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Intrinsic reproductive isolation occurs when genetic differences between populations disrupt the development of hybrid organisms, preventing gene flow and enforcing speciation. 1-4 While prior studies have examined the genetic origins of hybrid incompatibility, 5-18 the effects of incompatible factors on development remain poorly understood. Here, we investigate the mechanistic basis of hybrid incompatibility in Caenorhabditis nematodes by capitalizing on the ability of C. brenneri females to produce embryos after mating with males from several other species. Contrary to expectations, hybrid incompatibility was evident immediately after fertilization, suggesting that post-fertilization barriers to hybridization originate from physical incompatibility between sperm and oocyte-derived factors rather than from zygotic transcription, which starts after the 4-cell stage. 19-22 Sperm deliver chromatin, which expands to form a pronucleus, and a pair of centrioles, which form centrosomes that attach to the sperm-derived pronucleus and signal to establish the embryo's anterior-posterior axis. 23,24 In C. brenneri oocytes fertilized with C. elegans sperm, sperm pronuclear expansion was compromised, frequent centrosome detachment was observed, and cortical polarity was disrupted. Live imaging revealed that defective polar body extrusion contributes to defects in mitotic spindle morphology. C. brenneri oocytes fertilized with C. remanei or C. sp. 48 sperm showed similar defects, and their severity and frequency increased with phylogenetic distance. Defective expansion of the sperm-derived pronucleus and unreliable polar body extrusion immediately after fertilization generally underlie the inviability of hybrid embryos in this clade. These results indicate that physical mismatches between sperm and oocyte-derived structures may be a primary mechanism of hybrid incompatibility., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Kinesin-5 motors play an essential role during mitotic spindle assembly in many organisms 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 : they crosslink antiparallel spindle microtubules, step toward plus ends, and slide the microtubules apart. 12 , 13 , 14 , 15 , 16 , 17 This activity separates the spindle poles and chromosomes. Kinesin-5s are not only plus-end-directed but can walk or be carried toward MT minus ends, 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 where they show enhanced localization. 3 , 5 , 7 , 27 , 29 , 32 The kinesin-5 C-terminal tail interacts with and regulates the motor, affecting structure, motility, and sliding force of purified kinesin-5 35 , 36 , 37 along with motility and spindle assembly in cells. 27 , 38 , 39 The tail contains phosphorylation sites, particularly in the conserved BimC box. 6 , 7 , 40 , 41 , 42 , 43 , 44 Nine mitotic tail phosphorylation sites were identified in the kinesin-5 motor of the fission yeast Schizosaccharomyces pombe, 45 , 46 , 47 , 48 suggesting that multi-site phosphorylation may regulate kinesin-5s. Here, we show that mutating all nine sites to either alanine or glutamate causes temperature-sensitive lethality due to a failure of bipolar spindle assembly. We characterize kinesin-5 localization and sliding force in the spindle based on Cut7-dependent microtubule minus-end protrusions in cells lacking kinesin-14 motors. 39 , 49 , 50 , 51 , 52 Imaging and computational modeling show that Cut7p simultaneously moves toward the minus ends of protrusion MTs and the plus ends of spindle midzone MTs. Phosphorylation mutants show dramatic decreases in protrusions and sliding force. Comparison to a model of force to create protrusions suggests that tail truncation and phosphorylation mutants decrease Cut7p sliding force similarly to tail-truncated human Eg5. 36 Our results show that C-terminal tail phosphorylation is required for kinesin-5/Cut7 sliding force and bipolar spindle assembly in fission yeast., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Amino acid transporters play a vital role in cellular homeostasis by maintaining protein synthesis. L-type amino acid transporter 1 (LAT1/SLC7A5/CD98lc) is a major transporter of large neutral amino acids in cancer cells because of its predominant expression. Although amino acid restriction with various amino acid analog treatments is known to induce mitotic defects, the involvement of amino acid transporters in cell division remains unclear. In this study, we identified that LAT1 is responsible for mitotic progression in a transport activity-independent manner. LAT1 knockdown activates the spindle assembly checkpoint, leading to a delay in metaphase. LAT1 maintains proper spindle orientation with confinement of the lateral cortex localization of the NuMA protein, which mediates the pulling force against the mitotic spindle toward the lateral cortex. Unexpectedly, JPH203, an inhibitor of LAT1 amino acid transport activity, does not affect mitotic progression. Moreover, the transport activity-deficient LAT1 mutant maintains the proper spindle orientation and mitotic progression. LAT1 forms a heterodimer with CD98 (SLC3A2/CD98hc) both in interphase and mitosis. Although CD98 knockdown decreases the plasma membrane localization of LAT1, it does not affect mitotic progression. LAT1 is localized to the Golgi and ER not only at the plasma membrane in interphase, and promotes Golgi unlinking during the mitotic entry, leading to centrosome maturation. These results suggest that LAT1 supports mitotic progression in an amino acid transport activity-independent manner and that Golgi-localized LAT1 is important for mitotic progression through the acceleration of Golgi unlinking and centrosome maturation. These findings reveal a novel LAT1 function in mitosis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Aurora-A is an essential cell-cycle kinase with critical roles in mitotic entry and spindle dynamics. These functions require binding partners such as CEP192 and TPX2, which modulate both kinase activity and localisation of Aurora-A. Here we investigate the structure and role of the centrosomal Aurora-A:CEP192 complex in the wider molecular network. We find that CEP192 wraps around Aurora-A, occupies the binding sites for mitotic spindle-associated partners, and thus competes with them. Comparison of two different Aurora-A conformations reveals how CEP192 modifies kinase activity through the site used for TPX2-mediated activation. Deleting the Aurora-A-binding interface in CEP192 prevents centrosomal accumulation of Aurora-A, curtails its activation-loop phosphorylation, and reduces spindle-bound TPX2:Aurora-A complexes, resulting in error-prone mitosis. Thus, by supplying the pool of phosphorylated Aurora-A necessary for TPX2 binding, CEP192:Aurora-A complexes regulate spindle function. We propose an evolutionarily conserved spatial hierarchy, which protects genome integrity through fine-tuning and correctly localising Aurora-A activity., (© 2024. The Author(s).)

