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The 14-3-3 family of proteins are conserved across eukaryotes and serve myriad important regulatory functions in the cell. Homo- and hetero-dimers of these proteins mainly recognize their ligands via conserved motifs to modulate the localization and functions of those effector ligands. In most of the genetic backgrounds of Saccharomyces cerevisiae, disruption of both 14-3-3 homologs (Bmh1 and Bmh2) are either lethal or cells survive with severe growth defects, including gross chromosomal missegregation and prolonged cell cycle arrest. To elucidate their contributions to chromosome segregation, in this work, we investigated their centromere- and kinetochore-related functions of Bmh1 and Bmh2. Analysis of appropriate deletion mutants shows that Bmh isoforms have cumulative and non-shared isoform-specific contributions in maintaining the proper integrity of the kinetochore ensemble. Consequently, Bmh mutant cells exhibited perturbations in kinetochore–microtubule (KT–MT) dynamics, characterized by kinetochore declustering, mis-localization of kinetochore proteins and Mad2-mediated transient G2/M arrest. These defects also caused an asynchronous chromosome congression in bmh mutants during metaphase. In summary, this report advances the knowledge on contributions of budding yeast 14-3-3 proteins in chromosome segregation by demonstrating their roles in kinetochore integrity and chromosome congression. [ABSTRACT FROM AUTHOR]

Mitosis is a crucial stage in the cell cycle, controlled by a vast network of regulators responding to multiple internal and external factors. The fission yeast Schizosaccharomyces pombe demonstrates catastrophic mitotic phenotypes due to mutations or drug treatments. One of the factors provoking catastrophic mitosis is a disturbed lipid metabolism, resulting from, for example, mutations in the acetyl-CoA/biotin carboxylase (cut6), fatty acid synthase (fas2, also known as lsd1) or transcriptional regulator of lipid metabolism (cbf11) genes, as well as treatment with inhibitors of fatty acid synthesis. It has been previously shown that mitotic fidelity in lipid metabolism mutants can be partially rescued by ammonium chloride supplementation. In this study, we demonstrate that mitotic fidelity can be improved by multiple nitrogen sources. Moreover, this improvement is not limited to lipid metabolism disturbances but also applies to a number of unrelated mitotic mutants. Interestingly, the partial rescue is not achieved by restoring the lipid metabolism state, but rather indirectly. Our results highlight a novel role for nitrogen availability in mitotic fidelity. [ABSTRACT FROM AUTHOR]

Division plane positioning is crucial for proper growth and development in many organisms. In plants, the division plane is established before mitosis, by accumulation of a cytoskeletal structure called the preprophase band (PPB). The PPB is thought to be essential for recruitment of division site-localized proteins, which remain at the division site after the PPB disassembles. Here, we show that the division site-localized protein TANGLED1 (TAN1) is recruited independently of the PPB to the cell cortex by the plant cytokinetic machinery, the phragmoplast, from experiments using both the PPBdefectivemutant discordia1 (dcd1) and chemical treatments that disrupt the phragmoplast in maize. TAN1 recruitment to de novo sites on the cortex is partially dependent on intact actin filaments and themyosin XI motor protein OPAQUE1 (O1). These data imply a yet unknown role for TAN1 and possibly other division site-localized proteins during the last stages of cell division when the phragmoplast touches the cell cortex to complete cytokinesis. [ABSTRACT FROM AUTHOR]

Dinoflagellates are marine organisms that undergo seasonal proliferation events known as algal blooms. Vegetative cell proliferation is a main contributing factor in these events. However, mechanistical understanding of mitosis and cytokinesis in dinoflagellates remains rudimentary. Using an optimized immunofluorescence protocol, we analysed changes in microtubule organization occurring during the mitotic cycle of the toxic dinoflagellate Ostreopsis cf. ovata. We find that the flagella and the cortical microtubule array persist throughout the mitotic cycle. Two cytoplasmic microtubule bundles originate from the ventral area, where the basal bodies are located - a cortical bundle and a cytoplasmic bundle. The latter associates with the nucleus in the cell centre before mitosis and with the acentrosomal extranuclear spindle during mitosis. Analysis of tubulin post-translational modifications identifies two populations of spindle microtubules - polar acetylated microtubules, whose length is constant, and central tyrosinated microtubules, which elongate during chromosome segregation. During cell division a microtubule-rich structure forms along the dorsal-ventral axis, associated with the site of cytokinesis, consistent with a cytokinetic mechanism that is independent of the actomyosin ring typical of animal and yeast cells. [ABSTRACT FROM AUTHOR]

In this article we reflect on the life and work of Andrew Fry, a renowned molecular cell biologist and a cherished member of the scientific community at the University of Leicester, UK, who passed away on 30th April 2024 at the age of 57. His groundbreaking work on the cellular mechanisms of Never in Mitosis gene-A related kinases (Neks) made an indelible mark on the field. Alongside his scientific achievements, Andrew was an exceptional mentor, a thoughtful academic leader and a dependable collaborator. To understand what motivated Andrew, we first need to look into his background. [ABSTRACT FROM AUTHOR]

DPF3, along with other subunits, is a well-known component of the BAF chromatin remodeling complex, which plays a key role in regulating chromatin remodeling activity and gene expression. Here, we elucidated a non-canonical localization and role for DPF3. We showed that DPF3 dynamically localizes to the centriolar satellites in interphase and to the centrosome, spindle midzone and bridging fiber area, and midbodies during mitosis. Loss of DPF3 causes kinetochore fiber instability, unstable kinetochore-microtubule attachment and defects in chromosome alignment, resulting in altered mitotic progression, cell death and genomic instability. In addition, we also demonstrated that DPF3 localizes to centriolar satellites at the base of primary cilia and is required for ciliogenesis by regulating axoneme extension. Taken together, these findings uncover a moonlighting dual function for DPF3 during mitosis and ciliogenesis. [ABSTRACT FROM AUTHOR]

The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprising a large vesicle (bladder) linked with a tubular reticulum (spongiome). CVCs provide key osmoregulatory roles across diverse eukaryotic lineages, but probing the mechanisms underlying their structure and function is hampered by the limited tools available for in vivo analysis. In the experimentally tractable ciliate Tetrahymena thermophila, we describe four proteins that, as endogenously tagged constructs, localize specifically to distinct CVC zones. The DOPEY homolog Dop1p and the CORVET subunit Vps8Dp localize both to the bladder and spongiome but with different local distributions that are sensitive to osmotic perturbation, whereas the lipid scramblase Scr7p colocalizes with Vps8Dp. The H+-ATPase subunit Vma4 is spongiome specific. The live imaging permitted by these probes revealed dynamics at multiple scales including rapid exchange of CVC-localized and soluble protein pools versus lateral diffusion in the spongiome, spongiome extension and branching, and CVC formation during mitosis. Although the association with DOP1 and VPS8D implicate the CVC in endosomal trafficking, both the bladder and spongiome might be isolated from bulk endocytic input. [ABSTRACT FROM AUTHOR]

