Showing papers on "Metaphase published in 2016"

A USP28-53BP1-p53-p21 signaling axis arrests growth after centrosome loss or prolonged mitosis.

Abstract: Precise regulation of centrosome number is critical for accurate chromosome segregation and the maintenance of genomic integrity. In nontransformed cells, centrosome loss triggers a p53-dependent surveillance pathway that protects against genome instability by blocking cell growth. However, the mechanism by which p53 is activated in response to centrosome loss remains unknown. Here, we have used genome-wide CRISPR/Cas9 knockout screens to identify a USP28-53BP1-p53-p21 signaling axis at the core of the centrosome surveillance pathway. We show that USP28 and 53BP1 act to stabilize p53 after centrosome loss and demonstrate this function to be independent of their previously characterized role in the DNA damage response. Surprisingly, the USP28-53BP1-p53-p21 signaling pathway is also required to arrest cell growth after a prolonged prometaphase. We therefore propose that centrosome loss or a prolonged mitosis activate a common signaling pathway that acts to prevent the growth of cells that have an increased propensity for mitotic errors.

A force-generating machinery maintains the spindle at the cell center during mitosis

Abstract: The position and orientation of the mitotic spindle is precisely regulated to ensure the accurate partition of the cytoplasm between daughter cells and the correct localization of the daughters within growing tissue. Using magnetic tweezers to perturb the position of the spindle in intact cells, we discovered a force-generating machinery that maintains the spindle at the cell center during metaphase and anaphase in one- and two-cell Caenorhabditis elegans embryos. The forces increase with the number of microtubules and are larger in smaller cells. The machinery is rigid enough to suppress thermal fluctuations to ensure precise localization of the mitotic spindle, yet compliant enough to allow molecular force generators to fine-tune the position of the mitotic spindle to facilitate asymmetric division.

Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy

Abstract: Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging and mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and noninvasive illumination of the newly developed lattice light-sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and found that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and was relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light-sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and multicellular assemblies.

The human SKA complex drives the metaphase-anaphase cell cycle transition by recruiting protein phosphatase 1 to kinetochores

Abstract: When one cell divides into two daughter cells it is critical that both new cells inherit an entire copy of the genetic material. This process is called mitosis, and it involves the duplicated chromosomes lining up in the middle of the cell before being pulled apart into the two newly forming cells. Many different proteins control mitosis because mistakes during cell division can lead to cells with too much or too little DNA, which can lead to cancers and other diseases. Mitosis is mainly regulated by enzymes called kinases and phosphatases. Kinases add phosphate groups on to other proteins, which often changes their activity or localization within the cell. Phosphatases counteract the kinases by removing the phosphate groups. During mitosis, kinases and phosphatases accumulate at a specific region of the chromosomes called kinetochores. The kinetochores play two key roles: they are the regions from which the chromosomes are pulled apart and also serve as control centers for regulating the sequence of events in mitosis. To date, mitosis is best understood in yeast cells and less is known about the more complex process in human cells. Previous research had shown that human cells need a group of proteins called the Ska complex to undergo mitosis, because without this complex the chromosomes remain in the middle of the cell and do not separate. Sivakumar et al. – who include two of the researchers involved in the previous research – have now explored the Ska complex’s role in human cells in more detail. The experiments showed that the Ska complex binds to and recruits a phosphatase called PP1 to the chromosomes; this is not how PP1 is brought to the kinetochores in yeast. When enough PP1 is concentrated around the kinetochores this gives the human cell the signal to pull the chromosomes apart and finish mitosis. Future work could ask how PP1 brings about the last stages of mitosis; for example, by finding all the proteins from which PP1 removes phosphate groups. Lastly, further studies could also explore the possibility that the Ska complex performs other tasks that are crucial for the division of human cells.

Unattached kinetochores rather than intrakinetochore tension arrest mitosis in taxol-treated cells

Abstract: Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression.

The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes

Abstract: Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore’s fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies.

