Segregation Defects Research Articles

The microtubule-severing enzyme spastin regulates spindle dynamics to promote chromosome segregation in Trypanosoma brucei.

Microtubule-severing enzymes play essential roles in regulating diverse cellular processes, including mitosis and cytokinesis, by modulating microtubule dynamics. In the early branching protozoan parasite Trypanosoma brucei , microtubule-severing enzymes are involved in cytokinesis and flagellum length control during different life cycle stages, but none of them have been found to regulate mitosis in any life cycle form. Here, we report the biochemical and functional characterization of the microtubule-severing enzyme spastin in the procyclic form of T. brucei . We demonstrate that spastin catalyzes microtubule severing in vitro and ectopic overexpression of spastin disrupts spindle microtubules in vivo in trypanosome cells, leading to defective chromosome segregation. Knockdown of spastin impairs spindle integrity and disrupts chromosome alignment in metaphase and chromosome segregation in anaphase. We further show that the function of spastin requires the catalytic AAA-ATPase domain, the microtubule-binding domain, and the microtubule interacting and trafficking domain, and that the association of spastin with spindle depends on the microtubule-binding domain. Together, these results uncover an essential role for spastin in chromosome segregation by regulating spindle dynamics in this unicellular eukaryote.

Sequestration of ribosomal subunits as inactive 80S by targeting eIF6 limits mitotic exit and cancer progression.

Moderating the pool of active ribosomal subunits is critical for maintaining global translation rates. A factor crucial for modulating the 60S ribosomal subunit is eukaryotic translation initiation factor-6 (eIF6). Release of eIF6 from the 60S subunit is essential to permit 60S interactions with the 40S subunit. Here, using the eIF6-N106S mutant, we show that disrupting eIF6 interaction with the 60S subunit leads to an increase in vacant 80S ribosomes. It further highlights a dichotomy in the anti-association activity of eIF6 that is distinct from its role in 60S subunit biogenesis and shows that nucleolar localization of eIF6 is not dependent on BCCIP chaperone and uL14. Limiting active ribosomal pools markedly deregulates translation especially in mitosis and leads to chromosome segregation defects, mitotic exit delays and mitotic catastrophe. Ribo-seq analysis of eIF6-N106S mutant shows a significant downregulation in the translation efficiencies of mitotic factors and specifically transcripts with long 3' untranslated regions. eIF6-N106S mutation also limits cancer invasion, and this role is correlated with overexpression of eIF6 only in high-grade invasive cancers suggesting that deregulation of eIF6 is probably not an early event in cancers. Thus, this study highlights the segregation of eIF6 functions and its role in moderating 80S ribosome availability for translation, mitosis and cancer progression.

Mutations in tumor suppressor genes Vhl and Rassf1a cause DNA damage, chromosomal instability and induce gene expression changes characteristic of clear cell renal cell carcinoma.

RASSF1A is frequently biallelically inactivated in clear cell renal cell carcinoma (ccRCC) due to loss of chromosome 3p and promoter hypermethylation. Here we investigated the cellular and molecular consequences of single and combined deletion of the Rassf1a and Vhl tumor suppressor genes to model the common ccRCC genotype of combined loss of function of RASSF1A and VHL. In mouse embryonic fibroblasts and in primary kidney epithelial cells, double deletion of Rassf1a and Vhl caused chromosomal segregation defects and increased formation of micronuclei, demonstrating that pVHL and RASSF1A function to maintain genomic integrity. Combined Rassf1a and Vhl deletion in kidney epithelial cells in vivo increased proliferation and caused mild tubular disorganization, but did not lead to the development of kidney tumors. Single cell RNA-sequencing unexpectedly revealed that Rassf1a or Vhl deletion both induce the expression of an overlapping set of genes in a sub-population of proximal tubule cells. Many of these genes are also upregulated in the Vhl/Trp53/Rb1 deficient mouse model of ccRCC. In other subsets of proximal tubule cells, combined Vhl/Rassf1a deletion induced the expression of additional genes that were not upregulated in each of the single knockouts. The expression of the human homologues of Rassf1a-regulated genes correlate negatively with RASSF1 expression levels in human ccRCC. Our results suggest that the loss of RASSF1A function establishes a ccRCC-characteristic gene expression pattern.