Sister chromatid cohesion is a multi-step process implemented throughout the cell cycle to ensure the correct transmission of chromosomes to daughter cells. Although cohesion establishment and mitotic cohesion dissolution have been extensively explored, the regulation of cohesin loading is still poorly understood. Here, we report that the methyltransferase NSD3 is essential for mitotic sister chromatid cohesion before mitosis entry. NSD3 interacts with the cohesin loader complex kollerin (composed of NIPBL and MAU2) and promotes the chromatin recruitment of MAU2 and cohesin at mitotic exit. We also show that NSD3 associates with chromatin in early anaphase, prior to the recruitment of MAU2 and RAD21, and dissociates from chromatin when prophase begins. Among the two NSD3 isoforms present in somatic cells, the long isoform is responsible for regulating kollerin and cohesin chromatin-loading, and its methyltransferase activity is required for efficient sister chromatid cohesion. Based on these observations, we propose that NSD3-dependent methylation contributes to sister chromatid cohesion by ensuring proper kollerin recruitment and thus cohesin loading. [ABSTRACT FROM AUTHOR]

Transient changes in intracellular pH (pHi) regulate normal cell behaviors, but roles for spatiotemporal pHi dynamics in single-cell behaviors remain unclear. Here, we mapped single-cell spatiotemporal pHi dynamics during mammalian cell cycle progression both with and without cell cycle synchronization. We found that single-cell pHi is dynamic throughout the cell cycle: pHi decreases at G1/S, increases in mid-S, decreases at late S, increases at G2/M and rapidly decreases during mitosis. Importantly, although pHi is highly dynamic in dividing cells, non-dividing cells have attenuated pHi dynamics. Using two independent pHi manipulation methods, we found that low pHi inhibits completion of S phase whereas high pHi promotes both S/G2 and G2/M transitions. Our data also suggest that low pHi cues G1 exit, with decreased pHi shortening G1 and increased pHi elongating G1. Furthermore, dynamic pHi is required for S phase timing, as high pHi elongates S phase and low pHi inhibits S/G2 transition. This work reveals that spatiotemporal pHi dynamics are necessary for cell cycle progression at multiple phase transitions in single human cells. [ABSTRACT FROM AUTHOR]

Finely tuned regulation of transport protein localization is vital for epithelial function. The Na+-HCO3- co-transporter NBCn1 (also known as SLC4A7) is a key contributor to epithelial pH homeostasis, yet the regulation of its subcellular localization is not understood. Here, we show that a predicted N-terminal β-sheet and short C-terminal α-helical motif are essential for NBCn1 plasma membrane localization in epithelial cells. This localization was abolished by cell-cell contact disruption, and co-immunoprecipitation (co-IP) and proximity ligation (PLA) revealed NBCn1 interaction with E-cadherin and DLG1, linking it to adherens junctions and the Scribble complex. NBCn1 also interacted with RhoA and localized to lamellipodia and filopodia in migrating cells. Finally, analysis of native and GFP-tagged NBCn1 localization, subcellular fractionation, co-IP with Arl13B and CEP164, and PLA of NBCn1 and tubulin in mitotic spindles led to the surprising conclusion that NBCn1 additionally localizes to centrosomes and primary cilia in non-dividing, polarized epithelial cells, and to the spindle, centrosomes and midbodies during mitosis. We propose that NBCn1 traffics between lateral junctions, the leading edge and cell division machinery in Rab11 endosomes, adding new insight to the role of NBCn1 in cell cycle progression. [ABSTRACT FROM AUTHOR]

Cell division requires dramatic reorganization of the cell cortex, which is primarily driven by the actomyosin network. We previously reported that protocadherin 7 (PCDH7) gets enriched at the cell surface during mitosis, which is required to build up the full mitotic rounding pressure. Here, we report that PCDH7 interacts with and is palmitoylated by the palmitoyltransferase, ZDHHC5. PCDH7 and ZDHHC5 colocalize at the mitotic cell surface and translocate to the cleavage furrow during cytokinesis. The localization of PCDH7 depends on the palmitoylation activity of ZDHHC5. Silencing PCDH7 increases the percentage of multinucleated cells and the duration of mitosis. Loss of PCDH7 expression correlates with reduced levels of active RhoA and phospho-myosin at the cleavage furrow. This work uncovers a palmitoylation-dependent translocation mechanism for PCDH7, which contributes to the reorganization of the cortical cytoskeleton during cell division. [ABSTRACT FROM AUTHOR]

Mitotic cell division requires that kinetochores form microtubule attachments that can segregate chromosomes and control mitotic progression via the spindle assembly checkpoint. During prometaphase, kinetochores shed a domain called the fibrous corona as microtubule attachments form. This shedding is mediated, in part, by the minus-end directed motor dynein, which 'strips' cargoes along K-fibre microtubules. Despite its essentiality, little is known about how dynein stripping is regulated and how it responds to attachment maturation. Lis1 (also known as PAFAH1B1) is a conserved dynein regulator that is mutated in the neurodevelopmental disease lissencephaly. Here, we have combined loss-of-function studies, high-resolution imaging and separation-of-function mutants to define how Lis1 contributes to dynein-mediated corona stripping in HeLa cells. Cells depleted of Lis1 fail to disassemble the corona and showa delay in metaphase as a result of persistent checkpoint activation. Furthermore, we find that although kinetochore-tethered Lis1-dynein is required for error-free microtubule attachment, the contribution of Lis1 to corona disassembly can be mediated by a cytoplasmic pool. These findings support the idea that Lis1 drives dynein function at kinetochores to ensure corona disassembly and prevent chromosome mis-segregation. [ABSTRACT FROM AUTHOR]