Cytoskeleton-associated protein 2-like (CKAP2L) is a paralogue of cytoskeleton-associated protein 2 (CKAP2). We characterized the expression pattern, subcellular localization, and microtubule-stabilizing properties of human CKAP2L. The levels of both CKAP2L transcript and protein were cell cycle phase-dependent, peaking during the G 2 /M phase and relatively high in certain human tissues, including testis, intestine, and spleen. CKAP2L protein was detectable in all human cancer cell lines we tested. CKAP2L localized to the mitotic spindle apparatus during mitosis, as reported previously. During interphase, however, CKAP2L localized mainly to the nucleus. Ectopic overexpression of CKAP2L resulted in 'microtubule bundling', and, consequently, an elevated CKAP2L level led to prolonged mitosis. These findings support the mitotic role of CKAP2L during the human cell cycle., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The Canadian prairie ecosystem is subjected to abiotic and biotic conditions that induce plants to produce secondary metabolites that affect mammalian physiology. Extracts prepared from certain plant species native to Canadian prairie and montane cordillera ecosystems have previously been shown to have anti-mitotic activity on human cancer cell lines. In this study, we investigated the glacier lily, Erythronium grandiflorum (Liliaceae), in which the species was the most phylogenetically distant from Asteraceae and had anti-mitotic activity. When added to cell lines, E. grandiflorum extracts induced rounded cell morphology and arrested cells in the G2/M phase of the cell cycle. Of the cells that displayed a rounded phenotype, all were positive for phospho-histone H3 and contained a distorted mitotic spindle. This anti-mitotic activity was distinct from that of the compound colchicine, which has been previously isolated from the Liliaceae family. By biology-guided fractionation, we isolated the natural product (+)-6-tuliposide A and are the first to report its anti-mitotic activity. These results reveal a chemical motif in secondary metabolites and expand the range of Canadian prairie plants with anti-mitotic activity that can become new scientific tools or used in the development of anti-proliferative medicines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Bis(2-ethylhexyl) phthalate is the most abundant phthalate used as plasticizer to soften plastics and polymers included in medical devices. Human and environmental exposure may occur because DEHP is not chemically bound to plastics and can easily leach out of the materials. This phthalate is classified as reproductive toxicant and possible carcinogen to humans. The genotoxic potential has still to be clarified, but there are indications suggesting that DEHP may have aneugenic effects. To further investigate DEHP genotoxicity, the cytochalasin-block micronucleus assay was applied and combined with the CREST staining to characterise micronucleus content and gain insights on its genotoxic mode of action. Chromosomal damage was also analysed in metaphase and ana-telophase cells and the morphology of the mitotic spindle was investigated to evaluate the possible involvement of this cellular apparatus as a target of DEHP. Our findings indicated that DEHP induced a statistically significant increase in the frequency of micronuclei as well as in the frequency of CREST-positive micronuclei. Consistently, disturbance of chromosome segregation and induction of numerical chromosome changes were observed together with changes in spindle morphology, formation of multipolar spindles and alteration of the microtubule network. Experiments performed without metabolic activation demonstrated a direct action of DEHP on chromosome segregation not mediated by its metabolites. In conclusion, there is consistent evidence for an aneugenic activity of DEHP. A thresholded genotoxic activity was identified for DEHP, disclosing possible implications for risk assessment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The widespread use of radiofrequency (RF) sources, ranging from household appliances to telecommunications devices and military equipment, raises concerns among people and regulatory agencies about the potential health risks of RF exposure. Consequently, several in vitro and in vivo studies have been done to investigate the biological effects, in particular non-thermal, of this non-ionizing radiation. To date, this issue is still being debated due to the controversial results that have been reported. Furthermore, the impact of different RF signal modulations on biological systems remains poorly investigated. The present in vitro study aims to evaluate the cytotoxicity and genotoxicity of continuous or pulsed 1.6 GHz RF in human dermal fibroblasts (HDF)., Methods: HDF cultures were exposed to continuous and pulsed 1.6 GHz RF, for 2 h, with Specific Absorption Rate (SAR) of 0.4 W/kg. The potential biological effects of 1.6 GHz RF on HDF were assessed with a multi-methodological approach, analyzing the effects on cell cycle, ultrastructure, protein expression, mitotic spindle, CREST stained micronuclei, chromosome segregation and γ-H2AX/53BP1 foci., Results: 1.6 GHz RF exposure modified proteins expression and morphology of HDF. Specifically, the expression of different heat-shock proteins (HSP) (i.e., HSP-90, HSP-60, and HSP-25) and phospho-AKT were affected. In addition, both continuous and pulsed RF modified the cytoskeletal organization in HDF and increased the number of lysosomes, while the formation of autophagosomes was observed only after pulsed RF exposure. Mitotic spindle anomalies were also found after exposure. However, no significant effect was observed on cell cycle, chromosome segregation, CREST-stained micronuclei and γ-H2AX/53BP1 foci., Conclusion: The results of the present study show the absence of genotoxic damage in 1.6 GHz RF exposed HDF and, although mitotic spindle alterations were observed, they did not have an aneugenic effect. On the other hand, changes in some proteins expression and cell ultrastructure in exposed HDF suggest that RF can potentially induce cell alterations at the morphological and molecular levels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Massaro, De Sanctis, Franchini, Regalbuto, Alfano, Focaccetti, Benvenuto, Cifaldi, Sgura, Berardinelli, Marinaccio, Barbato, Rossi, Nardozi, Masuelli, Bei and Lista.)

Precise segregation of chromosomes during mitosis requires assembly of a bipolar mitotic spindle followed by correct attachment of microtubules to the kinetochores. This highly spatiotemporally organized process is controlled by various mitotic kinases and molecular motors. We have recently shown that Casein Kinase 1 (CK1) promotes timely progression through mitosis by phosphorylating FAM110A leading to its enrichment at spindle poles. However, the mechanism by which FAM110A exerts its function in mitosis is unknown. Using structure prediction and a set of deletion mutants, we mapped here the interaction of the N- and C-terminal domains of FAM110A with actin and tubulin, respectively. Next, we found that the FAM110A-Δ40-61 mutant deficient in actin binding failed to rescue defects in chromosomal alignment caused by depletion of endogenous FAM110A. Depletion of FAM110A impaired assembly of F-actin in the proximity of spindle poles and was rescued by expression of the wild-type FAM110A, but not the FAM110A-Δ40-61 mutant. Purified FAM110A promoted binding of F-actin to microtubules as well as bundling of actin filaments in vitro. Finally, we found that the inhibition of CK1 impaired spindle actin formation and delayed progression through mitosis. We propose that CK1 and FAM110A promote timely progression through mitosis by mediating the interaction between spindle microtubules and filamentous actin to ensure proper mitotic spindle formation., Competing Interests: Competing interests statement:The authors declare no competing interest.

Confined cell migration hampers genome integrity and activates the ATR and ATM mechano-transduction pathways. We investigated whether the mechanical stress generated by metastatic interstitial migration contributes to the enhanced chromosomal instability observed in metastatic tumor cells. We employed live cell imaging, micro-fluidic approaches, and scRNA-seq to follow the fate of tumor cells experiencing confined migration. We found that, despite functional ATR, ATM, and spindle assembly checkpoint (SAC) pathways, tumor cells dividing across constriction frequently exhibited altered spindle pole organization, chromosome mis-segregations, micronuclei formation, chromosome fragility, high gene copy number variation, and transcriptional de-regulation and up-regulation of c-MYC oncogenic transcriptional signature via c-MYC locus amplifications. In vivo tumor settings showed that malignant cells populating metastatic foci or infiltrating the interstitial stroma gave rise to cells expressing high levels of c-MYC. Altogether, our data suggest that mechanical stress during metastatic migration contributes to override the checkpoint controls and boosts genotoxic and oncogenic events. Our findings may explain why cancer aneuploidy often does not correlate with mutations in SAC genes and why c-MYC amplification is strongly linked to metastatic tumors., Competing Interests: Competing interests statement:M.P. is of the board of Directors and stakeholder of CheckmAb s.r.l. and is a recipient of grants under a research agreement with Bristol-Myers Squibb and Macomics.