Docetaxel response in BRCA1,p53-deficient mammary tumor cells is affected by Huntingtin and BAP1

Taxanes are frequently used anticancer drugs known to kill tumor cells by inducing mitotic aberrations and segregation defects. A defining feature of specific cancers, notably triple-negative breast cancer (TNBC) and particularly those deficient in BRCA1, is chromosomal instability (CIN). Here, we focused on understanding the mechanisms of docetaxel-induced cytotoxicity, especially in the context of BRCA1-deficient TNBC. Using functional genetic screens in CIN+ cells, we identified genes that mediate docetaxel response and found an interaction between Huntingtin (HTT) and BRCA1-associated protein-1 (BAP1). We employed Brca1-/-;p53-/- mammary tumor cells, derived from genetically engineered mouse tumors that closely mimic the human disease, to investigate the role of these genes in CIN+ BRCA1-deficient cells. Specifically, we observed that loss of HTT sensitizes CIN+ BRCA1-deficient mammary tumor cells to docetaxel by shortening mitotic spindle poles and increasing spindle multipolarity. In contrast, BAP1 depletion protected cells against these spindle aberrations by restoring spindle length and enhancing mitotic clustering of the extra centrosomes. In conclusion, our findings shed light on the roles of HTT and BAP1 in controlling mitotic spindle multipolarity and centrosome clustering, specifically in the absence of BRCA1. This affects the response to microtubule-targeting agents and suggests that further studies of the interaction of these genes with the mitotic spindle may provide useful insights into how to target CIN+ cells, particularly in the challenging therapeutic landscape of BRCA1-deficient TNBC.

The interplay of the translocase activity and protein recruitment function of PICH in ultrafine anaphase bridge resolution and genomic stability.

Incomplete sister centromere decatenation results in centromeric ultrafine anaphase bridges (UFBs). PICH (PLK1-interacting checkpoint helicase), a DNA translocase, plays a crucial role in UFB resolution by recruiting UFB-binding proteins and stimulating topoisomerase IIα. However, the involvement of distinct PICH functions in UFB resolution remains ambiguous. Here, we demonstrate that PICH depletion in non-transformed diploid cells induces DNA damage, micronuclei formation, p53 activation, G1-phase delay and cell death. Whole-genome sequencing reveals that segregation defects induced by PICH depletion cause chromosomal rearrangements, including translocations and inversions, emphasizing its significance in preserving genomic integrity. Furthermore, a PICH mutant that impairs UFB recruitment of BLM and RIF1 partially inhibits UFB resolution while a translocase-inactive mutant (PICHK128A) fails to resolve UFBs. Notably, expression of PICHK128A inhibits single-stranded UFB formation and induces hypocondensed chromosomes. We propose that PICH's translocase activity plays a dual role in promoting UFB resolution by facilitating the generation of single-stranded UFBs and stimulating topoisomerase IIα.

The histone H3.3 K27M mutation suppresses Ser31phosphorylation and mitotic fidelity, which can directly drive gliomagenesis.

Serine 31 is a phospho-site unique to the histone H3.3 variant; mitotic phospho-Ser31 is restricted to pericentromeric heterochromatin, and disruption of phospho-Ser31 results in chromosome segregation defects and loss of p53-dependant G1 cell-cycle arrest.1,2,3,4 Ser31 is proximal to the H3.3 lysine 27-to-methionine (K27M) mutation that drives ∼80% of pediatric diffuse midline gliomas.5,6,7,8,9,10,11,12 Here, we show that expression of the H3.3 K27M mutant in normal, diploid cells results in increased chromosome missegregation and failure to arrest in the following G1. Expression of a non-phosphorylatable S31A mutant also drives chromosome missegregation, while the expression of a double K27M+ phosphomimetic S31E mutant restores mitotic fidelity and the p53 response to chromosome missegregation. We show that patient-derived H3.3 K27M tumor cells have decreased mitotic Ser31 phosphorylation and increased frequency of chromosome missegregation. CRISPR reversion of the K27M mutation to wild type (WT) restores phospho-Ser31 levels and results in a decrease in chromosome missegregation. However, inserting an S31A mutation by CRISPR into these revertant cells disrupts mitotic fidelity. Invitro and invivo analyses reveal that Chk1-the mitotic Ser31 kinase-is preferentially retained at pericentromeres in K27M-expressing tumor cells, compared with MLysine27-to-methionine mutation (M27K) isogenic revertants, correlating with both diminished phospho-Ser31 and mitotic defects. Interestingly, whereas M27K revertant cells do not form xenograft tumors in mice, H3.3 S31A cells do, similar to those formed by H3.3 K27M cells. Replication-competent avian leukosis virus splice-acceptor (RCAS)/cellular receptor for subgroup A avian sarcoma and leukosis virus (TVA) mice expressing S31A also form diffuse midline gliomas morphologically indistinguishable from K27M tumors. Together, our results reveal that the H3.3 K27M mutant alters H3.3 Ser31 phosphorylation, which, in turn, has profound impacts on chromosome segregation/cell-cycle regulation.