Mitosis is a fundamental and highly regulated process that acts to faithfully segregate chromosomes into two identical daughter cells. Localization of gene transcripts involved in mitosis to the mitotic spindle might be an evolutionarily conserved mechanism to ensure that mitosis occurs in a timely manner. We identified many RNA transcripts that encode proteins involved in mitosis localized at the mitotic spindles in dividing sea urchin embryos and mammalian cells. Disruption of microtubule polymerization, kinesin-1 or dynein results in lack of spindle localization of these transcripts in the sea urchin embryo. Furthermore, results indicate that the cytoplasmic polyadenylation element (CPE) within the 3'UTR of the Aurora B transcript, a recognition sequence for CPEB, is essential for RNA localization to the mitotic spindle in the sea urchin embryo. Blocking this sequence results in arrested development during early cleavage stages, suggesting that RNA localization to the mitotic spindle might be a regulatory mechanism of cell division that is important for early development. [ABSTRACT FROM AUTHOR]

Kinesin-5 motors are essential to separate mitotic spindle poles and assemble a bipolar spindle in many organisms. These motors crosslink and slide apart antiparallel microtubules via microtubule plus-end-directed motility. However, kinesin-5 localization is enhanced away from antiparallel overlaps. Increasing evidence suggests this localization occurs due to bidirectional motility or trafficking. The purified fission-yeast kinesin-5 protein Cut7 moves bidirectionally, but bidirectionality has not been shown in cells, and the function of the minus-end-directed movement is unknown. Here, we characterized the motility of Cut7 on bipolar and monopolar spindles and observed movement toward both plus- and minus-ends of microtubules. Notably, the activity of the motor increased at anaphase B onset. Perturbations to microtubule dynamics only modestly changed Cut7 movement, whereas Cut7 mutation reduced movement. These results suggest that the directed motility of Cut7 contributes to the movement of the motor. Comparison of the Cut7 mutant and human Eg5 (also known as KIF11) localization suggest a new hypothesis for the function of minus-end-directed motility and spindle-pole localization of kinesin-5s. [ABSTRACT FROM AUTHOR]

The precise regulation of microtubule length during mitosis is essential to assemble and position the mitotic spindle and segregate chromosomes. The kinesin-13 Kif2C or MCAK acts as a potent microtubule depolymerase that diffuses short distances on microtubules, whereas the kinesin-8 Kif18b is a processive motor with weak depolymerase activity. However, the individual activities of these factors cannot explain the dramatic increase in microtubule dynamics in mitosis. Using in vitro reconstitution and single-molecule imaging, we demonstrate that Kif18b, MCAK and the plus-end tracking protein EB3 (also known as MAPRE3) act in an integrated manner to potently promote microtubule depolymerization at very low concentrations. We find that Kif18b can transport EB3 and MCAK and promotes their accumulation to microtubule plus ends through multivalent weak interactions. Together, our work defines the mechanistic basis for a cooperative Kif18b-MCAK-EB network at microtubule plus ends, that acts to efficiently shorten and regulate microtubules in mitosis, essential for correct chromosome segregation. [ABSTRACT FROM AUTHOR]

Nuclear envelope (NE) expansion must be controlled to maintain nuclear shape and function. The nuclear membrane expands massively during closed mitosis, enabling chromosome segregation within an intact NE. Phosphatidic acid (PA) and diacylglycerol (DG) can both serve as biosynthetic precursors for membrane lipid synthesis. How they are regulated in time and space and what the implications are of changes in their flux for mitotic fidelity are largely unknown. Using genetically encoded PA andDGprobes, we show that DG is depleted from the inner nuclear membrane during mitosis in the fission yeast Schizosaccharomyces pombe, but PA does not accumulate, indicating that it is rerouted to membrane synthesis. We demonstrate that DG-to-PA conversion catalyzed by the diacylglycerol kinase Dgk1 (also known as Ptp4) and direct glycerophospholipid synthesis from DG by diacylglycerol cholinephosphotransferase/ethanolaminephosphotransferase Ept1 reinforce NE expansion. We conclude that DG consumption through both the de novo pathway and the Kennedy pathway fuels a spike in glycerophospholipid biosynthesis, controlling NE expansion and, ultimately, mitotic fidelity. [ABSTRACT FROM AUTHOR]

Cancer cells have heterogeneous fitness, and this heterogeneity stems from genetic and epigenetic sources. Here, we sought to assess the contribution of asymmetric mitosis (AM) and time on the variability of fitness in sister cells. Around one quarter of sisters had differences in fitness, assessed as the intermitotic time (IMT), from 330 to 510 min. Phenotypes related to fitness, such as ERK activity (herein referring to ERK1 and ERK2, also known as MAPK3 and MAPK1, respectively), DNA damage and nuclear morphological phenotypes were also asymmetric at mitosis or turned asymmetric over the course of the cell cycle. The ERK activity of mother cell was found to influence the ERK activity and the IMT of the daughter cells, and cells with ERK asymmetry at mitosis produced more offspring with AMs, suggesting heritability of the AM phenotype for ERK activity. Our findings demonstrate how variabilities in sister cells can be generated, contributing to the phenotype heterogeneities in tumor cells. [ABSTRACT FROM AUTHOR]

53BP1 (also known as TP53BP1) is a key mediator of the nonhomologous end joining (NHEJ) DNA repair pathway, which is the primary repair pathway in interphase cells. However, the mitotic functions of 53BP1 are less well understood. Here, we describe 53BP1 mitotic stress bodies (MSBs) formed in cancer cell lines in response to delayed mitosis. These bodies displayed liquid--liquid phase separation characteristics, were close to centromeres, and included lamin A/C and the DNA repair protein RIF1. After release from mitotic arrest, 53BP1 MSBs decreased in number and moved away from the chromatin. Using GFP fusion constructs, we found that the 53BP1 oligomerization domain region was required for MSB formation, and that inclusion of the 53BP1 N terminus increased MSB size. Exogenous expression of 53BP1 did not increase MSB size or number but did increase levels of MSB-free 53BP1. This was associated with slower mitotic progression, elevated levels of DNA damage and increased apoptosis, which is consistent with MSBs suppressing a mitotic surveillance by 53BP1 through sequestration. The 53BP1 MSBs, which were also found spontaneously in a subset of normally dividing cancer cells but not in non-transformed cells (ARPE-19), might facilitate the survival of cancer cells following aberrant mitoses. [ABSTRACT FROM AUTHOR]

Cell-division-plane orientation is critical for plant and animal development and growth. TANGLED1 (TAN1) and AUXININDUCED IN ROOT CULTURES 9 (AIR9) are division-sitelocalized microtubule-binding proteins required for division-plane positioning. The single mutants tan1 and air9 of Arabidopsis thaliana have minor or no noticeable phenotypes, but the tan1 air9 double mutant has synthetic phenotypes including stunted growth, misoriented divisions and aberrant cell-file rotation in the root differentiation zone. These data suggest that TAN1 plays a role in non-dividing cells. To determine whether TAN1 is required in elongating and differentiating cells in the tan1 air9 double mutant, we limited its expression to actively dividing cells using the G2/Mspecific promoter of the syntaxin KNOLLE (pKN:TAN1-YFP). Unexpectedly, in addition to rescuing division-plane defects, expression of pKN:TAN1-YFP rescued root growth and cell file rotation defects in the root-differentiation zone in tan1 air9 double mutants. This suggests that defects that occur in the meristematic zone later affect the organization of elongating and differentiating cells. [ABSTRACT FROM AUTHOR]