Chromosome Bridges Maintain Kinetochore-Microtubule Attachment throughout Mitosis and Rarely Break during Anaphase

Abstract: Accurate chromosome segregation during cell division is essential to maintain genome stability, and chromosome segregation errors are causally linked to genetic disorders and cancer. An anaphase chromosome bridge is a particular chromosome segregation error observed in cells that enter mitosis with fused chromosomes/sister chromatids. The widely accepted Breakage/Fusion/Bridge cycle model proposes that anaphase chromosome bridges break during mitosis to generate chromosome ends that will fuse during the following cell cycle, thus forming new bridges that will break, and so on. However, various studies have also shown a link between chromosome bridges and aneuploidy and/or polyploidy. In this study, we investigated the behavior and properties of chromosome bridges during mitosis, with the idea to gain insight into the potential mechanism underlying chromosome bridge-induced aneuploidy. We find that only a small number of chromosome bridges break during anaphase, whereas the rest persist through mitosis into the subsequent cell cycle. We also find that the microtubule bundles (k-fibers) bound to bridge kinetochores are not prone to breakage/detachment, thus supporting the conclusion that k-fiber detachment is not the cause of chromosome bridge-induced aneuploidy. Instead, our data suggest that while the microtubules bound to the kinetochores of normally segregating chromosomes shorten substantially during anaphase, the k-fibers bound to bridge kinetochores shorten only slightly, and may even lengthen, during anaphase. This causes some of the bridge kinetochores/chromosomes to lag behind in a position that is proximal to the cell/spindle equator and may cause the bridged chromosomes to be segregated into the same daughter nucleus or to form a micronucleus.

A noncatalytic function of the topoisomerase II CTD in Aurora B recruitment to inner centromeres during mitosis

Abstract: Faithful chromosome segregation depends on the precise timing of chromatid separation, which is enforced by checkpoint signals generated at kinetochores. Here, we provide evidence that the C-terminal domain (CTD) of DNA topoisomerase IIα (Topo II) provides a novel function at inner centromeres of kinetochores in mitosis. We find that the yeast CTD is required for recruitment of the tension checkpoint kinase Ipl1/Aurora B to inner centromeres in metaphase but is not required in interphase. Conserved CTD SUMOylation sites are required for Ipl1 recruitment. This inner-centromere CTD function is distinct from the catalytic activity of Topo II. Genetic and biochemical evidence suggests that Topo II recruits Ipl1 via the Haspin-histone H3 threonine 3 phosphorylation pathway. Finally, Topo II and Sgo1 are equally important for Ipl1 recruitment to inner centromeres. This indicates H3 T3-Phos/H2A T120-Phos is a universal epigenetic signature that defines the eukaryotic inner centromere and provides the binding site for Ipl1/Aurora B.

The BTG4 and CAF1 complex prevents the spontaneous activation of eggs by deadenylating maternal mRNAs

Abstract: Once every menstrual cycle, eggs are ovulated into the oviduct where they await fertilization. The ovulated eggs are arrested in metaphase of the second meiotic division, and only complete meiosis upon fertilization. It is crucial that the maintenance of metaphase arrest is tightly controlled, because the spontaneous activation of the egg would preclude the development of a viable embryo (Zhang et al. 2015 J. Genet. Genomics 42 , 477–485. (doi:10.1016/j.jgg.2015.07.004); Combelles et al. 2011 Hum. Reprod. 26 , 545–552. (doi:10.1093/humrep/deq363); Escrich et al. 2011 J. Assist. Reprod. Genet. 28 , 111–117. (doi:10.1007/s10815-010-9493-5)). However, the mechanisms that control the meiotic arrest in mammalian eggs are only poorly understood. Here, we report that a complex of BTG4 and CAF1 safeguards metaphase II arrest in mammalian eggs by deadenylating maternal mRNAs. As a follow-up of our recent high content RNAi screen for meiotic genes (Pfender et al. 2015 Nature 524 , 239–242. (doi:10.1038/nature14568)), we identified Btg4 as an essential regulator of metaphase II arrest. Btg4- depleted eggs progress into anaphase II spontaneously before fertilization. BTG4 prevents the progression into anaphase by ensuring that the anaphase-promoting complex/cyclosome (APC/C) is completely inhibited during the arrest. The inhibition of the APC/C relies on EMI2 (Tang et al. 2010 Mol. Biol. Cell 21 , 2589–2597. (doi:10.1091/mbc.E09-08-0708); Ohe et al. 2010 Mol. Biol. Cell 21 , 905–913. (doi:10.1091/mbc.E09-11-0974)), whose expression is perturbed in the absence of BTG4. BTG4 controls protein expression during metaphase II arrest by forming a complex with the CAF1 deadenylase and we hypothesize that this complex is recruited to the mRNA via interactions between BTG4 and poly(A)-binding proteins. The BTG4–CAF1 complex drives the shortening of the poly(A) tails of a large number of transcripts at the MI–MII transition, and this wave of deadenylation is essential for the arrest in metaphase II. These findings establish a BTG4-dependent pathway for controlling poly(A) tail length during meiosis and identify an unexpected role for mRNA deadenylation in preventing the spontaneous activation of eggs.

