephalographic oscillations and the pathophysiology of sleep and neurodegenerative disorders. Here, we use a data-driven approach to examine the sleep, memory and default mode network connectivity phenotypes associated with sleep spindle architecture in older adults (mean age = 66 years). Participants were recruited from a specialist clinic for early diagnosis and intervention for cognitive decline, with a proportion showing mild cognitive deficits on neuropsychological testing. In a sample of 88 people who underwent memory assessment, overnight polysomnography and resting-state fMRI, a k-means cluster analysis was applied to spindle measures of interest: fast spindle density, spindle duration and spindle amplitude. This resulted in three clusters, characterised by preserved spindle architecture with higher fast spindle density and longer spindle duration (Cluster 1), and alterations in spindle architecture (Clusters 2 and 3). These clusters were further characterised by reduced memory (Clusters 2 and 3) and nocturnal hypoxemia, associated with sleep apnea (Cluster 3). Resting-state fMRI analysis confirmed that default mode connectivity was related to spindle architecture, although directionality of this relationship differed across the cluster groups. Together, these results confirm a diversity in spindle architecture in older adults, associated with clinically meaningful phenotypes, including memory function and sleep apnea. They suggest that resting-state default mode connectivity during the awake state can be associated with sleep spindle architecture; however, this is highly dependent on clinical phenotype. Establishing relationships between clinical and neuroimaging features and sleep spindle alterations will advance our understanding of the bidirectional relationships between sleep changes and neurodegenerative diseases of ageing.

UHRF1 promotes spindle assembly and chromosome congression by catalyzing EG5 polyubiquitination.

UHRF1 is an epigenetic coordinator bridging DNA methylation and histone modifications. Additionally, UHRF1 regulates DNA replication and cell cycle, and its deletion induces G1/S or G2/M cell cycle arrest. The roles of UHRF1 in the regulation of G2/M transition remain poorly understood. UHRF1 depletion caused chromosome misalignment, thereby inducing cell cycle arrest at mitotic metaphase, and these cells exhibited the defects of spindle geometry, prominently manifested as shorter spindles. Mechanistically, UHRF1 protein directly interacts with EG5, a kinesin motor protein, during mitosis. Furthermore, UHRF1 induced EG5 polyubiquitination at the site of K1034 and further promoted the interaction of EG5 with spindle assembly factor TPX2, thereby ensuring accurate EG5 distribution to the spindles during metaphase. Our study clarifies a novel UHRF1 function as a nuclear protein catalyzing EG5 polyubiquitination for proper spindle architecture and faithful genomic transmission, which is independent of its roles in epigenetic regulation and DNA damage repair inside the nucleus. These findings revealed a previously unknown mechanism of UHRF1 in controlling mitotic spindle architecture and chromosome behavior and provided mechanistic evidence for UHRF1 deletion-mediated G2/M arrest.

Global SUMOylation in mouse oocytes maintains oocyte identity and regulates chromatin remodeling and transcriptional silencing at the end of folliculogenesis.

Meiotically competent oocytes in mammals undergo cyclic development during folliculogenesis. Oocytes within ovarian follicles are transcriptionally active, producing and storing transcripts required for oocyte growth, somatic cell communication and early embryogenesis. Transcription ceases as oocytes transition from growth to maturation and does not resume until zygotic genome activation. Although SUMOylation, a post-translational modification, plays multifaceted roles in transcriptional regulation, its involvement during oocyte development remains poorly understood. In this study, we generated an oocyte-specific knockout of Ube2i, encoding the SUMO E2 enzyme UBE2I, using Zp3-cre+ to determine how loss of oocyte SUMOylation during folliculogenesis affects oocyte development. Ube2i Zp3-cre+ female knockout mice were sterile, with oocyte defects in meiotic competence, spindle architecture and chromosome alignment, and a premature arrest in metaphase I. Additionally, fully grown Ube2i Zp3-cre+ oocytes exhibited sustained transcriptional activity but downregulated maternal effect genes and prematurely activated genes and retrotransposons typically associated with zygotic genome activation. These findings demonstrate that UBE2I is required for the acquisition of key hallmarks of oocyte development during folliculogenesis, and highlight UBE2I as a previously unreported orchestrator of transcriptional regulation in mouse oocytes.