Dephosphorylation of lamin A, which triggers nuclear lamina reconstitution, is crucial for the completion of mitosis. However, the specific phosphatase and regulatorymechanismthat allowtimely lamin A dephosphorylation remain unclear. Here, we report that RepoMan (also known as CDCA2), a regulatory subunit of protein phosphatase 1γ (PP1γ) is transiently modified with SUMO-2 at K762 during late telophase. SUMOylation of RepoMan markedly enhanced its binding affinity with lamin A. Moreover, SUMOylated RepoMan contributes to lamin A recruitment to telophase chromosomes and dephosphorylation of themitotic lamin A phosphorylation. Expression of a SUMO-2mutant that has a defective interaction with the SUMO-interacting motif (SIM) resulted in failure of the lamin A and RepoMan association, along with abrogation of lamin A dephosphorylation and subsequent nuclear lamina formation. These findings strongly suggest that RepoMan recruits lamin A through SUMO-SIM interaction. Thus, transient SUMOylation of RepoMan plays an important role in the spatiotemporal regulation of lamin A dephosphorylation and the subsequent nuclear lamina formation at the end of mitosis. [ABSTRACT FROM AUTHOR]

Polo-like kinase 1 (PLK-1) is present in centrosomes, the nuclear envelope, and kinetochores and plays a significant role in meiosis and mitosis. PLK-1 depletion or inhibition has severe consequences for spindle assembly, spindle assembly checkpoint (SAC) activation, chromosome segregation, and cytokinesis. BUB-1 targets PLK-1 to the outer kinetochore and, in mammals, the inner kinetochore PLK1 targeting is mediated by the constitutive centromere associated network (CCAN). BUB1-targeted PLK-1 plays a key role in SAC activation and a SAC-independent role through targeting CDC-20. In contrast, whether there is a specific, non-redundant role for inner kinetochore targeted PLK-1 is unknown. Here, we used the C. elegans embryo to study the role of inner kinetochore PLK-1. We found that CENP-C, the sole CCAN component in C. elegans and other species, targets PLK-1 to the inner kinetochore during prometaphase and metaphase. Disruption of the CENP-C/PLK-1 interaction leads to an imbalance in kinetochore components and a defect in chromosome congression, without affecting CDC-20 recruitment. These findings indicate that PLK-1 kinetochore recruitment by CENP-C has at least partially distinct functions than outer kinetochore PLK-1, providing a platform for a better understanding of the different roles played by PLK-1 during mitosis., (© 2024. Published by The Company of Biologists Ltd.)

Chromosome segregation errors caused by centromere malfunction can lead to chromosome instability and aneuploidy. In Caenorhabditis elegans, the Argonaute protein CSR-1 is essential for proper chromosome segregation, although the specific mechanisms are not fully understood. Here, we investigated how CSR-1 regulates centromere and kinetochore function in C. elegans embryos. We found that depletion of CSR-1 results in defects in mitotic progression and chromosome positioning relative to the spindle pole. Knockdown of CSR-1 does not affect mRNA and protein levels of the centromeric histone H3 variant and CENP-A homolog HCP-3 but does increase the localization of HCP-3 and some kinetochore proteins to the mitotic chromosomes. Such elevation of HCP-3 chromatin localization depends on EGO-1, which is an upstream factor in the CSR-1 RNA interference (RNAi) pathway, and PIWI domain activity of CSR-1. Our results suggest that CSR-1 restricts the level of HCP-3 at the holocentromeres, prevents erroneous kinetochore assembly and thereby promotes accurate chromosome segregation. Our work sheds light on the role of CSR-1 in regulating deposition of HCP-3 on chromatin and centromere function in embryos., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The eukaryotic cell cycle is driven by the activity of cyclin-dependent kinases (CDKs). CDK activity rises over 50-fold during the cell cycle, from a low level in G1 to a high level in mitosis. However, it is not known whether the entire range of CDK activity is necessary for cell cycle progression, or whether cells can tolerate a reduction in CDK activity level. Here, in fission yeast, we show that sublethal CDK inhibition lengthens the time cells spend in mitosis but does not cause misordering of mitotic events. Maximum attainable CDK activity exceeds the amount necessary for mitosis, and thus forms a CDK activity buffer between sufficient and maximal possible CDK activities. This CDK activity buffer is needed for mitotic completion when CDK activity is compromised, and CDK inhibition only becomes lethal to cells when this buffer is exhausted. Finally, we explore what factors influence this CDK activity buffer, and find that it is influenced by CDK-counteracting phosphatases. Therefore, maximum attainable CDK activity is not necessary for mitosis but provides robustness to CDK activity reduction to ensure mitotic completion. [ABSTRACT FROM AUTHOR]

Mitotic kinesin-like protein 2 (MKLP2; also known as KIF20A) is a motor protein with a well-established function in promoting cytokinesis. However, our results with siRNAs targeting MKLP2 and small-molecule inhibitors of MKLP2 (MKLP2i) suggest that it also has a function earlier in mitosis, prior to anaphase. In this study, we provide direct evidence that MKLP2 facilitates chromosome congression in prometaphase. We employed live imaging to observe HeLa cells with fluorescently tagged histones treated with MKLP2i and discovered a pronounced chromosome congression defect. We show that MKLP2 facilitates error correction, as inhibited cells have a significant increase in unstable, syntelic kinetochore–microtubule attachments. We find that the aberrant attachments are accompanied by elevated Aurora kinase (A and B) activity and phosphorylation of the downstream target HEC1 (also known as NDC80) at Ser55. Finally, we show that MKLP2 inhibition results in aneuploidy, confirming that MKLP2 safeguards cells against chromosomal instability. [ABSTRACT FROM AUTHOR]

