More than Two Populations of Microtubules Comprise the Dynamic Mitotic Spindle

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 to reflect kinetochore versus non-kinetochore microtubules, respectively. Here, we test this categorization by disrupting kinetochores through depletion of the Ndc80 complex. In the absence of functional kinetochores, microtubule dynamics still exhibit slow and fast turnover populations, though the proportion of each population and the timings of turnover are altered. Importantly, the data obtained following Hec1/Ndc80 depletion suggests other sub-populations, in addition to kinetochore microtubules, contribute to the slow turnover population. Further manipulation of spindle microtubules revealed a complex landscape. For example, while Aurora B kinase functions to destabilize kinetochore bound microtubules it may also stabilize certain slow turnover, non-kinetochore 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.

Comparative transcriptome and DNA methylation analysis in temperature-sensitive genic male sterile wheat BS366

Background Known as the prerequisite component for the heterosis breeding system, the male sterile line determines the hybrid yield and seed purity. Therefore, a deep understanding of the mechanism and gene network that leads to male sterility is crucial. BS366, a temperature-sensitive genic male sterile (TGMS) line, is male sterile under cold conditions (12 °C with 12 h of daylight) but fertile under normal temperature (20 °C with 12 h of daylight). Results During meiosis, BS366 was defective in forming tetrads and dyads due to the abnormal cell plate. During pollen development, unusual vacuolated pollen that could not accumulate starch grains at the binucleate stage was also observed. Transcriptome analysis revealed that genes involved in the meiotic process, such as sister chromatid segregation and microtubule-based movement, were repressed, while genes involved in DNA and histone methylation were induced in BS366 under cold conditions. MethylRAD was used for reduced DNA methylation sequencing of BS366 spikes under both cold and control conditions. The differentially methylated sites (DMSs) located in the gene region were mainly involved in carbohydrate and fatty acid metabolism, lipid metabolism, and transport. Differentially expressed and methylated genes were mainly involved in cell division. Conclusions These results indicated that the methylation of genes involved in carbon metabolism or fatty acid metabolism might contribute to male sterility in BS366 spikes, providing novel insight into the molecular mechanism of wheat male sterility.

Distinctive Kinesin-14 Motors Associate with Midzone Microtubules to Construct Mitotic Spindles with Two Convergent Poles in Arabidopsis

Microtubule (MT) motors in the Kinesin-14 subfamily proliferated in photosynthetic organisms and they often incorporated sequences bearing novel structural features. To gain insights into the functions of diversified Kinesin-14 motors from an evolutionary perspective, we performed phylogenetic analyses across different eukaryotic kingdoms. Compared to fungi that have a single class of Kinesin-14, the early divergent protist Giardia possesses two classes and the motile green alga Chlamydomonas produces four classes (Kinesin-14A to Kinesin-14D). The fifth class Kinesin-14E first appeared among immotile green algae and the sixth Kinesin-14F emerged in mosses, concomitantly with the display of 3D growth. The conservation of Kinesin-14D from green algae prompted us to investigate its function in Arabidopsis in which three such motors functioned in cell cycle-dependent manners. They localized on selective spindle MTs and/or sometimes kinetochore-like structures, and later all became conspicuous on MT bundles in the spindle midzone following sister chromatid segregation. Genetic dissection of Kinesin-14D1 showed that its loss led to hypersensitivity to low doses of the MT-depolymerizing herbicide oryzalin. Kinesin-14D1 association with the midzone MTs in both prophase and mitotic spindles. The oryzalin treatment left behind discrete kinetochore fibers attached to randomly positioned chromosomes in the mitotic kinesin-14d1 cells but prevented the pole convergence of bipolar mitotic spindles. This function of Kinesin-14D1 in the spindle midzone is likely dependent on an MT-binding domain at the C-terminus to the catalytic motor domain. Therefore, our results revealed a novel Kinesin-14D-dependent mechanism that regulates the formation of bipolar spindle apparatus with converged acentrosomal poles.