Globally, ∼850 million individuals suffer from some form of kidney disease. This staggering figure underscores the importance of continued research and innovation in the field of nephrology to develop effective treatments and improve overall global kidney health. In current research, the polo-like kinase (Plk) family has emerged as a group of highly conserved enzyme kinases vital for proper cell cycle regulation. Plks are defined by their N-terminal kinase domain and C-terminal polo-box domain, which regulate their catalytic activity, subcellular localization, and substrate recognition. Among the Plk family members, Plk1 has garnered significant attention due to its pivotal role in regulating multiple mitotic processes, particularly in the kidneys. It is a crucial serine-threonine (Ser-Thr) kinase involved in cell division and genomic stability. In this review, we delve into the types and functions of Plks, focusing on Plk1's significance in processes such as cell proliferation, spindle assembly, and DNA damage repair. The review also underscores Plk1's vital contributions to maintaining kidney homeostasis, elucidating its involvement in nuclear envelope breakdown, anaphase-promoting complex/cyclosome activation, and the regulation of mRNA translation machinery. Furthermore, the review discusses how Plk1 contributes to the development and progression of kidney diseases, emphasizing its overexpression in conditions such as acute kidney injury, chronic kidney disease, and so forth. It also highlights the importance of exploring Plk1 modulators as targeted therapies for kidney diseases in future. This review will help in understanding the role of Plk1 in kidney disease development, paving the way for the discovery and development of novel therapeutic approaches to manage kidney diseases effectively., (© 2024 John Wiley & Sons Ltd.)

Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis and has a role in localizing CENP-E, but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild-type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time-lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Long-term inhibition of CNEP-E via GSK923295 recapitulates CTCF knockdown abnormal mitotic spindles with polar chromosomes and increased nuclear sizes. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild-type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting that population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus likely through its known role in recruiting CENP-E., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The mitotic spindle contains many bundles of microtubules (MTs) including midzones and kinetochore fibers, but little is known about how bundled structures are formed. Here, we show that the chromosomal passenger complex (CPC) purified from Escherichia coli undergoes liquid-liquid demixing in vitro. An emergent property of the resultant condensates is to generate parallel MT bundles when incubated with free tubulin and GTP in vitro. We demonstrate that MT bundles emerge from CPC droplets with protruding minus ends that then grow into long and tapered MT structures. During this growth, we found that the CPC in these condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for liquid-liquid demixing or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data demonstrate that an in vitro biochemical activity to produce MT bundles emerges after the concentration of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle. Moreover, these data suggest that cells contain MT-organizing centers that generate MT bundles that emerge with the opposite polarity from centrosomes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Polarised epithelial cell divisions represent a fundamental mechanism for tissue maintenance and morphogenesis. Morphological and mechanical changes in the plasma membrane influence the organisation and crosstalk of microtubules and actin at the cell cortex, thereby regulating the mitotic spindle machinery and chromosome segregation. Yet, the precise mechanisms linking plasma membrane remodelling to cell polarity and cortical cytoskeleton dynamics to ensure accurate execution of mitosis in mammalian epithelial cells remain poorly understood. Here, we manipulated the density of mammary epithelial cells in culture, which led to several mitotic defects. Perturbation of cell-cell adhesion formation impairs the dynamics of the plasma membrane, affecting the shape and size of mitotic cells and resulting in defects in mitotic progression and the generation of daughter cells with aberrant architecture. In these conditions, F- actin-astral microtubule crosstalk is impaired, leading to mitotic spindle misassembly and misorientation, which in turn contributes to chromosome mis-segregation. Mechanistically, we identify S100 Ca2+-binding protein A11 (S100A11) as a key membrane-associated regulator that forms a complex with E-cadherin (CDH1) and the leucine-glycine-asparagine repeat protein LGN (also known as GPSM2) to coordinate plasma membrane remodelling with E-cadherin-mediated cell adhesion and LGN-dependent mitotic spindle machinery. Thus, plasma membrane-mediated maintenance of mammalian epithelial cell identity is crucial for correct execution of polarised cell divisions, genome maintenance and safeguarding tissue integrity., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

High-risk neuroblastoma is a very aggressive pediatric cancer, accounting for ~15% of childhood cancer mortality. Therefore, novel therapeutic strategies for the treatment of neuroblastoma are urgently sought. Here, we focused on the potential implications of the Dual-specificity tYrosine-Regulated Kinase (DYRK) family and downstream signaling pathways. We used bioinformatic analysis of public datasets from neuroblastoma cohorts and cell lines to search correlations between patient survival and expression of DYRK kinases. Additionally, we performed biochemical, molecular, and cellular approaches to validate and characterize our observations, as well as an in vivo orthotopic murine model of neuroblastoma. We identified the DYRK3 kinase as a critical mediator of neuroblastoma cell proliferation and in vivo tumor growth. DYRK3 has recently emerged as a key regulator of several biomolecular condensates and has been linked to the hypoxic response of neuroblastoma cells. Our data suggest a role for DYRK3 as a regulator of the neuroblastoma-specific protein CAMKV, which is also required for neuroblastoma cell proliferation. CAMKV is a very understudied member of the Ca 2+ /calmodulin-dependent protein kinase family, originally described as a pseudokinase. We show that CAMKV is phosphorylated by DYRK3, and that inhibition of DYRK3 kinase activity induces CAMKV aggregation, probably mediated by its highly disordered C-terminal half. Importantly, we provide evidence that the DYRK3/CAMKV signaling module could play an important role for the function of the mitotic spindle during cell division. Our data strongly support the idea that inhibition of DYRK3 and/or CAMKV in neuroblastoma cells could constitute an innovative and highly specific intervention to fight against this dreadful cancer.

During eukaryotic cell division a microtubule-based structure, the mitotic spindle, aligns and segregates chromosomes between daughter cells. Understanding how this cellular structure is assembled and coordinated in space and in time requires measuring microtubule dynamics and visualizing spindle assembly with high temporal and spatial resolution. Visualization is often achieved by the introduction and the detection of molecular probes and fluorescence microscopy. Microtubules and mitotic spindles are highly conserved across eukaryotes; however, several technical limitations have restricted these investigations to only a few species. The ability to monitor microtubule and chromosome choreography in a wide range of species is fundamental to reveal conserved mechanisms or unravel unconventional strategies that certain forms of life have developed to ensure faithful partitioning of chromosomes during cell division. Here, we describe a technique based on injection of purified proteins that enables the visualization of microtubules and chromosomes with a high contrast in several divergent marine embryos. We also provide analysis methods and tools to extract microtubule dynamics and monitor spindle assembly. These techniques can be adapted to a wide variety of species in order to measure microtubule dynamics and spindle assembly kinetics when genetic tools are not available or in parallel to the development of such techniques in non-model organisms., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The tripartite attachment complex (TAC) of the single mitochondrion of trypanosomes allows precise segregation of its single nucleoid mitochondrial genome during cytokinesis. It couples the segregation of the duplicated mitochondrial genome to the segregation of the basal bodies of the flagella. Here, we provide a model of the molecular architecture of the TAC that explains how its eight essential subunits connect the basal body, across the mitochondrial membranes, with the mitochondrial genome. We also discuss how the TAC subunits are imported into the mitochondrion and how they assemble to form a new TAC. Finally, we present a comparative analysis of the trypanosomal TAC with open and closed mitotic spindles, which reveals conserved concepts between these diverse DNA segregation systems., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Shwachman-Diamond syndrome (SDS) is an autosomal recessive inherited disorder caused by biallelic mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS protein is involved in ribosome biogenesis; therefore SDS is classified as a ribosomopathy. SBDS is localized at mitotic spindles and stabilizes microtubules. Previously, we showed that SBDS interacts with ring finger protein 2 (RNF2) and is degraded through RNF2-dependent ubiquitination. In this study, we investigated when and where SBDS interacts with RNF2 and the effects of the interaction on cells. We found that SBDS co-localized with RNF2 on centrosomal microtubules in the mitotic phase (M phase), whereas SBDS and RNF2 localized to the nucleolus and nucleoplasm in the interphase, respectively. The microtubule-binding assay revealed that SBDS interacted directly with microtubules and RNF2 interacted with SBDS bound to microtubules. In addition, SBDS was ubiquitinated and degraded by RNF2 during the M phase. Moreover, RNF2 overexpression accelerated mitotic progression. These findings suggest that SBDS delays mitotic progression, and RNF2 releases cells from suppression through the ubiquitination and subsequent degradation of SBDS. The interaction between SBDS and RNF2 at mitotic spindles might be involved in mitotic progression as a novel regulatory cascade., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The endoplasmic reticulum (ER) undergoes a remarkable transition in morphology during cell division to aid in the proper portioning of the ER. However, whether changes in ER behaviors modulate mitotic events is less clear. Like many animal embryos, the early Drosophila embryo undergoes rapid cleavage cycles in a lipid-rich environment. Here, we show that mitotic spindle formation, centrosomal maturation, and ER condensation occur with similar time frames in the early syncytium. In a screen for Rab family GTPases that display dynamic function at these stages, we identified Rab1. Rab1 disruption led to an enhanced buildup of ER at the spindle poles and produced an intriguing 'mini-spindle' phenotype. ER accumulation around the mitotic space negatively correlates with spindle length/intensity. Importantly, centrosomal maturation is defective in these embryos, as mitotic recruitment of key centrosomal proteins is weakened after Rab1 disruption. Finally, division failures and ER overaccumulation is rescued by Dynein inhibition, demonstrating that Dynein is essential for ER spindle recruitment. These results reveal that ER levels must be carefully tuned during mitotic processes to ensure proper assembly of the division machinery., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Accurate placement of the cleavage furrow is crucial for successful cell division. Recent advancements have revealed that diverse mechanisms have evolved across different branches of the phylogenetic tree. Here, we employed Dictyostelium cells to validate previous models. We observed that during metaphase and early anaphase, mitotic spindles exhibited random rotary movements which ceased when the spindle elongated by approximately 7 μm. At this point, astral microtubules reached the polar cell cortex and fixed the spindle axis, causing cells to elongate by extending polar pseudopods and divide along the spindle axis. Therefore, the position of the furrow is determined when the spindle orientation is fixed. The distal ends of astral microtubules stimulate the extension of pseudopods at the polar cortex. One signal for pseudopod extension may be phosphatidylinositol trisphosphate in the cell membrane, but there appears to be another unknown signal. At the onset of polar pseudopod extension, cortical flow began from both poles toward the equator. We suggest that polar stimulation by astral microtubules determines the furrow position, induces polar pseudopod extension and cortical flow, and accumulates the elements necessary for the construction of the contractile ring., (© 2023 Wiley Periodicals LLC.)