Novel pof1 mutation suppresses the sensitivity to DNA replication inhibitor in fission yeast RecQ helicase mutant.

Homologous recombination is vital for DNA double-strand break repair. Dysfunction in homologous recombination can lead to cell death, mutations, and cancer. In fission yeast (Schizosaccharomyces pombe), RecQ helicase Rqh1 resolves recombination intermediates. We found that rqh1-hd strain impaired growth in media containing hydroxyurea and thiabendazole. Using this condition, we identified a novel pof1 mutation (pof1-A81T) that suppress the poor growth of the rqh1-hd strain on the plate containing hydroxyurea and thiabendazole. Compared to rqh1-hd, rqh1-hd pof1-A81T cells displayed reduced Replication Protein A foci on chromosome bridges after hydroxyurea treatment. This suggests that pof1-A81T mutation suppresses the accumulation of recombination intermediates in hydroxyurea-treated rqh1-hd cells. Additionally, pof1-A81T mutation rescued the segregation defect of nucleolar protein Gar2 observed in hydroxyurea-treated rqh1-hd cells, potentially by mitigating recombination intermediate accumulation in rDNA. These results suggest that the pof1-A81T mutation suppresses the accumulation of recombination intermediates, particularly in rDNA, and alleviates the rqh1 deficiency phenotype in S. pombe.

Enhanced Efficiency and Intrinsic Stability of Wide-Bandgap Perovskite Solar Cells Through Dimethylamine-Based Cation Engineering.

High efficiency and stable wide-bandgap (WBG) perovskite solar cells (PSCs) are crucial for the development of perovskite-based tandem solar cells. However, the efficiency and stability of WBG PSCs are compromised by significant phase segregation and surface defects. In this study, we introduce a cation engineering strategy for WBG perovskite, employing a two-step sequential method that incorporates dimethylamine hydroiodide (DMAI) into the lead halide complex during the initial step. The addition of DMAI modifies the crystal structure and grain growth of the perovskite film, leading to improved crystal quality, reduced photo-induced halide segregation, decreased defect density, and enhanced charge carrier mobility. Consequently, we achieved a champion power conversion efficiency (PCE) of 21.9 % for 1.68 eV WBG PSCs. Furthermore, the stability of PSCs based on DMA-doped perovskite was significantly improved. After 1500 hours of exposure to ambient air, the unencapsulated device retained an impressive 80.6 % of its initial efficiency. This result highlights the substantial potential for stable and efficient WBG PSCs.

Exploring the role of oxidative stress and mitochondrial dysfunction in β-damascone-induced aneuploidy

BackgroundThe rose ketone β-damascone (β-Dam) elicits positive results in the in vitro micronucleus (MN) assay using human lymphocytes, but shows negative outcomes in the Ames test and combined in vivo MN and comet assays. This has led to the interpretation that the in vitro MN result is a misleading positive result. Oxidative stress has been suggested as an indirect mode of action (MoA) for in vitro MN formation, with the α, β-unsaturated carbonyl moiety of the β-Dam chemical structure expected to cause misleading positive results through this MoA. In this study, we investigated the role of oxidative stress in β-Dam-induced in vitro MN formation by co-treatment with the antioxidant N-acetyl-l-cysteine (NAC), thereby highlighting a possible link between mitochondrial dysfunction and aneugenicity.Resultsβ-Dam induced MN formation in both CHL/IU and BEAS-2B cells, with the response completely inhibited by co-treatment with NAC. Moreover, β-Dam induced oxidative stress-related reporter activity in the ToxTracker assay and increased reactive oxygen species levels, while decreasing glutathione levels, in BEAS-2B cells in the high-content analysis. All of these effects were suppressed by NAC co-treatment. These findings indicate that β-Dam elicits oxidative stress, which causes DNA damage and ultimately leads to MN induction. However, no significant DNA damage-related reporter activities were observed in the ToxTracker assay, nor was there an increased number of γH2AX foci in the high-content analysis. These data suggest that MN formation is not a DNA-reactive MoA. Considering recent reports of aneuploidy resulting from chromosome segregation defects caused by mitochondrial dysfunction, we investigated if β-Dam could cause such dysfunction. We observed that the mitochondrial membrane potential was dose-dependently impaired in BEAS-2B cells exposed to β-Dam.ConclusionsThese findings suggest that the oxidative stress induced by β-Dam exposure may be explained through an aneugenic MoA via mitochondrial dysfunction, thereby contributing to MN formation in mammalian cells.