Lysosomes exert pleiotropic functions to maintain cellular homeostasis and degrade autophagy cargo. Despite the great advances that have boosted our understanding of autophagy and lysosomes in both physiology and pathology, their function in mitosis is still controversial. During mitosis, most organelles are reshaped or repurposed to allow the correct distribution of chromosomes. Mitotic entry is accompanied by a reduction in sites of autophagy initiation, supporting the idea of an inhibition of autophagy to protect the genetic material against harmful degradation. However, there is accumulating evidence revealing the requirement of selective autophagy and functional lysosomes for a faithful chromosome segregation. Degradation is the most-studied lysosomal activity, but recently described alternative functions that operate in mitosis highlight the lysosomes as guardians of mitotic progression. Because the involvement of autophagy in mitosis remains controversial, it is important to consider the specific contribution of signalling cascades, the functions of autophagic proteins and the multiple roles of lysosomes, as three entangled, but independent, factors controlling genomic stability. In this Review, we discuss the latest advances in this area and highlight the therapeutic potential of targeting autophagy for drug development. [ABSTRACT FROM AUTHOR]

Proper mitotic spindle orientation depends on the correct anchorage of astral microtubules to the cortex. It relies on the remodeling of the cell cortex, a process not fully understood. Annexin A2 (Anx2; also known as ANXA2) is a protein known to be involved in cortical domain remodeling. Here, we report that in HeLa cell early mitosis, Anx2 recruits the scaffold protein Ahnak at the cell cortex facing spindle poles, and the distribution of both proteins is controlled by cell adhesion. Depletion of either protein or impaired cortical Ahnak localization result in delayed anaphase onset and unstable spindle anchoring, which leads to altered spindle orientation. We find that Ahnak is present in a complex with dynein-dynactin. Furthermore, Ahnak and Anx2 are required for correct dynein and NuMA (also known as NUMA1) cortical localization and dynamics. We propose that the Ahnak-Anx2 complex influences the cortical organization of the astral microtubule-anchoring complex, and thereby mitotic spindle positioning in human cells. [ABSTRACT FROM AUTHOR]

Centrioles are central structural elements of centrosomes and cilia. In human cells, daughter centrioles are assembled adjacent to existing centrioles in S-phase and reach their full functionality with the formation of distal and subdistal appendages one-and-a-half cell cycles later, as they exit their second mitosis. Current models postulate that the centriolar protein centrobin acts as placeholder for distal appendage proteins that must be removed to complete distal appendage formation. Here, we investigated, in non-transformed human epithelial RPE1 cells, the mechanisms controlling centrobin removal and its effect on distal appendage formation. Our data are consistent with a speculative model in which centrobin is removed from older centrioles due to a higher affinity for the newly born daughter centrioles, under the control of the centrosomal kinase PLK1. This removal also depends on the presence of subdistal appendage proteins on the oldest centriole. Removing centrobin, however, is not required for the recruitment of distal appendage proteins, even though this process is equally dependent on PLK1. We conclude that PLK1 kinase regulates centrobin removal and distal appendage formation during centriole maturation via separate pathways. [ABSTRACT FROM AUTHOR]

The centromere serves as the binding site for the kinetochore and is essential for the faithful segregation of chromosomes throughout cell division. The point centromere in yeast is encoded by a ~115 bp specific DNA sequence, whereas regional centromeres range from 6-10 kbp in fission yeast to 5-10 Mbp in humans. Understanding the physical structure of centromere chromatin (pericentromere in yeast), defined as the chromatin between sister kinetochores, will provide fundamental insights into how centromere DNA is woven into a stiff spring that is able to resist microtubule pulling forces during mitosis. One hallmark of the pericentromere is the enrichment of the structural maintenance of chromosome (SMC) proteins cohesin and condensin. Based on studies from population approaches (ChIP-seq and Hi-C) and experimentally obtained images of fluorescent probes of pericentromeric structure, as well as quantitative comparisons between simulations and experimental results, we suggest a mechanism for building tension between sister kinetochores. We propose that the centromere is a chromatin bottlebrush that is organized by the loop-extruding proteins condensin and cohesin. The bottlebrush arrangement provides a biophysical means to transform pericentromeric chromatin into a spring due to the steric repulsion between radial loops. We argue that the bottlebrush is an organizing principle for chromosome organization that has emerged from multiple approaches in the field. [ABSTRACT FROM AUTHOR]

PCTAIRE1 (also known as CDK16) is a serine-threonine kinase implicated in physiological processes like neuronal development, vesicle trafficking, spermatogenesis and cell proliferation. However, its exact role in cell division remains unclear. In this study, using a library screening approach, we identified PCTAIRE1 among several candidates that resisted mitotic arrest and mitotic cell death induced by polyomavirus small T (PolST) expression in mammalian cells. Our study showed that PCTAIRE1 is a mitotic kinase that localizes at centrosomes during G2 and at spindle poles as the cells enter mitosis, and then at the midbody during cytokinesis. We also report that PCTAIRE1 protein levels fluctuate through the cell cycle and reach their peak at mitosis, during which there is an increase in PCTAIRE1 phosphorylation as well. Interestingly, knockdown of PCTAIRE1 resulted in aberrant mitosis by interfering with spindle assembly and chromosome segregation. Further, we found that PCTAIRE1 promotes resistance of cancer cells to antimitotic drugs, and this underscores the significance of PCTAIRE1 as a potential drug target for overcoming chemotherapeutic resistance. Taken together, these studies establish PCTAIRE1 as a critical mediator of mitotic progression and highlight its role in chemotherapeutic resistance. This article has an associated First Person interview with the first author of the paper. [ABSTRACT FROM AUTHOR]

The microtubules of the mitotic spindle mediate chromosome alignment to the metaphase plate, then sister chromatid segregation to the spindle poles in anaphase. Previous analyses of spindle microtubule kinetics utilizing fluorescence dissipation after photoactivation described two main populations, a slow and a fast turnover population, and these were ascribed as reflecting kinetochore versus non-kinetochore microtubules, respectively. Here, we test this categorization by disrupting kinetochores through depletion of the Ndc80 complex in U2OS cells. In the absence of functional kinetochores, microtubule dynamics still exhibit slow and fast turnover populations, although the proportion of each population and the timings of turnover are altered. Importantly, the data obtained following Hec1 (also known as Ndc80) depletion suggests that other subpopulations, in addition to kinetochore microtubules, contribute to the slow turnover population. Further manipulation of spindle microtubules revealed a complex landscape. For example, although Aurora B kinase functions to destabilize kinetochore bound microtubules it might also stabilize certain slow turnover nonkinetochore microtubules. Dissection of the dynamics of microtubule populations provides a greater understanding of mitotic spindle kinetics and insight into their roles in facilitating chromosome attachment, movement and segregation during mitosis. [ABSTRACT FROM AUTHOR]

