G1 Nuclei Research Articles

Characterization of cell-to-cell variation in nuclear transport rates and identification of its sources

Nuclear transport is an essential part of eukaryotic cell function. Here, we present scFRAP, a model-assisted fluorescent recovery after photobleaching (FRAP)- based method to determine nuclear import and export rates independently in individual live cells. To overcome the inherent noise of single-cell measurements, we performed sequential FRAPs on the same cell. We found large cell-to-cell variation in transport rates within isogenic yeast populations. For passive transport, the variability in NPC number might explain most of the variability. Using this approach, we studied mother-daughter cell asymmetry in the active nuclear shuttling of the transcription factor Ace2, which is specifically concentrated in daughter cell nuclei in early G1. Rather than reduced export in the daughter cell, as previously hypothesized, we found that this asymmetry is mainly due to an increased import in daughters. These results shed light on cell-to-cell variation in cellular dynamics and its sources.

A proposed unified interphase nucleus chromosome structure: Preliminary preponderance of evidence

Cryoelectron tomography of the cell nucleus using scanning transmission electron microscopy and deconvolution processing technology has highlighted a large-scale, 100- to 300-nm interphase chromosome structure, which is present throughout the nucleus. This study further documents and analyzes these chromosome structures. The paper is divided into four parts: 1) evidence (preliminary) for a unified interphase chromosome structure; 2) a proposed unified interphase chromosome architecture; 3) organization as chromosome territories (e.g., fitting the 46 human chromosomes into a 10-μm-diameter nucleus); and 4) structure unification into a polytene chromosome architecture and lampbrush chromosomes. Finally, the paper concludes with a living light microscopy cell study showing that the G1 nucleus contains very similar structures throughout. The main finding is that this chromosome structure appears to coil the 11-nm nucleosome fiber into a defined hollow structure, analogous to a Slinky helical spring [https://en.wikipedia.org/wiki/Slinky; motif used in Bowerman etal., eLife 10, e65587 (2021)]. This Slinky architecture can be used to build chromosome territories, extended to the polytene chromosome structure, as well as to the structure of lampbrush chromosomes.

Mechanical constraints to cell-cycle progression in a pseudostratified epithelium

As organs and tissues approach their normal size during development or regeneration, growth slows down, and cell proliferation progressively comes to a halt. Among the various processes suggested to contribute to growth termination,1-10 mechanical feedback, perhaps via adherens junctions, has been suggested to play a role.11-14 However, since adherens junctions are only present in a narrow plane of the subapical region, other structures are likely needed to sense mechanical stresses along the apical-basal (A-B) axis, especially in a thick pseudostratified epithelium. This could be achieved by nuclei, which have been implicated in mechanotransduction in tissue culture.15 In addition, mechanical constraints imposed by nuclear crowding and spatial confinement could affect interkinetic nuclear migration (IKNM),16 which allows G2 nuclei to reach the apical surface, where they normally undergo mitosis.17-25 To explore how mechanical constraints affect IKNM, we devised an individual-based model that treats nuclei as deformable objects constrained by the cell cortex and the presence of other nuclei. The model predicts changes in the proportion of cell-cycle phases during growth, which we validate with the cell-cycle phase reporter FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator).26 However, this model does not preclude indefinite growth, leading us to postulate that nuclei must migrate basally to access a putative basal signal required for S phase entry. With this refinement, our updated model accounts for the observed progressive slowing down of growth and explains how pseudostratified epithelia reach a stereotypical thickness upon completion of growth.

Excess TPX2 Interferes with Microtubule Disassembly and Nuclei Reformation at Mitotic Exit.

The microtubule-associated protein TPX2 is a key mitotic regulator that contributes through distinct pathways to spindle assembly. A well-characterised function of TPX2 is the activation, stabilisation and spindle localisation of the Aurora-A kinase. High levels of TPX2 are reported in tumours and the effects of its overexpression have been investigated in cancer cell lines, while little is known in non-transformed cells. Here we studied TPX2 overexpression in hTERT RPE-1 cells, using either the full length TPX2 or a truncated form unable to bind Aurora-A, to identify effects that are dependent—or independent—on its interaction with the kinase. We observe significant defects in mitotic spindle assembly and progression through mitosis that are more severe when overexpressed TPX2 is able to interact with Aurora-A. Furthermore, we describe a peculiar, and Aurora-A-interaction-independent, phenotype in telophase cells, with aberrantly stable microtubules interfering with nuclear reconstitution and the assembly of a continuous lamin B1 network, resulting in daughter cells displaying doughnut-shaped nuclei. Our results using non-transformed cells thus reveal a previously uncharacterised consequence of abnormally high TPX2 levels on the correct microtubule cytoskeleton remodelling and G1 nuclei reformation, at the mitosis-to-interphase transition.

