NuMA interaction with chromatin is vital for proper chromosome decondensation at the mitotic exit

In this work, we have identified a novel function of an important mitotic regulator NuMA in chromatin decondensation and nuclear shape by directly interacting with the DNA.

Mitotic exit is controlled during anaphase by an Aurora B-Cyclin B1/Cdk1 crosstalk

SummaryAccording to the prevailing “clock” model, chromosome decondensation and nuclear envelope reassembly during mitotic exit are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the “ruler” model. Here we reconciled both models by showing that Cyclin B1 degradation continues during anaphase inDrosophila, mouse and human cells, including primary tissues. This required APC/CCdh1activity, and failure to degrade Cyclin B1 during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1 localization and half-life during anaphase depended on kinesin-6, which targets Aurora B to the spindle midzone. Mechanistically, we show that anaphase duration is regulated by Aurora B-mediated phosphorylation of Cyclin B1. We propose that a crosstalk between molecular “rulers” and “clocks” licenses mitotic exit only after proper chromosome separation.

On the origin of shape fluctuations of the cell nucleus

The nuclear envelope (NE) presents a physical boundary between the cytoplasm and the nucleoplasm, sandwiched in between two highly active systems inside the cell: cytoskeleton and chromatin. NE defines the shape and size of the cell nucleus, which increases during the cell cycle, accommodating for chromosome decondensation followed by genome duplication. In this work, we study nuclear shape fluctuations at short time scales of seconds in human cells. Using spinning disk confocal microscopy, we observe fast fluctuations of the NE, visualized by fluorescently labeled lamin A, and of the chromatin globule surface (CGS) underneath the NE, visualized by fluorescently labeled histone H2B. Our findings reveal that fluctuation amplitudes of both CGS and NE monotonously decrease during the cell cycle, serving as a reliable cell cycle stage indicator. Remarkably, we find that, while CGS and NE typically fluctuate in phase, they do exhibit localized regions of out-of-phase motion, which lead to separation of NE and CGS. To explore the mechanism behind these shape fluctuations, we use biochemical perturbations. We find the shape fluctuations of CGS and NE to be both thermally and actively driven, the latter caused by forces from chromatin and cytoskeleton. Such undulations might affect gene regulation as well as contribute to the anomalously high rates of nuclear transport by, e.g., stirring of molecules next to NE, or increasing flux of molecules through the nuclear pores.

95 OBSERVATION OF CHROMOSOME DECONDENSATION, HISTONE H3 MODIFICATION, AND HP1 PROTEIN IN MOUSE CLONED EMBRYOS FOLLOWING INHIBITION OF HISTONE DEACETYLATIONS AND CYCLIN-DEPENDENT KINASE

Low chromosome decondensation and hypoacetylation and hypermethylation of histones characterize the incomplete reprogramming of cloned embryos during the first mitotic prophase. Our study looks at the effect of histone deacetylase inhibitors (HDACi), Trichostatin A (TSA), APHA Compound 8 (APHA), and cyclin-dependent kinase inhibitor (CDKi) roscovitin (ROS), on nuclear reprogramming in cloned mouse embryos during the first mitotic prophase. Oocytes were collected from female B6D2F1 mouse cumulus cells collected from female B6D2F1 and ICR, and fibroblasts from male GFP-ICR. We performed somatic cell nuclear transfer (SCNT) using a piezo-actuated micromanipulator system; the SCNT oocytes were subsequently activated for 6 h with 5 mM SrCl2 in Ca2+-free CZB medium (CZB) supplemented individually with 100 nM TSA, 250 nM APHA, or 100 nM ROS or in combination with TSA+ROS or APHA+ROS. Activated SCNT embryos were then cultured in KSOM medium with the same concentrations of TSA, APHA, ROS, TSA+ROS, or APHA+ROS for 2 h. Following treatment, we cultured the cloned embryos in KSOM for in vitro development. In the first experiment, the levels of chromosome decondensation during the first mitotic prophase in these cloned embryos were performed, based on the ratio of nuclear volume, at 6 h and 10 h after activation. We calculated the average nuclear volume � SD for each treatment, and these were subsequently compared to control values (100%, without treatment). We next turned to examining the intensity of methyl H3-K9, acetyl H3-K9, and HP1β of cloned embryos at 6 h and 10 h after activation by immunostaining. Images of immunostained embryo nuclei were acquired using an OLYMPUS Fluoview FV 1000 confocal system. The distribution of fluorescence intensities from 5 different regions of the nucleus were determined using Fluoview FV 1.4a software. Student's t-test was used to calculate the significance of differences between groups in the experiment. We repeated each experiment 4 times to obtain 40 nuclear transfer embryos per treatment. The present results show that global histone hyperacetylation and hypomethylation can be characterized by an increase in H3-K9 acetylation and a decrease in H3-K9 methylation in the presence of HDAC inhibitors. The levels of all isoforms of HP1β, however, were not reduced following inhibition of HDAC. This was in contrast to the high decondensation observed in interphase chromatin, leading to a significant increase of pronuclear volumes and a decrease in H3-K9 methylation in cloned embryos in the presence of ROS, although we were unable to obtain hyperacetylation and HP1β did not change. From these results, we conclude that the combined inhibition of histone deacetylases and cyclin-dependent kinase for 8 h following NT caused an increase in somatic chromosome decondensation, hyperacetylation, and demethylation of histone H3-K9 during the first mitotic cell cycle in cloned mouse embryos. Our findings strongly suggest that there is no correlation of HP1β involved in hyperacetylation and demethylation of H3-K9.