Nuclear localization of MTHFD2 is required for correct mitosis progression

Subcellular compartmentalization of metabolic enzymes establishes a unique metabolic environment that elicits specific cellular functions. Indeed, the nuclear translocation of certain metabolic enzymes is required for epigenetic regulation and gene expression control. Here, we show that the nuclear localization of the mitochondrial enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) ensures mitosis progression. Nuclear MTHFD2 interacts with proteins involved in mitosis regulation and centromere stability, including the methyltransferases KMT5A and DNMT3B. Loss of MTHFD2 induces severe methylation defects and impedes correct mitosis completion. MTHFD2 deficient cells display chromosome congression and segregation defects and accumulate chromosomal aberrations. Blocking the catalytic nuclear function of MTHFD2 recapitulates the phenotype observed in MTHFD2 deficient cells, whereas restricting MTHFD2 to the nucleus is sufficient to ensure correct mitotic progression. Our discovery uncovers a nuclear role for MTHFD2, supporting the notion that translocation of metabolic enzymes to the nucleus is required to meet precise chromatin needs.

Phase transformation mechanism and microstructure of a Y-doped TiAl gas-atomized powders

In this study, the phase transformation mechanism of a yttrium-containing β-solidified TiAl alloy (Ti-43Al-9 V-0.3Y at.%), prepared by gas atomization, was systematically investigated. X-ray diffraction, electron backscatter diffraction, scanning electron microscopy, and transmission electron microscopy were utilized to comprehensively analyze the morphology and microstructure of powders with varying sizes as well as the form and distribution of yttrium and its influence on the phase transformation of the powders. The results show that the solidification phase structure of the powders exhibits significant variations: the ultra-fine powder consists of α’ martensite and remaining β phase, while the medium-sized powder is solely composed of β0 phase. The large-sized dendritic powder comprises β0, α’ martensite and α2 phase. With an increase in powder size, there is a corresponding increase in the content of α2 phase, whereas the content of martensite initially rises and subsequently declines. Additionally, yttrium is present in the form of multiscale Y-rich precipitates (YAl2 and Y2O3) within the matrix, and the segregation degree gradually increases with increasing powder size. The primary factors contributing to the disparity in solidification structure include cooling rate and segregation defects. A faster cooling rate and a higher supercooling degree will inhibit the β → α transition, while the Y-rich precipitated phase forms a pre-existing strain zone around it, providing an effective site for martensitic nucleation. In summary, these findings offer novel insights into the mechanism of phase transformation in yttrium-containing β-solidified TiAl alloy, thereby contributing to further advancements in the theory of rapid solidification for TiAl alloys.

Pachytene piRNAs control discrete meiotic events during spermatogenesis and restrict gene expression in space and time.

Pachytene piRNAs, a Piwi-interacting RNA subclass in mammals, are hypothesized to regulate non-transposon sequences during spermatogenesis. Caenorhabditis elegans piRNAs, the 21URNAs, are implicated in regulating coding sequences; the messenger RNA targets and biological processes they control during spermatogenesis are largely unknown. We demonstrate that loss of 21URNAs compromises homolog pairing and makes it permissive for nonhomologous synapsis resulting in defects in crossover formation and chromosome segregation during spermatogenesis. We identify Polo-like kinase 3 (PLK-3), among others, as a 21URNA target. 21URNA activity restricts PLK-3 protein to proliferative cells, and expansion of PLK-3 in pachytene overlaps with the meiotic defects. Removal of plk-3 results in quantitative genetic suppression of the meiotic defects. One discrete 21URNA inhibits PLK-3 expression in late pachytene cells. Together, these results suggest that the 21URNAs function as pachytene piRNAs during C. elegans spermatogenesis. We identify their targets and meiotic events and highlight the remarkable intricacy of this multi-effector mechanism during spermatogenesis.