High-content screen in human pluripotent cells identifies miRNA-regulated pathways controlling pluripotency and differentiation

BackgroundBy post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs or miR) play important biological functions. H1 embryonic stem cells (hESCs) and NTera-2 embryonal carcinoma cells (ECCs) are two of the most widely used human pluripotent model cell lines, sharing several characteristics, including the expression of miRNAs associated to the pluripotent state or with differentiation. However, how each of these miRNAs functionally impacts the biological properties of these cells has not been systematically evaluated.MethodsWe investigated the effects of 31 miRNAs on NTera-2 and H1 hESCs, by transfecting miRNA mimics. Following 3–4 days of culture, cells were stained for the pluripotency marker OCT4 and the G2 cell-cycle marker Cyclin B1, and nuclei and cytoplasm were co-stained with Hoechst and Cell Mask Blue, respectively. By using automated quantitative fluorescence microscopy (i.e., high-content screening (HCS)), we obtained several morphological and marker intensity measurements, in both cell compartments, allowing the generation of a multiparametric miR-induced phenotypic profile describing changes related to proliferation, cell cycle, pluripotency, and differentiation.ResultsDespite the overall similarities between both cell types, some miRNAs elicited cell-specific effects, while some related miRNAs induced contrasting effects in the same cell. By identifying transcripts predicted to be commonly targeted by miRNAs inducing similar effects (profiles grouped by hierarchical clustering), we were able to uncover potentially modulated signaling pathways and biological processes, likely mediating the effects of the microRNAs on the distinct groups identified. Specifically, we show that miR-363 contributes to pluripotency maintenance, at least in part, by targeting NOTCH1 and PSEN1 and inhibiting Notch-induced differentiation, a mechanism that could be implicated in naïve and primed pluripotent states.ConclusionsWe present the first multiparametric high-content microRNA functional screening in human pluripotent cells. Integration of this type of data with similar data obtained from siRNA screenings (using the same HCS assay) could provide a large-scale functional approach to identify and validate microRNA-mediated regulatory mechanisms controlling pluripotency and differentiation.

The mode of mitosis is dramatically modified by deletion of a single nuclear pore complex gene in Aspergillus nidulans

Nuclear pore complex (NPC) proteins (Nups) play multiple roles during mitosis. In this study we expand these roles and reveal that in Aspergillus nidulans, compromising the core Nup84-120 subcomplex of the NPC modifies the mitotic behavior of the nuclear envelope (NE). In wildtype cells, the NE undergoes simultaneous double pinching events to separate daughter nuclei during mitotic exit, whereas in Nup84-120 complex mutants, only one restriction of the NE is observed. Investigating the basis for this modified behavior of the NE in Nup deleted cells uncovered previously unrealized roles for core Nups in mitotic exit. During wildtype anaphase, the NE surrounds the two separating daughter DNA masses which typically flank the central nucleolus, to form three distinct nuclear compartments. In contrast, deletion of core Nups frequently results in early nucleolar eviction from the mitotic nucleus, in turn causing an uncharacteristic dumbbell-shaped NE morphology of anaphase nuclei with a nuclear membrane bridge connecting the two forming G1 nuclei. Importantly, the absence of the nucleolus between the separating daughter nuclei during anaphase delays chromosome segregation and progression into G1 as nuclei remain connected by chromatin bridges. Proteins localizing to late segregating chromosome arms are observed between forming daughter nuclei, and the mitotic spindle fails to resolve in a timely manner. These chromatin bridges are occupied by the Aurora kinase until nuclei have fully separated, suggesting involvement of Aurora in monitoring mitotic spindle and nuclear membrane resolution during mitotic exit. Our findings thus reveal a novel requirement for core Nups in mediating nucleolar positioning during mitosis, which dictates the pattern of NE fissions during karyokinesis and facilitates normal chromosome segregation. The findings additionally demonstrate that the mode of mitosis can be dramatically modified by deletion of a single NPC gene and reveals surprising fluidity in mitotic mechanisms.