The control of protein abundance is a fundamental regulatory mechanism during mitosis. The anaphase-promoting complex/cyclosome (APC/C) is the main protein ubiquitin ligase responsible for the temporal regulation of mitotic progression. It has been proposed that the APC/C might fulfil other functions, including assembly of the mitotic spindle. Here, we show that the APC/C localizes to centrosomes, the organizers of the eukaryotic microtubule cytoskeleton, specifically during mitosis. Recruitment of the APC/C to spindle poles requires the centrosomal protein Cep152, and we identified Cep152 as both an APC/C interaction partner and an APC/C substrate. Previous studies have shown that Cep152 forms a complex withCep57 and Cep63. The APC/C-mediated ubiquitylation of Cep152 at the centrosome releases Cep57 from this inhibitory complex and enables its interaction with pericentrin, a critical step in promoting microtubule nucleation. Thus, our study extends the function of the APC/C from being a regulator of mitosis to also acting as a positive governor of spindle assembly. The APC/C thereby integrates control of these two important processes in a temporal manner. [ABSTRACT FROM AUTHOR]

Some organelles cannot be synthesized anew, so they are segregated into daughter cells during cell division. In Saccharomyces cerevisiae, daughter cells bud from mother cells and are populated by organelles inherited from the mothers. To determine whether this organelle inheritance occurs in a stereotyped manner, we tracked organelles using fluorescence microscopy. We describe a program for organelle inheritance in budding yeast. The cortical endoplasmic reticulum (ER) and peroxisomes are inherited concomitantly with bud emergence. Next, vacuoles are inherited in small buds, followed closely by mitochondria. Finally, the nucleus and perinuclear ER are inherited when buds have nearly reached their maximal size. Because organelle inheritance timing correlates with bud morphology, which is coupled to the cell cycle, we tested whether disrupting the cell cycle alters organelle inheritance order. By arresting cell cycle progression but allowing continued bud growth, we determined that organelle inheritance still occurs when DNA replication is blocked, and that the general inheritance order is maintained. Thus, organelle inheritance follows a preferred order during polarized cell division and does not require completion of S-phase. [ABSTRACT FROM AUTHOR]

Centromere structure and function are defined by the epigenetic modification of histones at centromeric and pericentromeric chromatin. The constitutive heterochromatin found at pericentromeric regions is highly enriched for H3K9me3 and H4K20me3. Although misexpression of the methyltransferase enzymes that regulate these marks, Suv39 and Suv420, is common in disease, the consequences of such changes are not well understood. Our data show that increased centromere localization of Suv39 and Suv420 suppresses centromere transcription and compromises localization of the mitotic kinase Aurora B, decreasing microtubule dynamics and compromising chromosome alignment and segregation. We find that inhibition of Suv420 methyltransferase activity partially restores Aurora B localization to centromeres and that restoration of the Aurora B-containing chromosomal passenger complex to the centromere is sufficient to suppress mitotic errors that result when Suv420 and H4K20me3 is enriched at centromeres. Consistent with a role for Suv39 and Suv420 in negatively regulating Aurora B, high expression of these enzymes corresponds with increased sensitivity to Aurora kinase inhibition in human cancer cells, suggesting that increased H3K9 and H4K20 methylation may be an underappreciated source of chromosome mis-segregation in cancer. [ABSTRACT FROM AUTHOR]

Polo kinase (PLK1 in mammals) is a master cell cycle regulator that is recruited to various subcellular structures, often by its polo-box domain (PBD), which binds to phosphorylated S-pS/pT motifs. Polo/PLK1 kinases have multiple functions at centrioles and centrosomes, and we have previously shown that in Drosophila phosphorylated Sas-4 initiates Polo recruitment to newly formed centrioles, while phosphorylated Spd-2 recruits Polo to the pericentriolar material (PCM) that assembles around mother centrioles in mitosis. Here, we show that Ana1 (Cep295 in humans) also helps to recruit Polo to mother centrioles in Drosophila. If Ana1-dependent Polo recruitment is impaired, mother centrioles can still duplicate, disengage from their daughters and form functional cilia, but they can no longer efficiently assemble mitotic PCM or elongate during G2. We conclude that Ana1 helps recruit Polo to mother centrioles to specifically promote mitotic centrosome assembly and centriole elongation in G2, but not centriole duplication, centriole disengagement or cilia assembly. [ABSTRACT FROM AUTHOR]

Proper control of division orientation and symmetry, largely determined by spindle positioning, is essential to development and homeostasis. Spindle positioning has been extensively studied in cells dividing in two-dimensional (2D) environments and in epithelial tissues, where proteins such as NuMA (also known as NUMA1) orient division along the interphase long axis of the cell. However, little is known about how cells control spindle positioning in three-dimensional (3D) environments, such as early mammalian embryos and a variety of adult tissues. Here, we usemouseembryonic stemcells (ESCs), which grow in 3D colonies, as a model to investigate division in 3D. We observe that, at the periphery of 3D colonies, ESCs display high spindle mobility and divide asymmetrically. Our data suggest that enhanced spindle movements are due to unequal distribution of the cell-cell junction protein E-cadherin between future daughter cells. Interestingly, when cells progress towards differentiation, division becomes more symmetric, with more elongated shapes in metaphase and enhanced cortical NuMA recruitment in anaphase. Altogether, this study suggests that in 3D contexts, the geometry of the cell and its contacts with neighbors control division orientation and symmetry. [ABSTRACT FROM AUTHOR]

In eukaryotes, entry into and exit from mitosis is regulated, respectively, by the transient activation and inactivation of Cdk1. Taxol, an anti-microtubule anti-cancer drug, prevents microtubule– kinetochore attachments to induce spindle assembly checkpoint (SAC; also known as the mitotic checkpoint)-activated mitotic arrest. SAC activation causes mitotic arrest by chronically activating Cdk1. One consequence of prolonged Cdk1 activation is cell death. However, the cytoplasmic signal(s) that link SAC activation to the initiation of cell death remain unknown. We show here that activated Cdk1 forms a complex with the pro-apoptotic proteins Bax and Bak (also known as BAK1) during SAC-induced apoptosis. Bax- and Bakmediated delivery of activated Cdk1 to the mitochondrion is essential for the phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-xL (encoded by BCL2L1) and the induction of cell death. The interactions between a key cell cycle control protein and key proapoptotic proteins identify the Cdk1–Bax and Cdk1–Bak complexes as the long-sought-after cytoplasmic signal that couples SAC activation to the induction of apoptotic cell death. [ABSTRACT FROM AUTHOR]

Errors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin–kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule–kinetochore attachment. However, the molecular mechanisms by which astrin–kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule– kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule– kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process. [ABSTRACT FROM AUTHOR]