SF3B1 mutation and ATM deletion codrive leukemogenesis via centromeric R-loop dysregulation.

RNA splicing factor SF3B1 is recurrently mutated in various cancers, particularly in hematologic malignancies. We previously reported that coexpression of Sf3b1 mutation and Atm deletion in B cells, but not either lesion alone, leads to the onset of chronic lymphocytic leukemia (CLL) with CLL cells harboring chromosome amplification. However, the exact role of Sf3b1 mutation and Atm deletion in chromosomal instability (CIN) remains unclear. Here, we demonstrated that SF3B1 mutation promotes centromeric R-loop (cen-R-loop) accumulation, leading to increased chromosome oscillation, impaired chromosome segregation, altered spindle architecture, and aneuploidy, which could be alleviated by removal of cen-R-loop and exaggerated by deletion of ATM. Aberrant splicing of key genes involved in R-loop processing underlay augmentation of cen-R-loop, as overexpression of the normal isoform, but not the altered form, mitigated mitotic stress in SF3B1-mutant cells. Our study identifies a critical role of splice variants in linking RNA splicing dysregulation and CIN and highlights cen-R-loop augmentation as a key mechanism for leukemogenesis.

Phosphorylation controls spatial and temporalactivities of motor-PRC1 complexes to complete mitosis.

During mitosis, spindle architecture alters as chromosomes segregate into daughter cells. The microtubule crosslinker protein regulator of cytokinesis 1 (PRC1) is essential for spindle stability, chromosome segregation and completion of cytokinesis, but how it recruits motors to the central spindle to coordinate the segregation of chromosomes is unknown. Here, we combine structural and cell biology approaches to show that the human CENP-E motor, which is essential for chromosome capture and alignment by microtubules, binds to PRC1 through a conserved hydrophobic motif. This binding mechanism is also used by Kinesin-4 Kif4A:PRC1. Using in vitro reconstitution, we demonstrate that CENP-E slides antiparallel PRC1-crosslinked microtubules. We find that the regulation of CENP-E -PRC1 interaction is spatially and temporally coupled with relocalization to overlapping microtubules in anaphase. Finally, we demonstrate that the PRC1-microtubule motor interaction is essential in anaphase to control chromosome partitioning, retain central spindle integrity and ensure cytokinesis. Taken together our findings reveal the molecular basis for the cell cycle regulation of motor-PRC1 complexes to couple chromosome segregation and cytokinesis.

TET Family Members Are Integral to Porcine Oocyte Maturation and Parthenogenetic Pre-Implantation Embryogenesis.

The ten-eleven translocation (TET) enzyme family, which includes TET1/2/3, participates in active DNA demethylation in the eukaryotic genome; moreover, TET1/2/3 are functionally redundant in mice embryos. However, the combined effect of TET1/2/3 triple-gene knockdown or knockout on the porcine oocytes or embryos is still unclear. In this study, using Bobcat339, a specific small-molecule inhibitor of the TET family, we explored the effects of TET enzymes on oocyte maturation and early embryogenesis in pigs. Our results revealed that Bobcat339 treatment blocked porcine oocyte maturation and triggered early apoptosis. Furthermore, in the Bobcat339-treated oocytes, spindle architecture and chromosome alignment were disrupted, probably due to the huge loss of 5-hydroxymethylcytosine (5hmC)and concurrent increase in 5-methylcytosine (5mC). After Bobcat339 treatment, early parthenogenetic embryos exhibited abnormal 5mC and 5hmC levels, which resulted in compromised cleavage and blastocyst rate. The mRNA levels of EIF1A and DPPA2 (ZGA marker genes) were significantly decreased, which may explain why the embryos were arrested at the 4-cell stage after Bobcat339 treatment. In addition, the mRNA levels of pluripotency-related genes OCT4 and NANOG were declined after Bobcat339 treatment. RNA sequencing analysis revealed differentially expressed genes in Bobcat339-treated embryos at the 4-cell stage, which were significantly enriched in cell proliferation, cell component related to mitochondrion, and cell adhesion molecule binding. Our results indicated that TET proteins are essential for porcine oocyte maturation and early embryogenesis, and they act by mediating 5mC/5hmC levels and gene transcription.