Nr5a2 is dispensable for zygotic genome activation but essential for morula development.

Early embryogenesis is driven by transcription factors (TFs) that first activate the zygotic genome and then specify the lineages constituting the blastocyst. Although the TFs specifying the blastocyst's lineages are well characterized, those playing earlier roles remain poorly defined. Using mouse models of the TF Nr5a2, we show that Nr5a2-/- embryos arrest at the early morula stage and exhibit altered lineage specification, frequent mitotic failure, and substantial chromosome segregation defects. Although NR5A2 plays a minor but measurable role during zygotic genome activation, it predominantly acts as a master regulator at the eight-cell stage, controlling expression of lineage-specifying TFs and genes involved in mitosis, telomere maintenance, and DNA repair. We conclude that NR5A2 coordinates proliferation, genome stability, and lineage specification to ensure correct morula development.

Kinesin-7 CENP-E mediates centrosome organization and spindle assembly to regulate chromosome alignment and genome stability.

Chromosome congression and alignment are essential for cell cycle progression and genomic stability. Kinesin-7 CENP-E, a plus-end-directed kinesin motor, is required for chromosome biorientation, congression and alignment in cell division. However, it remains unclear how chromosomes are aligned and segregated in the absence of CENP-E in mitosis. In this study, we utilize the CRISPR-Cas9 gene editing method and high-throughput screening to establish CENP-E knockout cell lines and reveal that CENP-E deletion results in defects in chromosome congression, alignment and segregation, which further promotes aneuploidy and genomic instability in mitosis. Both CENP-E inhibition and deletion lead to the dispersion of spindle poles, the formation of the multipolar spindle and spindle disorganization, which indicates that CENP-E is necessary for the organization and maintenance of spindle poles. In addition, CENP-E heterozygous deletion in spleen tissues also leads to the accumulation of dividing lymphocytes and cell cycle arrest in vivo. Furthermore, CENP-E deletion also disrupts the localization of key kinetochore proteins and triggers the activation of the spindle assembly checkpoint. In summary, our findings demonstrate that CENP-E promotes kinetochore-microtubule attachment and spindle pole organization to regulate chromosome alignment and spindle assembly checkpoint during cell division.

Effect of Hot Isostatic Pressing and Solution Heat Treatment on the Microstructure and Mechanical Properties of Ti-6Al-4V Alloy Manufactured by Selective Laser Melting

A powder-bed-based additive manufacturing process called electron beam melting (EBM) is defined by high temperature gradients during solidification, which produces an extremely fine microstructure compared to the traditional cast material. However, porosity and segregation defects are still present on a smaller scale which may lead to a reduction in mechanical properties. It is important to have a better knowledge of the influence of post-fabrication treatments on the microstructure and mechanical characteristics before the use of additive manufacturing parts in specific applications. In this study, the effects of solution heat treatment (SHT) and hot isostatic pressing (HIP) on the microstructure and mechanical properties of Ti-6Al-4V alloy fabricated by the EBM process have been investigated. The SHT and HIP treatments can significantly improve the ductility of EBM Ti-6Al-4V due to the coarsening of α laths and the formation of β grains.

Retrotransposon addiction promotes centromere function via epigenetically activated small RNAs

Retrotransposons have invaded eukaryotic centromeres in cycles of repeat expansion and purging, but the function of centromeric retrotransposons has remained unclear. In Arabidopsis, centromeric ATHILA retrotransposons give rise to epigenetically activated short interfering RNAs in mutants in DECREASE IN DNA METHYLATION1 (DDM1). Here we show that mutants that lose both DDM1 and RNA-dependent RNA polymerase have pleiotropic developmental defects and mis-segregate chromosome 5 during mitosis. Fertility and segregation defects are epigenetically inherited with centromere 5, and can be rescued by directing artificial small RNAs to ATHILA5 retrotransposons that interrupt tandem satellite repeats. Epigenetically activated short interfering RNAs promote pericentromeric condensation, chromosome cohesion and chromosome segregation in mitosis. We propose that insertion of ATHILA silences centromeric transcription, while simultaneously making centromere function dependent on retrotransposon small RNAs in the absence of DDM1. Parallels are made with the fission yeast Schizosaccharomyces pombe, where chromosome cohesion depends on RNA interference, and with humans, where chromosome segregation depends on both RNA interference and HELLSDDM1.