The three-dimensional structure of chromatin is determined by the action of protein complexes of the structural maintenance of chromosome (SMC) family. Eukaryotic cells contain three SMC complexes, cohesin, condensin, and a complex of Smc5 and Smc6. Initially, cohesin was linked to sister chromatid cohesion, the process that ensures the fidelity of chromosome segregation in mitosis. In recent years, a second function in the organization of interphase chromatin into topologically associated domains has been determined, and loop extrusion has emerged as the leading mechanism of this process. Interestingly, fundamental mechanistic differences exist between mitotic tethering and loop extrusion. As distinct molecular switches that aim to suppress loop extrusion in different biological contexts have been identified, we hypothesize here that loop extrusion is the default biochemical activity of cohesin and that its suppression shifts cohesin into a tethering mode. With this model, we aim to provide an explanation for how loop extrusion and tethering can coexist in a single cohesin complex and also apply it to the other eukaryotic SMC complexes, describing both similarities and differences between them. Finally, we present model-derived molecular predictions that can be tested experimentally, thus offering a new perspective on the mechanisms by which SMC complexes shape the higher-order structure of chromatin. [ABSTRACT FROM AUTHOR]

Drosophila neural stem cells, or neuroblasts, rapidly proliferate during embryonic and larval development to populate the central nervous system. Neuroblasts divide asymmetrically to create cellular diversity, with each division producing one sibling cell that retains the neuroblast fate and another that differentiates into glia or neurons. This asymmetric outcome is mediated by the transient polarization of numerous factors to the cell cortex during mitosis. The powerful genetics and outstanding imaging tractability of the neuroblast make it an excellent model system for studying the mechanisms of cell polarity. This Cell Science at a Glance article and the accompanying poster explore the phases of the neuroblast polarity cycle and the regulatory circuits that control them. We discuss the key features of the cycle - the targeted recruitment of proteins to specific regions of the plasma membrane and multiple phases of highly dynamic actomyosin-dependent cortical flows that pattern both protein distribution and membrane structure., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

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.)

During the initial stages of mitosis, multiple mechanisms drive centrosome separation and positioning. How they are coordinated to promote centrosome migration to opposite sides of the nucleus remains unclear. Here, we present Trackosome, an open-source image analysis software for tracking centrosomes and reconstructing nuclear and cellular membranes, based on volumetric live-imaging data. The toolbox runs in MATLAB and provides a graphical user interface for easy access to the tracking and analysis algorithms. It provides detailed quantification of the spatiotemporal relationships between centrosomes, nuclear envelope and cellular membrane, and can also be used to measure the dynamic fluctuations of the nuclear envelope. These fluctuations are important because they are related to the mechanical forces exerted on the nucleus by its adjacent cytoskeletal structures. Unlike previous algorithms based on circular or elliptical approximations, Trackosome measures membrane movement in a model-free condition, making it viable for irregularly shaped nuclei. Using Trackosome, we demonstrate significant correlations between the movements of the centrosomes, and identify specific oscillation modes of the nuclear envelope. Overall, Trackosome is a powerful tool that can be used to help unravel new elements in the spatiotemporal dynamics of subcellular structures. [ABSTRACT FROM AUTHOR]

The centrosome, which consists of centrioles and pericentriolar material (PCM), becomes mature and assembles mitotic spindles by increasing the number of microtubules (MTs) emanating from the PCM. Among the molecules involved in centrosome maturation, Cep192 and Aurora A (AurA, also known as AURKA) are primarily responsible for recruitment of γ-tubulin and MT nucleators, whereas pericentrin (PCNT) is required for PCM organization. However, the role of Cep215 (also known as CDK5RAP2) in centrosome maturation remains elusive. Cep215 possesses binding domains for γ-tubulin, PCNT and MT motors that transport acentrosomal MTs towards the centrosome. We identify a mitosis-specific centrosome-targeting domain of Cep215 (215N) that interacts with Cep192 and phosphorylated AurA (pAurA). Cep192 is essential for targeting 215N to centrosomes, and centrosomal localization of 215N and pAurA is mutually dependent. Cep215 has a relatively minor role in γ-tubulin recruitment to the mitotic centrosome. However, it has been shown previously that this protein is important for connecting mitotic centrosomes to spindle poles. Based on the results of rescue experiments using versions of Cep215 with different domain deletions, we conclude that Cep215 plays a role in maintaining the structural integrity of the spindle pole by providing a platform for the molecules involved in centrosome maturation. [ABSTRACT FROM AUTHOR]

Nucleoporin Nup153 is a multifunctional protein and a known binding partner of mitotic checkpoint protein Mad1 (also known as MAD1L1). The functional relevance of their interaction has remained elusive. Here, we have further dissected the interface and functional interplay of Nup153 and Mad1. Using in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is a prerequisite for the Nup153–Mad1 association. Time-lapse microscopy revealed that depletion of Mad1 delayed recruitment of Nup153 to anaphase chromatin, which was often accompanied by a prolongation of anaphase. Furthermore, as seen by electron microscopic and three-dimensional structured illumination investigations, Nup153 and Mad1 depletion led to alterations in NE architecture, characterised by a change of membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between inner and outer nuclear membranes. Nup153 depletion, but not Mad1 depletion, caused defects in interphase NPC assembly, with partial displacement of cytoplasmic nucleoporins and a reduction in NPC density. Taken together, our results suggest that Nup153 has separable roles in NE and NPC formation: in post-mitotic NE re-formation in concert with Mad1 and in interphase NPC assembly, independent of Mad1. [ABSTRACT FROM AUTHOR]

Polo-like kinase 1 (PLK1) dynamically changes its localization and plays important roles in proper mitotic progression. In particular, strict control of cytoplasmic PLK1 is needed to prevent mitotic defects. However, the regulation of cytoplasmic PLK1 is not fully understood. In this study, we show that CEP76, a centriolar protein, physically interacts with PLK1 and tightly controls the activation of cytoplasmic PLK1 during mitosis in human cells. We found that removal of centrosomes induced ectopic aggregation of PLK1, which is highly phosphorylated, in the cytoplasm during mitosis. Importantly, a targeted RNAi screen revealed that depletion of CEP76 resulted in a similar phenotype. In addition, depletion of CEP76 caused defective spindle orientation and mitotic delay. Moreover, the formation of ectopic PLK1 aggregates and defective spindle orientation were significantly suppressed by the inhibition of PLK1 kinase activity. Overall, these results demonstrate that CEP76 suppresses the aberrant activation of cytoplasmic PLK1 for proper mitotic progression. [ABSTRACT FROM AUTHOR]