The selective continued linkage of centromeres from mitosis to interphase in the absence of mammalian separase

Separase is an evolutionarily conserved protease that is essential for chromosome segregation and cleaves cohesin Scc1/Rad21, which joins the sister chromatids together. Although mammalian separase also functions in chromosome segregation, our understanding of this process in mammals is still incomplete. We generated separase knockout mice, reporting an essential function for mammalian separase. Separase-deficient mouse embryonic fibroblasts exhibited severely restrained increases in cell number, polyploid chromosomes, and amplified centrosomes. Chromosome spreads demonstrated that multiple chromosomes connected to a centromeric region. Live observation demonstrated that the chromosomes of separase-deficient cells condensed, but failed to segregate, although subsequent cytokinesis and chromosome decondensation proceeded normally. These results establish that mammalian separase is essential for the separation of centromeres, but not of the arm regions of chromosomes. Other cell cycle events, such as mitotic exit, DNA replication, and centrosome duplication appear to occur normally. We also demonstrated that heterozygous separase-deficient cells exhibited severely restrained increases in cell number with apparently normal mitosis in the absence of securin, which is an inhibitory partner of separase.

Decondensation of Xenopus sperm chromatin using Saccharomyces cerevisiae whole-cell extractsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

Biochemical studies using highly condensed Xenopus sperm chromatin and protein extracts prepared from multiple systems have lead to the identification of conserved proteins involved in chromosome decondensation. However, mutations to these proteins are unavailable as the systems used are not amenable to genetic studies. We took a genetic approach to isolating chromosome decondensation mutants by incubating Xenopus sperm chromatin with whole-cell extracts prepared from the Hartwell library of random temperature sensitive (ts) yeast cells. We show that decondensation of Xenopus sperm chromatin using wild type yeast extracts was rapid, ATP- and extract-dependent, and resistant to heat, N-ethylmaleimide, protease K, RNase A, and micrococcal nuclease. From 100 mutant extracts screened, we obtained one strain, referred to as rmc4, that was chromosome decondensation defective. The mutant was slow growing and exhibited germination defects. Low concentrations of rmc4 extract would eventually decondense sperm heads, and fractionation of the mutant extract produced a decondensation competent fraction, suggesting the presence of an overactive inhibitor in rmc4 cells. We performed a multicopy suppressor screen that identified PDE2, a gene encoding a protein that inhibits protein kinase A (PKA) activity. As PKA was previously shown in human cells to maintain condensed chromatin, our results suggest that PKA activity is elevated in rmc4 cells, causing a decondensation defect. Thus, our experiments reveal that yeast encodes an evolutionarily conserved chromosome decondensation activity that can be genetically manipulated.

PNUTS enhances in vitro chromosome decondensation in a PP1-dependent manner

PP1 (protein phosphatase-1) is a serine/threonine phosphatase involved in mitosis exit and chromosome decondensation. In the present study, we characterize the subcellular and subnuclear localization of PNUTS (PP1 nuclear targeting subunit), a nuclear regulatory subunit of PP1, and report a stimulatory role of PNUTS in the decondensation of prometaphase chromosomes in two in vitro systems. In interphase, PNUTS co-fractionates, together with a fraction of nuclear PP1, primarily with micrococcal nuclease-soluble chromatin. Immunofluorescence analysis shows that PNUTS is targeted to the reforming nuclei in telophase following the assembly of nuclear membranes and concomitantly with chromatin decondensation. In interphase cytosolic extract, ATP-dependent decondensation of prometaphase chromosomes is blocked by PP1-specific inhibitors. In contrast, a recombinant PNUTS(309–691) fragment accelerates chromosome decondensation. This decondensation-promoting activity requires the consensus RVXF PP1-binding motif of PNUTS, whereas a secondary, inhibitory PP1-binding site is dispensable. In a defined buffer system, PNUTS(309–691) also elicits decondensation in an exogenous PP1-dependent manner and, as in the cytosolic extract, a W401A (Thr401→Ala) mutation that destroys PP1 binding abolishes this activity. The results illustrate an involvement of the PNUTS:PP1 holoenzyme in chromosome decondensation in vitro and argue that PNUTS functions as a PP1-targeting subunit in this process. We hypothesize that targeting of PNUTS to reforming nuclei in telophase may be a part of a signalling event promoting chromatin decondensation as cells re-enter interphase.