The Cdc14 Phosphatase Controls Resolution of Recombination Intermediates and Crossover Formation during Meiosis

Meiotic defects derived from incorrect DNA repair during gametogenesis can lead to mutations, aneuploidies and infertility. The coordinated resolution of meiotic recombination intermediates is required for crossover formation, ultimately necessary for the accurate completion of both rounds of chromosome segregation. Numerous master kinases orchestrate the correct assembly and activity of the repair machinery. Although much less is known, the reversal of phosphorylation events in meiosis must also be key to coordinate the timing and functionality of repair enzymes. Cdc14 is a crucial phosphatase required for the dephosphorylation of multiple CDK1 targets in many eukaryotes. Mutations that inactivate this phosphatase lead to meiotic failure, but until now it was unknown if Cdc14 plays a direct role in meiotic recombination. Here, we show that the elimination of Cdc14 leads to severe defects in the processing and resolution of recombination intermediates, causing a drastic depletion in crossovers when other repair pathways are compromised. We also show that Cdc14 is required for the correct activity and localization of the Holliday Junction resolvase Yen1/GEN1. We reveal that Cdc14 regulates Yen1 activity from meiosis I onwards, and this function is essential for crossover resolution in the absence of other repair pathways. We also demonstrate that Cdc14 and Yen1 are required to safeguard sister chromatid segregation during the second meiotic division, a late action that is independent of the earlier role in crossover formation. Thus, this work uncovers previously undescribed functions of the evolutionary conserved Cdc14 phosphatase in the regulation of meiotic recombination.

RhoA- and Cdc42-induced antagonistic forces underlie symmetry breaking and spindle rotation in mouse oocytes

Mammalian oocyte meiotic divisions are highly asymmetric and produce a large haploid gamete and 2 small polar bodies. This relies on the ability of the cell to break symmetry and position its spindle close to the cortex before anaphase occurs. In metaphase II–arrested mouse oocytes, the spindle is actively maintained close and parallel to the cortex, until fertilization triggers sister chromatid segregation and the rotation of the spindle. The latter must indeed reorient perpendicular to the cortex to enable cytokinesis ring closure at the base of the polar body. However, the mechanisms underlying symmetry breaking and spindle rotation have remained elusive. In this study, we show that spindle rotation results from 2 antagonistic forces. First, an inward contraction of the cytokinesis furrow dependent on RhoA signaling, and second, an outward attraction exerted on both sets of chromatids by a Ran/Cdc42-dependent polarization of the actomyosin cortex. By combining live segmentation and tracking with numerical modeling, we demonstrate that this configuration becomes unstable as the ingression progresses. This leads to spontaneous symmetry breaking, which implies that neither the rotation direction nor the set of chromatids that eventually gets discarded are biologically predetermined.

Non-Random Sister Chromatid Segregation in Human Tissue Stem Cells

The loss of genetic fidelity in tissue stem cells is considered a significant cause of human aging and carcinogenesis. Many cellular mechanisms are well accepted for limiting mutations caused by replication errors and DNA damage. However, one mechanism, non-random sister chromatid segregation, remains controversial. This atypical pattern of chromosome segregation is restricted to asymmetrically self-renewing cells. Though first confirmed in murine cells, non-random segregation was originally proposed by Cairns as an important genetic fidelity mechanism in human tissues. We investigated human hepatic stem cells expanded by suppression of asymmetric cell kinetics (SACK) for evidence of non-random sister chromatid segregation. Cell kinetics and time-lapse microscopy analyses established that an ex vivo expanded human hepatic stem cell strain possessed SACK agent-suppressible asymmetric cell kinetics. Complementary DNA strand-labeling experiments revealed that cells in hepatic stem cell cultures segregated sister chromatids non-randomly. The number of cells cosegregating sister chromatids with the oldest “immortal DNA strands” was greater under conditions that increased asymmetric self-renewal kinetics. Detection of this mechanism in a human tissue stem cell strain increases support for Cairns’ proposal that non-random sister chromatid segregation operates in human tissue stem cells to limit carcinogenesis.