Nucleolin is a multifunctional RNA-binding protein that resides predominantly not only in the nucleolus, but also in multiple other subcellular pools in the cytoplasm in mammalian cells, and is best known for its roles in ribosome biogenesis, RNA stability, and translation. During early mitosis, nucleolin is required for equatorial mitotic chromosome alignment prior to metaphase. Using high resolution fluorescence imaging, we reveal that nucleolin is required for multiple centrosome-associated functions at the G2-prophase boundary. Nucleolin depletion led to dissociation of the centrosomes from the G2 nuclear envelope, a delay in the onset of nuclear envelope breakdown, reduced inter-centrosome separation, and longer metaphase spindles. Our results reveal novel roles for nucleolin in early mammalian mitosis, establishing multiple important functions for nucleolin during mammalian cell division., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mechanical force generation plays an essential role in many cellular functions, including mitosis. Actomyosin contractile forces mediate changes in cell shape in mitosis and are implicated in mitotic spindle integrity via cortical tension. An unbiased screen of 150 small molecules that impact actin organization and 32 anti-mitotic drugs identified two molecular targets, Rho kinase (ROCK) and tropomyosin 3.1/2 (Tpm3.1/2), whose inhibition has the greatest impact on mitotic cortical tension. The converse was found for compounds that depolymerize microtubules. Tpm3.1/2 forms a co-polymer with mitotic cortical actin filaments, and its inhibition prevents rescue of multipolar spindles induced by anti-microtubule chemotherapeutics. We examined the role of mitotic cortical tension in this rescue mechanism. Inhibition of ROCK and Tpm3.1/2 and knockdown (KD) of cortical nonmuscle myosin 2A (NM2A), all of which reduce cortical tension, inhibited rescue of multipolar mitotic spindles, further implicating cortical tension in the rescue mechanism. GEF-H1 released from microtubules by depolymerization increased cortical tension through the RhoA pathway, and its KD also inhibited rescue of multipolar mitotic spindles. We conclude that microtubule depolymerization by anti-cancer drugs induces cortical-tension-based rescue to ensure integrity of the mitotic bipolar spindle mediated via the RhoA pathway. Central to this mechanism is the dependence of NM2A on Tpm3.1/2 to produce the functional engagement of actin filaments responsible for cortical tension., Competing Interests: Declaration of interests P.W.G. and E.C.H. are directors and shareholders of TroBio Therapeutics Pty Ltd., a company that is commercializing anti-tropomyosin drugs for the treatment of cancer. Further, their labs receive funding from TroBio to evaluate anti-tropomyosin drug candidates., (Copyright © 2023 Elsevier Inc. All rights reserved.)

For the analysis of cellular architecture during mitosis, nanometer resolution is needed to visualize the organization of microtubules in spindles. Here, we present a detailed protocol that can be used to produce 3D reconstructions of whole mitotic spindles in cells grown in culture. For this, we attach mammalian cells enriched in mitotic stages to sapphire discs. Our protocol further involves cryo-immobilization by high-pressure freezing, freeze-substitution, and resin embedding. We then use fluorescence light microscopy to stage select mitotic cells in the resin-embedded samples. This is followed by large-scale electron tomography to reconstruct the selected and staged mitotic spindles in 3D. The generated and stitched electron tomograms are then used to semi-automatically segment the microtubules for subsequent quantitative analysis of spindle organization. Thus, by providing a detailed correlative light and electron microscopy (CLEM) approach, we give cell biologists a toolset to streamline the 3D visualization and analysis of spindle microtubules (http://kiewisz.shinyapps.io/asga). In addition, we refer to a recently launched platform that allows for an interactive display of the 3D-reconstructed mitotic spindles (https://cfci.shinyapps.io/ASGA_3DViewer/). Key features • High-throughput screening of mitotic cells by correlative light and electron microscopy (CLEM). • Serial-section electron tomography of selected cells. • Visualization of mitotic spindles in 3D and quantitative analysis of microtubule organization., Competing Interests: Competing interestsAll authors disclose no competing interests., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY license.)

Purpose: Abnormalities in the mitotic spindle have been linked to a variety of cancers. Data on their role in the onset, progression, and treatment of lung adenocarcinoma (LUAD) need to be explored., Methods: The data were retrieved from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Molecular Signatures Database (MSigDB), for the training cohort, external validation cohort, and the hallmark mitotic spindle gene set, respectively. Mitotic spindle genes linked to LUAD prognosis were identified and intersected with differentially expressed up-regulated genes in the training cohort. Nomogram prediction models were built based on least absolute shrinkage and selection operator (LASSO) regression, univariate cox, and multivariate cox analyses. The seven-gene immunological score was examined, as well as the correlation of immune checkpoints. The DLGAP5 and KIF15 expression in BEAS-2B, A549, H1299, H1975, and PC-9 cell lines was validated with western blot (WB)., Results: A total of 965 differentially expressed up-regulated genes in the training cohort intersected with 51 mitotic spindle genes associated with LUAD prognosis. Finally, the seven-gene risk score was determined and integrated with clinical characteristics to construct the nomogram model. Immune cell correlation analysis revealed a negative correlation between seven-gene expression with B cell, endothelial cell (excluding LMNB1), and T cell CD8 + (p < 0.05). However, the seven-gene expression was positively correlated with multiple immune checkpoints (p < 0.05). The expression of DLGAP5 and KIF15 were significantly higher in A549, H1299, H1975, and PC-9 cell lines than that in BEAS-2B cell line., Conclusion: High expression of the seven genes is positively correlated with poor prognosis of LUAD, and these genes are promising as prospective immunotherapy targets., (© 2023. The Author(s).)

AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis., (© 2023. The Author(s).)

Oncogenic Ras has been shown to change the way cancer cells divide by increasing the forces generated during mitotic rounding. In this way, Ras V12 enables cancer cells to divide across a wider range of mechanical environments than normal cells. Here, we identify a further role for oncogenic Ras-ERK signaling in division by showing that Ras V12 expression alters the shape, division orientation, and respreading dynamics of cells as they exit mitosis. Many of these effects appear to result from the impact of Ras V12 signaling on actomyosin contractility, because Ras V12 induces the severing of retraction fibers that normally guide spindle positioning and provide a memory of the interphase cell shape. In support of this idea, the Ras V12 phenotype is reversed by inhibition of actomyosin contractility and can be mimicked by the loss of cell-substrate adhesion during mitosis. Finally, we show that Ras V12 activation also perturbs division orientation in cells cultured in 2D epithelial monolayers and 3D spheroids. Thus, the induction of oncogenic Ras-ERK signaling leads to rapid changes in division orientation that, along with the effects of Ras V12 on cell growth and cell-cycle progression, are likely to disrupt epithelial tissue organization and contribute to cancer dissemination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Unlike normal cells, cancer cells frequently have multiple centrosomes that can cluster to form bipolar mitotic spindles and allow for successful cell division. Inhibiting centrosome clustering, therefore, holds therapeutic promise to promote cancer cell-specific cell death., Methods: We used confocal microscopy, real-time PCR, siRNA knockdown, and western blot to analyze centrosome clustering and declustering using normal lung bronchial epithelial and nonsmall-cell lung cancer (NSCLC) cell lines. Also, we used Ingenuity Pathway Analysis software to identify novel pathways associated with centrosome clustering., Results: In this study, we found that exposure to cigarette smoke condensate induces centrosome amplification and clustering in human lung epithelial cells. We observed a similar increase in centrosome amplification and clustering in unexposed NSCLC cell lines which may suggest a common underlying mechanism for lung carcinogenesis. We identified a cyclin D2-mediated centrosome clustering pathway that involves a sonic hedgehog-forkhead box protein M1 axis which is critical for mitosis. We also observed that cyclin D2 knockdown induced multipolar mitotic spindles that could eventually lead to cell death., Conclusions: Here we report a novel role of cyclin D2 in the regulation of centrosome clustering, which could allow the identification of tumors sensitive to cyclin D2 inhibitors. Our data reveal a pathway that can be targeted to inhibit centrosome clustering by interfering with the expression of cyclin D2-associated genes., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

γ-Tubulin ring complex (γ-TuRC), composed of γ-tubulin and multiple γ-tubulin complex proteins (GCPs), serves as the major microtubule nucleating complex in animal cells. However, several γ-TuRC-associated proteins have been shown to control its function. Centrosomal adaptor protein, TACC3, is one such γ-TuRC-interacting factor that is essential for proper mitotic spindle assembly across organisms. ch-TOG is another microtubule assembly promoting protein, which interacts with TACC3 and cooperates in mitotic spindle assembly. However, the mechanism how TACC3-ch-TOG interaction regulates microtubule assembly and the γ-TuRC functions at the centrosomes remain unclear. Here, we show that deletion of the ch-TOG-binding region in TACC3 enhances recruitment of the γ-TuRC proteins to centrosomes and aggravates spindle microtubule assembly in human cells. Loss of TACC3-ch-TOG binding imparts stabilization on TACC3 interaction with the γ-TuRC proteins and it does so by stimulating TACC3 phosphorylation and thereby enhancing phospho-TACC3 recruitment to the centrosomes. We also show that localization of ch-TOG at the centrosomes is substantially reduced and the same on the spindle microtubules is increased in its TACC3-unbound condition. Additional results reveal that ch-TOG depletion stimulates γ-tubulin localization on the spindles without significantly affecting the centrosomal γ-tubulin level. The results indicate that ch-TOG binding to TACC3 controls TACC3 phosphorylation and TACC3-mediated stabilization of the γ-TuRCs at the centrosomes. They also implicate that the spatio-temporal control of TACC3 phosphorylation via ch-TOG-binding ensures mitotic spindle assembly to the optimal level., (© 2023 The Author(s).)

Matrin3 is an RNA-binding protein that regulates diverse RNA-related processes, including mRNA splicing. Although Matrin3 has been intensively studied in neurodegenerative diseases, its function in cancer remains unclear. Here, we report Matrin3-mediated regulation of mitotic spindle dynamics in colorectal cancer (CRC) cells. We comprehensively identified RNAs bound and regulated by Matrin3 in CRC cells and focused on CDC14B, one of the top Matrin3 targets. Matrin3 knockdown results in increased inclusion of an exon containing a premature termination codon in the CDC14B transcript and simultaneous down-regulation of the standard CDC14B transcript. Knockdown of CDC14B phenocopies the defects in mitotic spindle dynamics upon Matrin3 knockdown, and the elongated and misoriented mitotic spindle observed upon Matrin3 knockdown are rescued upon overexpression of CDC14B, suggesting that CDC14B is a key downstream effector of Matrin3. Collectively, these data reveal a role for the Matrin3/CDC14B axis in control of mitotic spindle dynamics., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Cell division events require regulatory systems to ensure that events happen in a distinct order. The classic view of temporal control of the cell cycle posits that cells order events by linking them to changes in Cyclin Dependent Kinase (CDK) activities. However, a new paradigm is emerging from studies of anaphase where chromatids separate at the central metaphase plate and then move to opposite poles of the cell. These studies suggest that distinct events are ordered depending upon the location of each chromosome along its journey from the central metaphase plate to the elongated spindle poles. This system is dependent upon a gradient of Aurora B kinase activity that emerges during anaphase and acts as a spatial beacon to control numerous anaphase/telophase events and cytokinesis. Recent studies also suggest that Aurora A kinase activity specifies proximity of chromosomes or proteins to spindle poles during prometaphase. Together these studies argue that a key role for Aurora kinases is to provide spatial information that controls events depending upon the location of chromosomes or proteins along the mitotic spindle., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ali and Stukenberg.)

During mitosis, cells round up and utilize the interphase adhesion sites within the fibrous extracellular matrix (ECM) as guidance cues to orient the mitotic spindles. Here, using suspended ECM-mimicking nanofiber networks, we explore mitotic outcomes and error distribution for various interphase cell shapes. Elongated cells attached to single fibers through two focal adhesion clusters (FACs) at their extremities result in perfect spherical mitotic cell bodies that undergo significant 3-dimensional (3D) displacement while being held by retraction fibers (RFs). Increasing the number of parallel fibers increases FACs and retraction fiber-driven stability, leading to reduced 3D cell body movement, metaphase plate rotations, increased interkinetochore distances, and significantly faster division times. Interestingly, interphase kite shapes on a crosshatch pattern of four fibers undergo mitosis resembling single-fiber outcomes due to rounded bodies being primarily held in position by RFs from two perpendicular suspended fibers. We develop a cortex-astral microtubule analytical model to capture the retraction fiber dependence of the metaphase plate rotations. We observe that reduced orientational stability, on single fibers, results in increased monopolar mitotic defects, while multipolar defects become dominant as the number of adhered fibers increases. We use a stochastic Monte Carlo simulation of centrosome, chromosome, and membrane interactions to explain the relationship between the observed propensity of monopolar and multipolar defects and the geometry of RFs. Overall, we establish that while bipolar mitosis is robust in fibrous environments, the nature of division errors in fibrous microenvironments is governed by interphase cell shapes and adhesion geometries.