Reservoir Units Optimization in Pneumatic Spray Delivery-Based Fixed Spray System for Large-Scale Commercial Adaptation

A pneumatic spray delivery (PSD)-based solid set canopy delivery system (SSCDS) consists of in-line reservoirs and micro-emitter assemblies distributed throughout perennial crop canopies. The existing PSD-based SSCDS uses a large number of reservoirs, i.e., one unit per 3 m of linear spacing, which resulted in high installation and maintenance costs. These reservoirs also produces up to 25% post-spray chemical losses. Therefore, this study aimed to optimize the volumetric capacity and functionality of the existing reservoir for an efficient spray performance and the large-scale commercial adaptation of PSD-based SSCDS. Three reservoirs with volumetric capacities of 370 (1×), 740 (2×), and 1110 mL (3×) were developed to cover a spray span of 3.0, 6.1, and 9.1 m, respectively. Five system configurations with modified reservoirs and spray outlets were evaluated in the laboratory for pressure drop and spray uniformity. The three best system configurations were then field evaluated in a high-density apple orchard. These configurations had reservoirs with 1×, 2×, and 3× volumetric capacity and micro-emitters installed in a three-tier arrangement. Each replicate configuration was installed as a 77 m loop length encompassing 50 apple trees trained in a tall spindle architecture. A pair of water-sensitive paper (WSPs) samplers (25.4 × 25.4 mm) were placed on the abaxial and adaxial leaf surfaces in the bottom, middle, and top third of the canopy to evaluate the spray coverage (%). The PSD-based SSCDS showed no significant difference at the 5% level in terms of coverage among the three reservoir treatments. Coverage was more evenly distributed among the top, middle, and bottom zones for the 2× and 3× as compared to the 1× reservoir treatment. Overall, compared to the 1× reservoirs, the 2× and 3× reservoirs could potentially reduce the system costs by USD 20,000 and USD 23,410 ha−1, respectively, for tall spindle apple orchards and potentially reduce maintenance needs as well.

Pneumatic spray delivery-based solid set canopy delivery system for oblique banded leaf roller and codling moth control in a high-density modern apple orchard.

Pneumatic spray delivery (PSD)-based solid set canopy delivery systems (SSCDS) have demonstrated comparable spray deposition and reduced off-target drift compared with axial-fan airblast sprayers in high-density apple orchards. An important next step is to quantify whether PSD-based SSCDS can provide effective pest management. This study evaluated the biological efficacy of this fixed spray system variant and compared it with that of an axial-fan airblast sprayer. Partial field trials were conducted in a commercial apple orchard (cv. Jazz) trained in tall spindle architecture. Insecticides were applied at a rate of 935 L ha-1 (100 gallons per acre) for both application systems. Twenty-four hours after spraying, leaves and fruits were collected to prepare the laboratory bioassays enabling evaluation of efficacy against obliquebanded leafroller (OBLR) and codling moth (CM). OBLR mortality for SSCDS, airblast sprayer and untreated control treatments after 24 h of larval exposure was 91%, 98% and 4%, respectively and increased to 98%, 100% and 19% after 48 h. First-instar CM leaf bioassay mortality was 100% for SSCDS and airblast sprayer treatment, and 13% for the untreated control at 24 h post exposure. Larval CM mortality on fruit was 100% for SSCDS and airblast sprayer treatments, and 33% on the untreated control. Insecticides applied using SSCDS and an airblast sprayer had comparable larval mortality in all three assays, significantly higher than the untreated controls. These results suggest that PSD-based SSCDS may provide a viable alternative to axial-fan airblast sprayers in high-density apple orchards. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The phosphorylation and dephosphorylation switch of VCP/p97 regulates the architecture of centrosome and spindle.