Crystal structure of the Cenp-HIKHead-TW sub-module of the inner kinetochore CCAN complex

Kinetochores are large multi-subunit complexes that attach centromeric chromatin to microtubules of the mitotic spindle, enabling sister chromatid segregation in mitosis. The inner kinetochore constitutive centromere associated network (CCAN) complex assembles onto the centromere-specific Cenp-A nucleosome (Cenp-ANuc), thereby coupling the centromere to the microtubule-binding outer kinetochore. CCAN is a conserved 14–16 subunit complex composed of discrete modules. Here, we determined the crystal structure of the Saccharomyces cerevisiae Cenp-HIKHead-TW sub-module, revealing how Cenp-HIK and Cenp-TW interact at the conserved Cenp-HIKHead–Cenp-TW interface. A major interface is formed by the C-terminal anti-parallel α-helices of the histone fold extension (HFE) of the Cenp-T histone fold domain (HFD) combining with α-helix H3 of Cenp-K to create a compact three α-helical bundle. We fitted the Cenp-HIKHead-TW sub-module to the previously determined cryo-EM map of the S. cerevisiae CCAN–Cenp-ANuc complex. This showed that the HEAT repeat domain of Cenp-IHead and C-terminal HFD of Cenp-T of the Cenp-HIKHead-TW sub-module interact with the nucleosome DNA gyre at a site close to the Cenp-ANuc dyad axis. Our structure provides a framework for understanding how Cenp-T links centromeric Cenp-ANuc to the outer kinetochore through its HFD and N-terminal Ndc80-binding motif, respectively.

IRX3/5 regulate mitotic chromatid segregation and limb bud shape

ABSTRACTPattern formation is influenced by transcriptional regulation as well as by morphogenetic mechanisms that shape organ primordia, although factors that link these processes remain under-appreciated. Here we show that, apart from their established transcriptional roles in pattern formation, IRX3/5 help to shape the limb bud primordium by promoting the separation and intercalation of dividing mesodermal cells. Surprisingly, IRX3/5 are required for appropriate cell cycle progression and chromatid segregation during mitosis, possibly in a nontranscriptional manner. IRX3/5 associate with, promote the abundance of, and share overlapping functions with co-regulators of cell division such as the cohesin subunits SMC1, SMC3, NIPBL and CUX1. The findings imply that IRX3/5 coordinate early limb bud morphogenesis with skeletal pattern formation.

Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II

Partitioning of the genome in meiosis occurs through two highly specialized cell divisions, named meiosis I and II. Step-wise cohesin removal is required for chromosome segregation in meiosis I, and sister chromatid segregation in meiosis II. In meiosis I, mono-oriented sister kinetochores appear as fused together when examined by high resolution confocal microscopy, whereas they are clearly separated in meiosis II, when attachments are bipolar. It has been proposed that bipolar tension applied by the spindle is responsible for the physical separation of sister kinetochores, removal of cohesin protection and chromatid separation in meiosis II. We show here that this is not the case, and initial separation of sister kinetochores occurs already in anaphase I, when attachments are still monopolar, and independently of pericentromeric Sgo2 removal. This kinetochore individualization occurs also independently of spindle forces applied on sister kinetochores, but importantly, depends on cleavage activity of Separase. Crucially, without kinetochore individualization by Separase in meiosis I, oocytes separate bivalents into chromosomes and not sister chromatids in meiosis II, showing that whether centromeric cohesin is removed or not is determined by the kinetochore structure prior to meiosis II.

PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20

Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20 form the main ubiquitin E3 ligase for these proteins. APC/CCDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylations. Here we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilises cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1-phosphorylation defective CDC206A mutants. These CDC206A cells show a normal spindle checkpoint response, but once all chromosomes have aligned rapidly degrade cyclin B and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase by promoting APC/CCDC20-dependent destruction of cyclin B in human cells.