PTEN regulates EG5 to control spindle architecture and chromosome congression during mitosis

Abstract: Architectural integrity of the mitotic spindle is required for efficient chromosome congression and accurate chromosome segregation to ensure mitotic fidelity. Tumour suppressor PTEN has multiple functions in maintaining genome stability. Here we report an essential role of PTEN in mitosis through regulation of the mitotic kinesin motor EG5 for proper spindle architecture and chromosome congression. PTEN depletion results in chromosome misalignment in metaphase, often leading to catastrophic mitotic failure. In addition, metaphase cells lacking PTEN exhibit defects of spindle geometry, manifested prominently by shorter spindles. PTEN is associated and co-localized with EG5 during mitosis. PTEN deficiency induces aberrant EG5 phosphorylation and abrogates EG5 recruitment to the mitotic spindle apparatus, leading to spindle disorganization. These data demonstrate the functional interplay between PTEN and EG5 in controlling mitotic spindle structure and chromosome behaviour during mitosis. We propose that PTEN functions to equilibrate mitotic phosphorylation for proper spindle formation and faithful genomic transmission.

DNA damage response during mouse oocyte maturation

Abstract: Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II. Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation.

Chloroplast division checkpoint in eukaryotic algae.

Abstract: Chloroplasts evolved from a cyanobacterial endosymbiont. It is believed that the synchronization of endosymbiotic and host cell division, as is commonly seen in existing algae, was a critical step in establishing the permanent organelle. Algal cells typically contain one or only a small number of chloroplasts that divide once per host cell cycle. This division is based partly on the S-phase-specific expression of nucleus-encoded proteins that constitute the chloroplast-division machinery. In this study, using the red alga Cyanidioschyzon merolae, we show that cell-cycle progression is arrested at the prophase when chloroplast division is blocked before the formation of the chloroplast-division machinery by the overexpression of Filamenting temperature-sensitive (Fts) Z2-1 (Fts72-1), but the cell cycle progresses when chloroplast division is blocked during division-site constriction by the overexpression of either FtsZ2-1 or a dominant-negative form of dynamin-related protein 5B (DRP5B). In the cells arrested in the prophase, the increase in the cyclin B level and the migration of cyclin-dependent kinase B (CDKB) were blocked. These results suggest that chloroplast division restricts host cell-cycle progression so that the cell cycle progresses to the metaphase only when chloroplast division has commenced. Thus, chloroplast division and host cell-cycle progression are synchronized by an interactive restriction that takes place between the nucleus and the chloroplast. In addition, we observed a similar pattern of cell-cycle arrest upon the blockage of chloroplast division in the glaucophyte alga Cyanophora paradoxa, raising the possibility that the chloroplast division checkpoint contributed to the establishment of the permanent organelle.

Inhibiting the anaphase promoting complex/cyclosome induces a metaphase arrest and cell death in multiple myeloma cells.