The fate of the two daughter cells is intimately connected to their positioning, which is in turn regulated by cell junction remodelling and orientation of the mitotic spindle. How multiple cues are integrated to dictate the ultimate positioning of daughters is not clear. Here, we identify novel mechanisms of regulation of daughter positioning in single MCF10A cells. The polarity protein, Scribble cooperates with E-cadherin for sequential roles in daughter positioning. First Scribble stabilises E-cadherin at the mitotic cortex as well as the retraction fibres, to mediate spindle orientation. Second, Scribble re-locates to the junction between the two daughters to allow a new E-cadherin-based-interface to form between them, influencing the width of the nascent daughter-daughter junction and subsequent cell positioning. Thus, E-cadherin and Scribble dynamically relocate to different intracellular sites during cell division to orient the mitotic spindle and control placement of the daughter cells after cell division. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Kinetochores are molecular machines that power chromosome segregation during the mitotic and meiotic cell divisions of all eukaryotes. Aristotle explains how we think we have knowledge of a thing only when we have grasped its cause. In our case, to gain understanding of the kinetochore, the four causes correspond to questions that we must ask: ( a ) What are the constituent parts, ( b ) how does it assemble, ( c ) what is the structure and arrangement, and ( d ) what is the function? Here we outline the current blueprint for the assembly of a kinetochore, how functions are mapped onto this architecture, and how this is shaped by the underlying pericentromeric chromatin. The view of the kinetochore that we present is possible because an almost complete parts list of the kinetochore is now available alongside recent advances using in vitro reconstitution, structural biology, and genomics. In many organisms, each kinetochore binds to multiple microtubules, and we propose a model for how this ensemble-level architecture is organized, drawing on key insights from the simple one microtubule-one kinetochore setup in budding yeast and innovations that enable meiotic chromosome segregation.

Nuclear import receptors ensure the recognition and transport of proteins across the nuclear envelope into the nucleus. In addition, as diverse processes as mitosis, post-translational modifications at mitotic exit, ciliogenesis, and phase separation, all share a common need for regulation by nuclear import receptors - particularly importin beta-1 and importin beta-2/transportin - independent on nuclear import. In particular, 1) nuclear import receptors regulate the mitotic spindle after nuclear envelope breakdown, 2) they shield cargoes from unscheduled ubiquitination, regulating their timely proteolysis; 3) they regulate ciliary factors, crucial to cell communications and tissue architecture during development; and 4) they prevent phase separation of toxic proteins aggregates in neurons. The balance of nuclear import receptors to cargoes is critical in all these processes, albeit in opposite directions: overexpression of import receptors, as often found in cancer, inhibits cargoes and impairs downstream processes, motivating the therapeutic design of specific inhibitors. On the contrary, elevated expression is beneficial in neuronal contexts, where nuclear import receptors are regarded as potential therapeutic tools in counteracting the formation of aggregates that may cause neurodegeneration. This paradox demonstrates the amplitude of nuclear import receptors-dependent functions in different contexts and adds complexity in considering their therapeutic implications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Damizia, Altieri and Lavia.)

Cytokinesis is required to physically cleave a cell into two daughters at the end of mitosis. Decades of research have led to a comprehensive understanding of the core cytokinesis machinery and how it is regulated in animal cells, however this knowledge was generated using single cells cultured in vitro , or in early embryos before tissues develop. This raises the question of how cytokinesis is regulated in diverse animal cell types and developmental contexts. Recent studies of distinct cell types in the same organism or in similar cell types from different organisms have revealed striking differences in how cytokinesis is regulated, which includes different threshold requirements for the structural components and the mechanisms that regulate them. In this review, we highlight these differences with an emphasis on pathways that are independent of the mitotic spindle, and operate through signals associated with the cortex, kinetochores, or chromatin., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ozugergin and Piekny.)

Spc110 is an essential component of the spindle pole body (SPB), the yeast equivalent of the centrosome, that recruits the γ-tubulin complex to the nuclear side of the SPB to produce the microtubules that form the mitotic spindle. Here, we identified phosphosites S11 and S36 in maternally originated Spc110 and explored their functions in vivo. Yeast expressing non-phosphorylatable Spc110S11A had a distinct spindle phenotype characterised by higher levels of α-tubulin, which was frequently asymmetrically distributed between the two SPBs. Furthermore, expression of the double mutant Spc110S11AS36A had a delayed cell cycle progression. Specifically, the final steps of mitosis were delayed in Spc110S11AS36A cells, including expression and degradation of the mitotic cyclin Clb2, disassembling the mitotic spindle and re-localizing Cdc14 to the nucleoli, resulting in late mitotic exit and entry in G1. Thus, we propose that Spc110 phosphorylation at S11 and S36 is required to regulate timely cell cycle progression in budding yeast. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Centrosomes are the main microtubule-organizing center of the cell. They are normally formed by two centrioles, embedded in a cloud of proteins known as pericentriolar material (PCM). The PCM ascribes centrioles with their microtubule nucleation capacity. Toxoplasma gondii, the causative agent of toxoplasmosis, divides by endodyogeny. Successful cell division is critical for pathogenesis. The centrosome, one of the microtubule organizing centers of the cell, plays central roles in orchestrating the temporal and physical coordination of major organelle segregation and daughter cell formation during endodyogeny. The Toxoplasma centrosome is constituted by multiple domains: an outer core, distal from the nucleus; a middle core; and an inner core, proximal to the nucleus. This modular organization has been proposed to underlie T. gondii's cell division plasticity. However, the role of the inner core remains undeciphered. Here, we focus on understanding the function of the inner core by finely studying the localization and role of its only known molecular marker; TgCep250L1. We show that upon conditional degradation of TgCep250L1 parasites are unable to survive. Mutants exhibit severe nuclear segregation defects. In addition, the rest of the centrosome, defined by the position of the centrioles, disconnects from the nucleus. We explore the structural defects underlying these phenotypes by ultrastructure expansion microscopy. We show that TgCep250L1's location changes with respect to other markers, and these changes encompass the formation of the mitotic spindle. Moreover, we show that in the absence of TgCep250L1, the microtubule binding protein TgEB1, fails to localize at the mitotic spindle, while unsegregated nuclei accumulate at the residual body. Overall, our data support a model in which the inner core of the T. gondii centrosome critically participates in cell division by directly impacting the formation or stability of the mitotic spindle. IMPORTANCE Toxoplasma gondii parasites cause toxoplasmosis, arguably the most widespread and prevalent parasitosis of humans and animals. During the clinically relevant stage of its life cycle, the parasites divide by endodyogeny. In this mode of division, the nucleus, containing loosely packed chromatin and a virtually intact nuclear envelope, parcels into two daughter cells generated within a common mother cell cytoplasm. The centrosome is a microtubule-organizing center critical for orchestrating the multiple simultaneously occurring events of endodyogeny. It is organized in two distinct domains: the outer and inner cores. We demonstrate here that the inner core protein TgCEP250L1 is required for replication of T. gondii. Lack of TgCEP250L1 renders parasites able to form daughter cells, while unable to segregate their nuclei. We determine that, in the absence of TgCEP250L1, the mitotic spindle, which is responsible for karyokinesis, does not assemble. Our results support a role for the inner core in nucleation or stabilization of the mitotic spindle in T. gondii.