MiR-31-mediated local translation at the mitotic spindle is important for early development.

miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including β-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis.

Embryonic genome instability upon DNA replication timing program emergence

Faithful DNA replication is essential for genome integrity1–4. Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis5–8. However, the regulation of DNA replication remains poorly understood in early mammalian embryos. Here we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and identified an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing program, and the entire genome replicated gradually and uniformly using extremely slow-moving replication forks. In 4-cell embryos, a somatic-cell-like replication timing program commenced abruptly. However, the fork speed was still slow, S phase was extended, and markers of replication stress, DNA damage and repair increased. This was followed by an increase in break-type chromosome segregation errors specifically during the 4-to-8-cell division with breakpoints enriched in late-replicating regions. These errors were rescued by nucleoside supplementation, which accelerated fork speed and reduced the replication stress. By the 8-cell stage, forks gained speed, S phase was no longer extended and chromosome aberrations decreased. Thus, a transient period of genomic instability exists during normal mouse development, preceded by an S phase lacking coordination between replisome-level regulation and megabase-scale replication timing regulation, implicating a link between their coordination and genome stability.

CDK phosphorylation of Sfr1 downregulates Rad51 function in late-meiotic homolog invasions.

Meiosis is the developmental program that generates gametes. To produce healthy gametes, meiotic recombination creates reciprocal exchanges between each pair of homologous chromosomes that facilitate faithful chromosome segregation. Using fission yeast and biochemical, genetic, and cytological approaches, we have studied the role of CDK (cyclin-dependent kinase) in the control of Swi5-Sfr1, a Rad51-recombinase auxiliary factor involved in homolog invasion during recombination. We show that Sfr1 is a CDK target, and its phosphorylation downregulates Swi5-Sfr1 function in the meiotic prophase. Expression of a phospho-mimetic sfr1-7D mutant inhibits Rad51 binding, its robust chromosome loading, and subsequently decreases interhomolog recombination. On the other hand, the non-phosphorylatable sfr1-7A mutant alters Rad51 dynamics at late prophase, and exacerbates chromatin segregation defects and Rad51 retention observed in dbl2 deletion mutants when combined with them. We propose Sfr1 phospho-inhibition as a novel cell-cycle-dependent mechanism, which ensures timely resolution of recombination intermediates and successful chromosome distribution into the gametes. Furthermore, the N-terminal disordered part of Sfr1, an evolutionarily conserved feature, serves as a regulatory platform coordinating this phospho-regulation, protein localization and stability, with several CDK sites and regulatory sequences being conserved.

β-TrCP-Mediated Proteolysis of Mis18β Prevents Mislocalization of CENP-A and Chromosomal Instability.

Restricting the localization of evolutionarily conserved histone H3 variant CENP-A to the centromere is essential to prevent chromosomal instability (CIN), an important hallmark of cancers. Overexpressed CENP-A mislocalizes to non-centromeric regions and contributes to CIN in yeast, flies, and human cells. Centromeric localization of CENP-A is facilitated by the interaction of Mis18β with CENP-A specific chaperone HJURP. Cellular levels of Mis18β are regulated by β-transducin repeat containing protein (β-TrCP), an F-box protein of SCF (Skp1, Cullin, F-box) E3-ubiquitin ligase complex. Here, we show that defects in β-TrCP-mediated proteolysis of Mis18β contributes to the mislocalization of endogenous CENP-A and CIN in a triple-negative breast cancer (TNBC) cell line, MDA-MB-231. CENP-A mislocalization in β-TrCP depleted cells is dependent on high levels of Mis18β as depletion of Mis18β suppresses mislocalization of CENP-A in these cells. Consistent with these results, endogenous CENP-A is mislocalized in cells overexpressing Mis18β alone. In summary, our results show that β-TrCP-mediated degradation of Mis18β prevents mislocalization of CENP-A and CIN. We propose that deregulated expression of Mis18β may be one of the key mechanisms that contributes to chromosome segregation defects in cancers.