The proper orientation of centrosome and spindle is essential for genome stability; however, the mechanism that governs these processes remains elusive. Here, we demonstrated that polo-like kinase 1 (Plk1), a key mitotic kinase, phosphorylates residue Thr76 in VCP/p97 (an AAA-ATPase), at the centrosome from prophase to anaphase. This phosphorylation process recruits VCP to the centrosome and in this way, it regulates centrosome orientation. VCP exhibits strong co-localization with Eg5 (a mitotic kinesin motor), at the mitotic spindle, and the dephosphorylation of Thr76 in VCP is required for the enrichment of both VCP and Eg5 at the spindle, thus ensuring proper spindle architecture and chromosome segregation. We also showed that the phosphatase, PTEN, is responsible for the dephosphorylation of Thr76 in VCP; when PTEN was knocked down, the normal spread of VCP from the centrosome to the spindle was abolished. Cryo-EM structures of VCPT76A and VCPT76E, which represent dephosphorylated and phosphorylated states of VCP, respectively, revealed that the Thr76 phosphorylation modulates VCP by altering the inter-domain and inter-subunit interactions, and ultimately the nucleotide-binding pocket conformation. Interestingly, the tumor growth in nude mice implanted with VCPT76A-reconstituted cancer cells was significantly slower when compared with those implanted with VCPWT-reconstituted cancer cells. Collectively, our findings demonstrate that the phosphorylation and dephosphorylation switch of VCP regulates the architecture of centrosome and spindle for faithful chromosome segregation.

Non-centrosomal microtubules at kinetochores promote rapid chromosome biorientation during mitosis in human cells

Proper segregation of chromosomes during mitosis depends on "amphitelic attachments"-load-bearing connections of sister kinetochores to the opposite spindle poles via bundles of microtubules, termed as the "K-fibers." Current models of spindle assembly assume that K-fibers arise largely from stochastic capture of microtubules, which occurs at random times and locations and independently at sister kinetochores. We test this assumption by following the movements of all kinetochores in human cells and determine that most amphitelic attachments form synchronously at a specific stage of spindle assembly and within a spatially distinct domain. This biorientation domain is enriched in bundles of antiparallel microtubules, and perturbation of microtubule bundling changes the temporal and spatial dynamics of amphitelic attachment formation. Structural analyses indicate that interactions of kinetochores with microtubule bundles are mediated by non-centrosomal short microtubules that emanate from most kinetochores during early prometaphase. Computational analyses suggest that momentous molecular motor-driven interactions with antiparallel bundles rapidly convert these short microtubules into nascent K-fibers. Thus, load-bearing connections to the opposite spindle poles form simultaneously on sister kinetochores. In contrast to the uncoordinated sequential attachments of sister kinetochores expected in stochastic models of spindle assembly, our model envisions the formation of amphitelic attachments as a deterministic process in which the chromosomes connect with the spindle poles synchronously at a specific stage of spindle assembly and at a defined location determined by the spindle architecture. Experimental analyses of changes in the kinetochore behavior in cells with perturbed activity of molecular motors CenpE and dynein confirm the predictive power of the model.

Mammalian kinetochore-fibers regulate their length and dynamics individually, independent of spindle poles

At each cell division, the spindle builds itself from tubulin building blocks, with nanometer-scale components giving rise to a micron-scale structure. While many proteins modulating mammalian spindle length are known, how they work together to set spindle length remains poorly understood. To probe the role of spindle architecture in setting the length of spindle substructures, and vice versa, we inhibit dynein to generate spindles whose kinetochore-fibers (k-fibers) no longer focus and connect at poles.

R-Loop Associated Mitotic Stress Confers Vulnerabilities in Splicing Factor Mutant Leukemia