Abstract: The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase involved in cell cycle. During the metaphase-anaphase transition the APC/C is activated by Cdc20. The aim of this study is to elucidate the importance and therapeutic potential of APC/C and its co-activator Cdc20 in multiple myeloma (MM). Gene expression analysis revealed that Cdc20 was expressed at higher levels in gene expression-based high-risk MM patients. Moreover, high Cdc20 expression correlated with poor prognosis. Treatment of human myeloma cell lines with proTAME, an APC/C inhibitor, resulted in an accumulation of APC/CCdc20 substrate cyclin B1 and an accumulation of cells in metaphase. Moreover we observed a significant dose-dependent decrease in viability and increase in apoptosis in MM cells upon proTAME treatment. The induction of apoptosis was accompanied with caspase 3, 8, 9 and PARP cleavage. A similar metaphase arrest and induction of apoptosis were obtained with specific knockdown of Cdc20. In addition, we demonstrated the accumulation of Bim was partially responsible for the observed cell death. Combining proTAME with another APC/C inhibitor apcin or the alkylating agent melphalan resulted in enhanced anti-MM activity. This study suggests that the APC/C and its co-activator Cdc20 could be a new and promising target especially in high-risk MM patients.

The Light Intermediate Chain 2 Subpopulation of Dynein Regulates Mitotic Spindle Orientation

Abstract: Cytoplasmic dynein 1 is a multi-protein intracellular motor essential for mediating several mitotic functions, including the establishment of proper spindle orientation. The functional relevance and mechanistic distinctions between two discrete dynein subpopulations distinguished only by Light Intermediate Chain (LIC) homologues, LIC1 and LIC2 is unknown during mitosis. Here, we identify LIC2-dynein as the major mediator of proper spindle orientation and uncover its underlying molecular mechanism. Cortically localized dynein, essential for maintaining correct spindle orientation, consists majorly of LIC2-dynein, which interacts with cortical 14-3-3 e- ζ and Par3, conserved proteins required for orienting the spindle. LIC2-dynein is also responsible for the majority of dynein-mediated asymmetric poleward transport of NuMA, helping focus microtubule minus ends. In addition, LIC2-dynein dominates in equatorially aligning chromosomes at metaphase and in regulating mitotic spindle length. Key mitotic functions of LIC2 were remarkably conserved in and essential for early embryonic divisions and development in zebrafish. Thus LIC2-dynein exclusively engages with two major cortical pathways to govern spindle orientation. Overall, we identify a novel selectivity of molecular interactions between the two LICs in mitosis as the underlying basis for their uneven distribution of labour in ensuring proper spindle orientation.

Rapid Discovery of Pyrido[3,4-d]pyrimidine Inhibitors of Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization Approach

Abstract: Monopolar spindle 1 (MPS1) plays a central role in the transition of cells from metaphase to anaphase and is one of the main components of the spindle assembly checkpoint. Chromosomally unstable cancer cells rely heavily on MPS1 to cope with the stress arising from abnormal numbers of chromosomes and centrosomes and are thus more sensitive to MPS1 inhibition than normal cells. We report the discovery and optimization of a series of new pyrido[3,4-d]pyrimidine based inhibitors via a structure-based hybridization approach from our previously reported inhibitor CCT251455 and a modestly potent screening hit. Compounds in this novel series display excellent potency and selectivity for MPS1, which translates into biomarker modulation in an in vivo human tumor xenograft model.

Histone modifications define expression bias of homoeologous genomes in allotetraploid cotton