The human mitotic spindle is made of microtubules nucleated at centrosomes, at kinetochores, and from pre-existing microtubules by the augmin complex. However, it is unknown how the augmin-mediated nucleation affects distinct microtubule classes and thereby mitotic fidelity. Here, we use superresolution microscopy to analyze the previously indistinguishable microtubule arrangements within the crowded metaphase plate area and demonstrate that augmin is vital for the formation of uniformly arranged parallel units consisting of sister kinetochore fibers connected by a bridging fiber. This ordered geometry helps both prevent and resolve merotelic attachments. Whereas augmin-nucleated bridging fibers prevent merotelic attachments by creating a nearly parallel and highly bundled microtubule arrangement unfavorable for creating additional attachments, augmin-nucleated k-fibers produce robust force required to resolve errors during anaphase. STED microscopy revealed that bridging fibers were impaired twice as much as k-fibers following augmin depletion. The complete absence of bridging fibers from a significant portion of kinetochore pairs, especially in the inner part of the spindle, resulted in the specific reduction of the interkinetochore distance. Taken together, we propose a model where augmin promotes mitotic fidelity by generating assemblies consisting of bridging and kinetochore fibers that align sister kinetochores to face opposite poles, thereby preventing erroneous attachments., Competing Interests: VŠ, IK, MM, JS, IT No competing interests declared, (© 2022, Štimac, Koprivec et al.)

For over 100-years, genomic instability has been investigated as a central player in the pathogenesis of human cancer. Conceptually, genomic instability includes an array of alterations from small deletions/insertions to whole chromosome alterations, referred to as chromosome instability. Chromosome instability has a paradoxical impact in cancer. In most instances, the introduction of chromosome instability has a negative impact on cellular fitness whereas in cancer it is usually associated with a worse prognosis. One exception is the case of neuroblastoma, the most common solid tumor outside of the brain in children. Neuroblastoma tumors have two distinct patterns of genome instability: whole- chromosome aneuploidy, which is associated with a better prognosis, or segmental chromosomal alterations, which is a potent negative prognostic factor. Through a computational screen, we found that low levels of the de- ubiquitinating enzyme USP24 have a highly significant negative impact on survival in neuroblastoma. At the molecular level, USP24 loss leads to destabilization of the microtubule assembly factor CRMP2 - producing mitotic errors and leading to chromosome missegregation and whole-chromosome aneuploidy. This apparent paradox may be reconciled through a model in which whole chromosome aneuploidy leads to the subsequent development of segmental chromosome alterations. Here we review the mechanisms behind chromosome instability and the evidence for the progressive development of segmental alterations from existing numerical aneuploidy in support of a multi-step model of neuroblastoma progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Paolini, Hussain and Galardy.)

Early cellular patterning is a critical step of embryonic development that determines the proper progression of morphogenesis in all metazoans. It relies on a series of rapid reductive divisions occurring simultaneously with the specification of the fate of different subsets of cells. Multiple species developmental strategies emerged in the form of a unique cleavage pattern with stereotyped division geometries. Cleavage geometries have long been associated to the emergence of canonical developmental features such as cell cycle asynchrony, zygotic genome activation and fate specification. Yet, the direct causal role of division positioning on blastomere cell behavior remain partially understood. Oriented and/or asymmetric divisions define blastomere cell sizes, contacts and positions, with potential immediate impact on cellular decisions, lineage specification and morphogenesis. Division positions also instruct daughter cells polarity, mechanics and geometries, thereby influencing subsequent division events, in an emergent interplay that may pattern early embryos independently of firm deterministic genetic programs. We here review the recent literature which helped to delineate mechanisms and functions of division positioning in early embryos., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Schwann cells (SCs) migrate along peripheral axons and divide intensively to generate the right number of cells prior to axonal ensheathment; however, little is known regarding the temporal and molecular control of their division and its impact on myelination. We report that Sil, a spindle pole protein associated with autosomal recessive primary microcephaly, is required for temporal mitotic exit of SCs. In sil-deficient cassiopeia (csp-/-) mutants, SCs fail to radially sort and myelinate peripheral axons. Elevation of cAMP, but not Rac1 activity, in csp-/- restores myelin ensheathment. Most importantly, we show a significant decrease in laminin expression within csp-/- posterior lateral line nerve and that forcing Laminin 2 expression in csp-/- fully restores the ability of SCs to myelinate. Thus, we demonstrate an essential role for timely SC division in mediating laminin expression to orchestrate radial sorting and peripheral myelination in vivo., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The journey from a single fertilised cell to a multicellular organism is, at the most fundamental level, orchestrated by mitotic cell divisions. Both the rate and the orientation of cell divisions are important in ensuring the proper development of an embryo. Simultaneous with cell proliferation, embryonic cells constantly experience a wide range of mechanical forces from their surrounding tissue environment. Cells must be able to read and respond correctly to these forces since they are known to affect a multitude of biological functions, including cell divisions. The interplay between the mechanical environment and cell divisions is particularly crucial during embryogenesis when tissues undergo dynamic changes in their shape, architecture, and overall organisation to generate functional tissues and organs. Here we review our current understanding of the cellular mechanisms by which mechanical force regulates cell division and place this knowledge within the context of embryogenesis and tissue morphogenesis.