The RNA splicing factor SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL). Co-expression of mutant Sf3b1 and Atm deletionin murine B cellsresults in CLL development, in which CLL cells exhibit RNA splicing dysregulation, increased DNA damage and recurrent chromosomal amplification. These findings suggest a strong link between splicing factormutation, chromosomal instability (CIN), and oncogenesis, although the mechanism has yet to be elucidated.CIN in cells with defective RNA processing is triggered, at least in part, by excessive R-loop (RNA:DNA hybrid with displaced single strand DNA). Using dotblot assay, we observed R-loop augmentation in SF3B1 mutant (MT) compared to wild-type (WT) cells in both human cell lines and murine CLL cells. Along with R-loop accumulation, SF3B1 MT increased DNA double strand breaks (DSB) (measured by comet assay) and mis-segregated chromosomes during mitosis (quantified by immunofluorescence using anti-phospho-histone H3 and anti-αtubulin antibodies). Overexpression of R-loop resolving enzyme RNaseH1 in SF3B1 MT cell lines reduced DSB (t-test p-value <0.0001) and mitotic stress (t-test p-value <0.0001), confirming the causative role of R-loop in CIN.To elucidate the mechanisms underlying SF3B1 MT-associated CIN, we used DNA:RNA hybrid immunoprecipitation (DRIP) coupled with sequencing to map R-loop in SF3B1 MT and WT Nalm6 cells. Although SF3B1 MT caused minimal changes in overall R-loop distribution, it led to increased R-loop formation at centromeric regions. Proper centromeric R-loop (cen-R-loop) formation is essential for accurate chromosome segregation. Those R-loop are coated by phosphorylated replication protein A (p-RPA). p-RPA and centromere immunofluorescence (IF) co-staining revealed increased cen-R-loop in SF3B1 MT cells. Intriguingly, overexpression of RNaseH1 reduced both levels of centromeric p-RPA and percentage of cells with defective mitosis, suggesting SF3B1 MT associated cen-R-loop impacts CIN.To further discern the molecular underpinnings of R-loop mediated CIN, we investigated the effect of their accumulation on spindle geometry in metaphase cells. By analyzing the total body of chromosomes two-dimensional area, we observed one fold greater magnitude of chromosome oscillations in SF3B1 Nalm6 MT cells. Accordingly, compared to WT cells, SF3B1 MT cells displayed longer and wider spindles to accommodate an aneuploid chromosomal content. Removal of R-loops rescued mitotic spindle architecture and CIN in SF3B1 MT cells, highlighting a direct contribution of R-loop to kinetochore-microtubule organization and stability.As there is no evidence of strong binding of SF3B1 to centromeres, we tested the possibility that SF3B1 MT contributes to cen-R-loop formation through alternative splice variants. Through overlapping conserved SF3B1 MT-associated splice variants across isogenic cell lines and murine model with available R-loop interactome data, we identified SERBP1, which encodes an RNA-binding protein, as a target candidate . SF3B1 MT induces loss-of-function in SERBP1 through alternative splicing. Knockdown of SERBP1 triggered R-loop accumulation, increased centromeric p-RPA coating, and aberrant mitosis, which recapitulated the SF3B1 MT phenotype. Importantly, overexpression of SERBP1 WT alleviated both DSB and R-loop accumulation in SF3B1 MT cells. These observations strongly suggest that SF3B1 MT augments R-loop formation through dysregulated splicing of SERBP1 and possibly other genes involved in R-loop metabolism.We also investigated the possibility that the impact of RNA splicing defect on R-loop biology is a generalizable phenomenon. Examing a panel of leukemia-associated splicing factors (U2AF1, SRSF2, ZRSR2, RNU1), we found that these mutations all display R-loop dependent mitotic stress. Thus, mitotic stress caused by aberrant R-loop formation could be a convergent mechanism in splicing factor mutated leukemia cells.Altogether, we demonstrated that SF3B1 mutation promotes CIN by destabilizing mitotic spindles through aberrant R-loop accumulation at the centromere, via dysregulated splicing in genes such as SERBP1. Our study highlights an unrecognized role of cen-R-loop as a critical link between RNA splicing dysregulation and CIN, providing an opportunity for therapeutic targeting of R-loop in splicing factor mutant leukemias. DisclosuresWu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company.

Oblique circle method for measuring the curvature and twist of mitotic spindle microtubule bundles

The highly ordered spatial organization of microtubule bundles in the mitotic spindle is crucial for its proper functioning. The recent discovery of twisted shapes of microtubule bundles and spindle chirality suggests that the bundles extend along curved paths in three dimensions, rather than being confined to a plane. This, in turn, implies that rotational forces, i.e., torques, exist in the spindle in addition to the widely studied linear forces. However, studies of spindle architecture and forces are impeded by a lack of a robust method for the geometric quantification of microtubule bundles in the spindle. In this work, we describe a simple method for measuring and evaluating the shapes of microtubule bundles by characterizing them in terms of their curvature and twist. By using confocal microscopy, we obtain three-dimensional images of spindles, which allows us to trace the entire microtubule bundle. For each traced bundle, we first fit a plane and then fit a circle lying in that plane. With this robust method, we extract the curvature and twist, which represent the geometric information characteristic for each bundle. As the bundle shapes reflect the forces within them, this method is valuable for the understanding of forces that act on chromosomes during mitosis.