Abstract: Histone modifications regulate gene expression in eukaryotes, but their roles in gene expression changes in interspecific hybrids or allotetraploids are poorly understood. Histone modifications can be mapped by immunostaining of metaphase chromosomes at the single cell level and/or by chromatin immunoprecipitation-sequencing (ChIP-seq) for analyzing individual genes. Here, we comparatively analyzed immunostained metaphase chromosomes and ChIP-seq of individual genes, which revealed a chromatin basis for biased homoeologous gene expression in polyploids. We examined H3K4me3 density and transcriptome maps in root-tip cells of allotetraploid cotton (Gossypium hirsutum). The overall H3K4me3 levels were relatively equal between A and D chromosomes, which were consistent with equal numbers of expressed genes between the two subgenomes. However, intensities per chromosomal area were nearly twice as high in the D homeologs as in the A homeologs. Consistent with the cytological observation, ChIP-seq analysis showed that more D homeologs with biased H3K4me3 levels than A homeologs with biased modifications correlated with the greater number of the genes with D-biased expression than that with A-biased expression in most homeologous chromosome pairs. Two chromosomes displayed different expression levels compared with other chromosomes, which correlate with known translocations and may affect the local chromatin structure and expression levels for the genes involved. This example of genome-wide histone modifications that determine expression bias of homeologous genes in allopolyploids provides a molecular basis for the evolution and domestication of polyploid species, including many important crops.

Metaphase chromosome analysis by ligation-mediated PCR: Heritable chromatin structure and a comparison of active and inactive X chromosomes (cell memory/epigenetic mechanisms/heterochromatin/in vivo footprinting/mitosis)

Abstract: We report that ligation-mediated PCR (LMPCR) can be used for high-resolution study of metaphase chromosomes, and we discuss the role of metaphase chroma- tin structure in the preservation of differentiated cell states The X chromosome-linked human PGKI (phosphoglycerate kinase 1) promoter region was investigated, and euchromatic active X chromosome (Xa) metaphase chromatin was com- pared with interphase Xa chromatin and to heterochromatic inactive X chromosome (Xi) metaphase and interphase chro- matin We find that (i) good-quality data at single-nucleotide resolution can be obtained by LMPCR analysis of dimethyl sulfate-treated intact metaphase cells; (ii) transcription fac- tors present on the Xa promoter of interphase chromatin are not present on metaphase chromatin, establishing that the transcription complex on the PGKI promoter must form de novo each cell generation; and (iii) the dimethyl sulfate reactivity pattern of Xa and Xi chromatin at metaphase is very similar to that of naked DNA These results are discussed in the context of models for heritable chromatin structure and epigenetic mech- anisms for cell memory, and they are also relevant to more general aspects of chromatin structure and differences between euchromatin and heterochromatin

Epigenetic Histone Marks of Extended Meta-Polycentric Centromeres of Lathyrus and Pisum Chromosomes

Abstract: Species of the legume genera Lathyrus and Pisum possess chromosomes that exhibit a unique structure of their centromeric regions, which is clearly apparent during metaphase by the formation of extended primary constrictions which span up to a third of the length of the chromosome. In addition, these species express two different variants of the CenH3 protein which are co-localized in multiple domains along the poleward surface of the primary constrictions. Here we show that the constrictions represent a distinct type of chromatin differing from the chromosome arms. In metaphase, histone phosphorylation patterns including H3S10ph, H3S28ph and H3T3ph were observed along the entire constriction, in a way similar to holocentric chromosomes. On the other hand, distribution of phosphorylated H2AT120 was different from that previously reported from either, holocentric and monocentric chromosomes, occurring at chromatin surrounding but not overlapping CenH3 domains. Since some of these phosphorylations play a role in chromatid cohesion, it can be assumed that they facilitate correct chromosome segregation by ensuring that multiple separate CenH3 domains present on the same chromatid are oriented towards the same pole. The constrictions also displayed distinct patterns of histone methylation marks, being enriched in H3K9me2 and depleted in H3K4me3 and H3K27me2 compared to the chromosome arms. High resolution fluorescence microscopy revealed that although both CenH3 protein variants are present in all CenH3 domains detected on metaphase chromosomes, they are only partially co-localized while there are chromatin subdomains which are mostly made of only one CenH3 variant. Taken together, these data revealed specific features of extended primary constrictions of Lathyrus and Pisum and support the idea that they may represent an intermediate stage between monocentric and holocentric chromosomes.