During cell division, misaligned chromosomes are captured and aligned by motors before their segregation. The CENP-E motor is recruited to polar unattached kinetochores to facilitate chromosome alignment. The spindle checkpoint protein BubR1 (also known as BUB1B) has been reported as a CENP-E interacting partner, but the extent to which BubR1 contributes to CENP-E localization at kinetochores has remained controversial. Here we define the molecular determinants that specify the interaction between BubR1 and CENP-E. The basic C-terminal helix of BubR1 is necessary but not sufficient for CENP-E interaction, and a minimal key acidic patch on the kinetochore-targeting domain of CENP-E is also essential. We then demonstrate that BubR1 is required for the recruitment of CENPE to kinetochores to facilitate chromosome alignment. This BubR1– CENP-E axis is critical for alignment of chromosomes that have failed to congress through other pathways and recapitulates the major known function of CENP-E. Overall, our studies define the molecular basis and the function for CENP-E recruitment to BubR1 at kinetochores during mammalian mitosis. [ABSTRACT FROM AUTHOR]

Most eukaryotic centromeres are located within heterochromatic regions. Paradoxically, heterochromatin can also antagonize de novo centromere formation, and some centromeres lack it altogether. In order to investigate the importance of heterochromatin at centromeres, we used epigenetic engineering of a synthetic alphoidtetO human artificial chromosome (HAC), to which chimeric proteins can be targeted. By tethering the JMJD2D demethylase (also known as KDM4D), we removed heterochromatin mark H3K9me3 (histone 3 lysine 9 trimethylation) specifically from the HAC centromere. This caused no short-term defects, but long-term tethering reduced HAC centromere protein levels and triggeredHACmis-segregation.However, centromeric CENP-A was maintained at a reduced level. Furthermore, HAC centromere function was compatible with an alternative low- H3K9me3, high-H3K27me3 chromatin signature, as long as residual levels of H3K9me3 remained. When JMJD2D was released from the HAC, H3K9me3 levels recovered over several days back to initial levels along with CENP-A and CENP-C centromere levels, and mitotic segregation fidelity. Our results suggest that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory, and that a homeostatic pathway maintains heterochromatin at centromeres. [ABSTRACT FROM AUTHOR]

Cell division ends when two daughter cells physically separate via abscission, the cleavage of the intercellular bridge. It is not clear how the anti-parallel microtubule bundles bridging daughter cells are severed. Here, we present a novel abscission mechanism. We identified chromokinesin KIF4A, which is adjacent to the midbody during cytokinesis, as being required for efficient abscission. KIF4A is regulated by post-translational modifications. We evaluated modification of KIF4A by the ubiquitin-like protein SUMO. We mapped lysine 460 in KIF4A as the SUMO acceptor site and employed CRISPR-Cas9-mediated genome editing to block SUMO conjugation of endogenous KIF4A. Failure to SUMOylate this site in KIF4A delayed cytokinesis. SUMOylation of KIF4A enhanced the affinity for the microtubule destabilizer stathmin 1 (STMN1). We here present a new level of abscission regulation through the dynamic interactions between KIF4A and STMN1 as controlled by SUMO modification of KIF4A. [ABSTRACT FROM AUTHOR]

Regulation of proliferation, apoptosis and cell cycle is crucial for the physiology of germ cells. Their malfunction contributes to infertility and germ cell tumours. The kinesin KIF18A is an important regulator of those processes in animal germ cells. Post-transcriptional regulation of KIF18A has not been extensivelyexplored.Owing to the presence of PUM-binding elements (PBEs), KIF18A mRNA is a potential target of PUM proteins, where PUM refers to Pumilio proteins, RNAbinding proteins that act in post-transcriptional gene regulation. We conducted RNA co-immunoprecipitation combined with RT-qPCR, as well as luciferase reporter assays, by applying an appropriate luciferase construct encoding wild-type KIF18A 3'-UTR, upon PUM overexpression or knockdown in TCam-2 cells, representing human male germ cells. We found that KIF18A is repressed by PUM1 and PUM2. To study how this regulation influences KIF18A function, an MTS proliferation assay, and apoptosis and cell cycle analysis using flow cytometry, was performed upon KIF18A mRNA siRNA knockdown. KIF18A significantly influences proliferation, apoptosis and the cell cycle, with its effects being opposite to PUM effects. Repression by PUM proteins might represent one of mechanisms influencing KIF18A level in controlling proliferation, cell cycle and apoptosis in TCam-2 cells. [ABSTRACT FROM AUTHOR]

A novel 2,3-benzodiazepine-4 derivative, named 1g, has recently been shown to function as an anti-proliferative compound. We now show that it perturbs the formation of a functional mitotic spindle, inducing a spindle assembly checkpoint (SAC)-dependent arrest in human cells. Live analysis of individual microtubules indicates that 1g promotes a rapid and reversible reduction in microtubule growth. Unlike most anti-mitotic compounds, we found that 1g does not interfere directly with tubulin or perturb microtubule assembly in vitro. The observation that 1g also triggers a SAC-dependent mitotic delay associated with chromosome segregation in Drosophila neural stem cells, suggests that it targets a conserved microtubule regulation module in humans and flies. Altogether, our results indicate that 1g is a novel promising anti-mitotic drug with the unique properties of altering microtubule growth and mitotic spindle organization. [ABSTRACT FROM AUTHOR]

Chromosomal instability, one of the most prominent features of tumour cells, causes aneuploidy. Tetraploidy is thought to be an intermediate on the path to aneuploidy, but the mechanistic relationship between the two states is poorly understood. Here, we show that spindle polarity (e.g. bipolarity or multipolarity) in tetraploid cells depends on the level of functional phosphorylated Eg5, a mitotic kinesin, localised to the spindle. Multipolar spindles are formed in cells with high levels of phosphorylated Eg5. This process is suppressed by inhibition of Eg5 or expression of a nonphosphorylatable Eg5 mutant, as well as by changing the balance between opposing forces required for centrosome separation. Tetraploid cells with high levels of functional Eg5 give rise to a heterogeneous aneuploid population through multipolar division, whereas cells with low levels of functional Eg5 continue to undergo bipolar division and remain tetraploid. Furthermore, Eg5 protein levels correlate with ploidy status in tumour specimens. We provide a novel explanation for the tetraploid intermediate model, i.e. spindle polarity and subsequent tetraploid cell behaviour are determined by the balance of forces generated by mitotic kinesins at the spindle. [ABSTRACT FROM AUTHOR]