Chromosome alignment at the spindle equator promotes proper chromosome segregation and depends on pulling forces exerted at kinetochore fiber tips together with polar ejection forces. However, kinetochore fibers are also subjected to forces driving their poleward flux. Here we introduce a flux-driven centering model that relies on flux generated by forces within the overlaps of bridging and kinetochore fibers. This centering mechanism works so that the longer kinetochore fiber fluxes faster than the shorter one, moving the kinetochores toward the center. We develop speckle microscopy in human spindles and confirm the key prediction that kinetochore fiber flux is length dependent. Kinetochores are better centered when overlaps are shorter and the kinetochore fiber flux slower than the bridging fiber flux. We identify Kif18A and Kif4A as overlap and flux regulators and NuMA as a fiber coupler. Thus, length-dependent sliding forces exerted by the bridging fiber onto kinetochore fibers support chromosome alignment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Microtubules tightly regulate various cellular activities. Our understanding of microtubules is largely based on experiments using microtubule-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific microtubule populations, due to their slow effects on the entire pool of microtubules. To overcome this technological limitation, we have used chemo and optogenetics to disassemble specific microtubule subtypes, including tyrosinated microtubules, primary cilia, mitotic spindles, and intercellular bridges, by rapidly recruiting engineered microtubule-cleaving enzymes onto target microtubules in a reversible manner. Using this approach, we show that acute microtubule disassembly swiftly halts vesicular trafficking and lysosomal dynamics. It also immediately triggers Golgi and ER reorganization and slows the fusion/fission of mitochondria without affecting mitochondrial membrane potential. In addition, cell rigidity is increased after microtubule disruption owing to increased contractile stress fibers. Microtubule disruption furthermore prevents cell division, but does not cause cell death during interphase. Overall, the reported tools facilitate detailed analysis of how microtubules precisely regulate cellular architecture and functions., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Unscheduled tetraploidy is a metastable state that rapidly evolves into aneuploidy. Recent findings reported by Gemble et al. demonstrate that freshly formed tetraploid cells fail to accumulate the required amounts of DNA replication factors during the first G 1 phase after whole-genome duplication (WGD), culminating in genetic instability in the subsequent S phase and extensive karyotypic alterations., Competing Interests: Declaration of interests L.G. has been holding research contracts with Lytix Biopharma and Phosplatin, has received consulting/advisory honoraria from Boehringer Ingelheim, AstraZeneca, OmniSEQ, Onxeo, The Longevity Labs, Inzen, Sotio, Phosplatin, Noxopharm, and the Luke Heller TECPR2 Foundation, and owns Phosplatin stock options. All other authors have no conflicts to declare., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Plant cells form acentrosomal spindles with microtubules (MTs) converged toward two structurally undefined poles by employing MT minus end-directed Kinesin-14 motors. To date, it is unclear whether the convergent bipolar MT array assumes unified poles in plant spindles, and if so, how such a goal is achieved. Among six classes of Kinesin-14 motors in Arabidopsis thaliana , the Kinesin-14A motors ATK1 (KatA) and ATK5 share the essential function in spindle morphogenesis. To understand how the two functionally redundant Kinesin-14A motors contributed to the spindle assembly, we had ATK1-GFP and ATK5-GFP fusion proteins expressed in their corresponding null mutants and found that they were functionally comparable to their native forms. Although ATK1 was a nuclear protein and ATK5 cytoplasmic prior to nuclear envelop breakdown, at later mitotic stages, the two motors shared similar localization patterns of uniform association with both spindle and phragmoplast MTs. We found that ATK1 and ATK5 were rapidly concentrated toward unified polar foci when cells were under hyperosmotic conditions. Concomitantly, spindle poles became perfectly focused as if there were centrosome-like MT-organizing centers where ATK1 and ATK5 were highly enriched and at which kinetochore fibers pointed. The separation of ATK1/ATK5-highlighted MTs from those of kinetochore fibers suggested that the motors translocated interpolar MTs. Our protein purification and live-cell imaging results showed that ATK1 and ATK5 are associated with each other in vivo . The stress-induced spindle pole convergence was also accompanied by poleward accumulation of the MT nucleator γ-tubulin. These results led to the conclusion that the two Kinesin-14A motors formed oligomeric motor complexes that drove MT translocation toward the spindle pole to establish acentrosomal spindles with convergent poles., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hotta, Lee, Higaki, Hashimoto and Liu.)

Red blood cells are produced by terminal erythroid differentiation, which involves the dramatic morphological transformation of erythroblasts into enucleated reticulocytes. Microtubules are important for enucleation, but it is not known if the centrosome, a key microtubule-organizing center, is required as well. Mice lacking the conserved centrosome component, CDK5RAP2, are likely to have defective erythroid differentiation because they develop macrocytic anemia. Here, we show that fetal liver-derived, CDK5RAP2-deficient erythroid progenitors generate fewer and larger reticulocytes, hence recapitulating features of macrocytic anemia. In erythroblasts, but not in embryonic fibroblasts, loss of CDK5RAP2 or pharmacological depletion of centrosomes leads to highly aberrant spindle morphologies. Consistent with such cells exiting mitosis without chromosome segregation, tetraploidy is frequent in late-stage erythroblasts, thereby giving rise to fewer but larger reticulocytes than normal. Our results define a critical role for CDK5RAP2 and centrosomes in spindle formation specifically during blood production. We propose that disruption of centrosome and spindle function could contribute to the emergence of macrocytic anemias, for instance, due to nutritional deficiency or exposure to chemotherapy., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Uveal melanoma (UM) is the most common intraocular cancer in adults. Whilst treatment of primary UM (PUM) is often successful, around 50% of patients develop metastatic disease with poor outcomes, linked to chromosome 3 loss (monosomy 3, M3). Advances in understanding UM cell biology may indicate new therapeutic options. We report that UM exhibits centrosome abnormalities, which in other cancers are associated with increased invasiveness and worse prognosis, but also represent a potential Achilles' heel for cancer-specific therapeutics. Analysis of 75 PUM patient samples revealed both higher centrosome numbers and an increase in centrosomes with enlarged pericentriolar matrix (PCM) compared to surrounding normal tissue, both indicative of centrosome amplification. The PCM phenotype was significantly associated with M3 (t-test, p < 0.01). Centrosomes naturally enlarge as cells approach mitosis; however, whilst UM with higher mitotic scores had enlarged PCM regardless of genetic status, the PCM phenotype remained significantly associated with M3 in UM with low mitotic scores (ANOVA, p = 0.021) suggesting that this is independent of proliferation. Phenotypic analysis of patient-derived cultures and established UM lines revealed comparable levels of centrosome amplification in PUM cells to archetypal triple-negative breast cancer cell lines, whilst metastatic UM (MUM) cell lines had even higher levels. Importantly, many UM cells also exhibit centrosome clustering, a common strategy employed by other cancer cells with centrosome amplification to survive cell division. As UM samples with M3 display centrosome abnormalities indicative of amplification, this phenotype may contribute to the development of MUM, suggesting that centrosome de-clustering drugs may provide a novel therapeutic approach., (© 2022 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

The chromokinesin KIF22 generates forces that contribute to mitotic chromosome congression and alignment. Mutations in the α2 helix of the motor domain of KIF22 have been identified in patients with abnormal skeletal development, and we report the identification of a patient with a novel mutation in the KIF22 tail. We demonstrate that pathogenic mutations do not result in a loss of KIF22's functions in early mitosis. Instead, mutations disrupt chromosome segregation in anaphase, resulting in reduced proliferation, abnormal daughter cell nuclear morphology, and, in a subset of cells, cytokinesis failure. This phenotype could be explained by a failure of KIF22 to inactivate in anaphase. Consistent with this model, constitutive activation of the motor via a known site of phosphoregulation in the tail phenocopied the effects of pathogenic mutations. These results suggest that the motor domain α2 helix may be an important site for regulation of KIF22 activity at the metaphase to anaphase transition. In support of this conclusion, mimicking phosphorylation of α2 helix residue T158 also prevents inactivation of KIF22 in anaphase. These findings demonstrate the importance of both the head and tail of the motor in regulating the activity of KIF22 and offer insight into the cellular consequences of preventing KIF22 inactivation and disrupting force balance in anaphase., Competing Interests: AT, PB, NA, SS, DB, JL, EK, JS No competing interests declared, (© 2022, Thompson et al.)

The assembly of a functional kinetochore on centromeric chromatin is necessary to connect chromosomes to the mitotic spindle, ensuring accurate chromosome segregation. This connecting function of the kinetochore presents multiple internal and external structural challenges. A microtubule interacting outer kinetochore and centromeric chromatin interacting inner kinetochore effectively confront forces from the external spindle and centromere, respectively. While internally, special inner kinetochore proteins, defined as "linkers," simultaneously interact with centromeric chromatin and the outer kinetochore to enable association with the mitotic spindle. With the ability to simultaneously interact with outer kinetochore components and centromeric chromatin, linker proteins such as centromere protein (CENP)-C or CENP-T in vertebrates and, additionally CENP-Q Okp1 -U Ame1 in yeasts, also perform the function of force propagation within the kinetochore. Recent efforts have revealed an array of linker pathways strategies to effectively recruit the largely conserved outer kinetochore. In this review, we examine these linkages used to propagate force and recruit the outer kinetochore across evolution. Further, we look at their known regulatory pathways and implications on kinetochore structural diversity and plasticity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sridhar and Fukagawa.)