A novel homozygous splicing mutation of CASC5 causes primary microcephaly in a large Pakistani family

Abstract: Primary microcephaly is a disorder characterized by a small head and brain associated with impaired cognitive capabilities. Mutations in 13 different genes encoding centrosomal proteins and cell cycle regulators have been reported to cause the disease. CASC5, a gene encoding a protein important for kinetochore formation and proper chromosome segregation during mitosis, has been suggested to be associated with primary microcephaly-4 (MCPH4). This was based on one mutation only and circumstantial functional evidence. By combining homozygosity mapping and whole-exome sequencing in an MCPH family from Pakistan, we identified a second mutation (NM_170589.4;c.6673-19T>A) in CASC5. This mutation induced skipping of exon 25 of CASC5 resulting in a frameshift and the introduction of a premature stop codon (p.Met2225Ilefs*7). The C-terminally truncated protein lacks 118 amino acids that encompass the region responsible for the interaction with the hMIS12 complex, which is essential for proper chromosome alignment and segregation. Furthermore, we showed a down-regulation of CASC5 mRNA and reduction of the amount of CASC5 protein by quantitative RT-PCR and western blot analysis, respectively. As a further sign of functional deficits, we observed dispersed dots of CASC5 immunoreactive material outside the metaphase plate of dividing patient fibroblasts. Normally, CASC5 is a component of the kinetochore of metaphase chromosomes. A higher mitotic index in patient cells indicated a mitotic arrest in the cells carrying the mutation. We also observed lobulated and fragmented nuclei as well as micronuclei in the patient cells. Moreover, we detected an altered DNA damage response with higher levels of γH2AX and 53BP1 in mutant as compared to control fibroblasts. Our findings substantiate the proposed role of CASC5 for primary microcephaly and suggest that it also might be relevant for genome stability.

Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast

Abstract: Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism.

Unfolded-protein response–associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract

Abstract: Failure of lens fiber cell denucleation (LFCD) is associated with congenital cataracts, but the pathobiology awaits elucidation. Recent work has suggested that mechanisms that direct the unidirectional process of LFCD are analogous to the cyclic processes associated with mitosis. We found that lens-specific mutations that elicit an unfolded-protein response (UPR) in vivo accumulate p27(Cdkn1b), show cyclin-dependent kinase (Cdk)-1 inhibition, retain their LFC nuclei, and are cataractous. Although a UPR was not detected in lenses expressing K6W-Ub, they also accumulated p27 and showed failed LFCD. Induction of a UPR in human lens epithelial cells (HLECs) also induced accumulation of p27 associated with decreased levels of S-phase kinase-associated protein (Skp)-2, a ubiquitin ligase that regulates mitosis. These cells also showed decreased lamin A/C phosphorylation and metaphase arrest. The suppression of lamin A/C phosphorylation and metaphase transition induced by the UPR was rescued by knockdown of p27. Taken together, these data indicate that accumulation of p27, whether related to the UPR or not, prevents the phosphorylation of lamin A/C and LFCD in maturing LFCs in vivo, as well as in dividing HLECs. The former leads to cataract and the latter to metaphase arrest. These results suggest that accumulation of p27 is a common mechanism underlying retention of LFC nuclei.

EZH2 is required for mouse oocyte meiotic maturation by interacting with and stabilizing spindle assembly checkpoint protein BubRI

Abstract: Enhancer of zeste homolog 2 (EZH2) trimethylates histone H3 Lys 27 and plays key roles in a variety of biological processes Stability of spindle assembly checkpoint protein BubR1 is essential for mitosis in somatic cells and for meiosis in oocytes However, the role of EZH2 in oocyte meiotic maturation was unknown Here, we presented a mechanism underlying EZH2 control of BubR1 stability in the meiosis of mouse oocytes We identified a methyltransferase activity-independent function of EZH2 by demonstrating that EZH2 regulates spindle assembly and the polar body I extrusion EZH2 was increased with the oocyte progression from GVBD to MII, while EZH2 was concentrated on the chromosomes Interestingly, inhibition of EZH2 methyltranferase activity by DZNep or GSK343 did not affect oocyte meiotic maturation However, depletion of EZH2 by morpholino led to chromosome misalignment and abnormal spindle assembly Furthermore, ectopic expression of EZH2 led to oocyte meiotic maturation arrested at the MI stage followed by chromosome misalignment and aneuploidy Mechanistically, EZH2 directly interacted with and stabilized BubR1, an effect driving EZH2 into the concert of meiosis regulation Collectively, we provided a paradigm that EZH2 is required for mouse oocyte meiotic maturation

ATX-2, the C. elegans Ortholog of Human Ataxin-2, Regulates Centrosome Size and Microtubule Dynamics

Abstract: Centrosomes are critical sites for orchestrating microtubule dynamics, and exhibit dynamic changes in size during the cell cycle. As cells progress to mitosis, centrosomes recruit more microtubules (MT) to form mitotic bipolar spindles that ensure proper chromosome segregation. We report a new role for ATX-2, a C. elegans ortholog of Human Ataxin-2, in regulating centrosome size and MT dynamics. ATX-2, an RNA-binding protein, forms a complex with SZY-20 in an RNA-independent fashion. Depleting ATX-2 results in embryonic lethality and cytokinesis failure, and restores centrosome duplication to zyg-1 mutants. In this pathway, SZY-20 promotes ATX-2 abundance, which inversely correlates with centrosome size. Centrosomes depleted of ATX-2 exhibit elevated levels of centrosome factors (ZYG-1, SPD-5, γ-Tubulin), increasing MT nucleating activity but impeding MT growth. We show that ATX-2 influences MT behavior through γ-Tubulin at the centrosome. Our data suggest that RNA-binding proteins play an active role in controlling MT dynamics and provide insight into the control of proper centrosome size and MT dynamics.

Phyllanthus emblica Fruit Extract Activates Spindle Assembly Checkpoint, Prevents Mitotic Aberrations and Genomic Instability in Human Colon Epithelial NCM460 Cells

Abstract: The fruit of Phyllanthus emblica Linn. (PE) has been widely consumed as a functional food and folk medicine in Southeast Asia due to its remarkable nutritional and pharmacological effects. Previous research showed PE delays mitotic progress and increases genomic instability (GIN) in human colorectal cancer cells. This study aimed to investigate the similar effects of PE by the biomarkers related to spindle assembly checkpoint (SAC), mitotic aberrations and GIN in human NCM460 normal colon epithelial cells. Cells were treated with PE and harvested differently according to the biomarkers observed. Frequencies of micronuclei (MN), nucleoplasmic bridge (NPB) and nuclear bud (NB) in cytokinesis-block micronucleus assay were used as indicators of GIN. Mitotic aberrations were assessed by the biomarkers of chromosome misalignment, multipolar division, chromosome lagging and chromatin bridge. SAC activity was determined by anaphase-to- metaphase ratio (AMR) and the expression of core SAC gene budding uninhibited by benzimidazoles related 1 (BubR1). Compared with the control, PE-treated cells showed (1) decreased incidences of MN, NPB and NB (p < 0.01); (2) decreased frequencies of all mitotic aberration biomarkers (p < 0.01); and (3) decreased AMR (p < 0.01) and increased BubR1 expression (p < 0.001). The results revealed PE has the potential to protect human normal colon epithelial cells from mitotic and genomic damages partially by enhancing the function of SAC.

Auxin/AID versus conventional knockouts: distinguishing the roles of CENP-T/W in mitotic kinetochore assembly and stability

Abstract: Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1–3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and upon entry into mitosis. Interestingly, a putative super-complex involving Rod-Zw10-zwilch (RZZ complex), Spindly, Mad1/Mad2 and CENP-E requires the function of CENP-T/W during kinetochore assembly for its stable association with the outer kinetochore, but once assembled remains associated with chromosomes after stripping of CENP-T during mitosis. This study highlights the different roles core kinetochore components may play in the assembly of kinetochores (upon entry into mitosis) versus the maintenance of specific components (during mitosis).