Nuclear Actin Polymerized by mDia2 Confines Centromere Movement during CENP-A Loading

SummaryCentromeres are specialized chromosomal regions epigenetically defined by the histone H3 variant centromere protein A (CENP-A). CENP-A needs to be replenished in every cell cycle, but how new CENP-A is stably incorporated into centromeric chromatin remains unclear. We have discovered that a cytoskeletal protein, diaphanous formin mDia2, is essential for the stable incorporation of new CENP-A proteins into centromeric nucleosomes. Here we report that mDia2-mediated formation of dynamic and short nuclear actin filaments in G1 nucleus is required to maintain CENP-A levels at the centromere. Importantly, mDia2 and nuclear actin are required for constrained centromere movement during CENP-A loading, and depleting nuclear actin or MgcRacGAP, which lies upstream of mDia2, extends centromeric association of the CENP-A loading chaperone Holliday junction recognition protein (HJURP). Our findings thus suggest that nuclear actin polymerized by mDia2 contributes to the physical confinement of G1 centromeres so that HJURP-mediated CENP-A loading reactions can be productive, and centromere's epigenetic identity can be stably maintained.

Nup2 performs diverse interphase functions in Aspergillus nidulans.

The nuclear pore complex (NPC) protein Nup2 plays interphase nuclear transport roles and in Aspergillus nidulans also functions to bridge NPCs at mitotic chromatin for their faithful coinheritance to daughter G1 nuclei. In this study, we further investigate the interphase functions of Nup2 in A. nidulans. Although Nup2 is not required for nuclear import of all nuclear proteins after mitosis, it is required for normal G1 nuclear accumulation of the NPC nuclear basket–associated components Mad2 and Mlp1 as well as the THO complex protein Tho2. Targeting of Mlp1 to nuclei partially rescues the interphase delay seen in nup2 mutants indicating that some of the interphase defects in Nup2-deleted cells are due to Mlp1 mislocalization. Among the inner nuclear membrane proteins, Nup2 affects the localization of Ima1, orthologues of which are involved in nuclear movement. Interestingly, nup2 mutant G1 nuclei also exhibit an abnormally long period of extensive to-and-fro movement immediately after mitosis in a manner dependent on the microtubule cytoskeleton. This indicates that Nup2 is required to limit the transient postmitotic nuclear migration typical of many filamentous fungi. The findings reveal that Nup2 is a multifunctional protein that performs diverse functions during both interphase and mitosis in A. nidulans.

Holocentric chromosomes may be an apomorphy of Droseraceae

Holocentric chromosomes have evolved in various plant and animal taxa, which suggests they may confer a selective advantage in certain conditions, yet their adaptive potential has scarcely been studied. One of the reasons may reside in our insufficient knowledge of the phylogenetic distribution of holocentric chromosomes across eukaryotic phylogeny. In the present study, we focused on Droseraceae, a carnivorous plant family with an unknown chromosomal structure in monotypic genera Dionaea and Aldrovanda, and a closely related monotypic family Drosophyllaceae. We used flow cytometry to detect holocentric chromosomes by measuring changes in the ratio of the number of G2 nuclei to the number of G1 nuclei in response to gamma irradiation and determined chromosomal structures in Aldrovanda vesiculosa, Dionaea muscipula, Drosera tokaiensis, and Drosera ultramafica from Droseraceae and Drosophyllum lusitanicum from Drosophyllaceae. We confirmed monocentric chromosomes in D. lusitanicum and detected holocentric chromosomes in all four Droseraceae. Our novel finding of holocentric chromosomes in monotypic genera Aldrovanda and Dionaea suggests that all Droseraceae may be holocentric, but to confirm that further research is needed due to previously reported conflicting results in Drosera rotundifolia.

Suppression of targeting of Dbf4-dependent kinase to pre-replicative complex in G0 nuclei.

Intact G0 nuclei isolated from quiescent cells are not capable of DNA replication in interphase Xenopus egg extracts, which allow efficient replication of permeabilized G0 nuclei. Previous studies have shown multiple control mechanisms for maintaining the quiescent state, but DNA replication inhibition of intact G0 nuclei in the extracts remains poorly understood. Here, we showed that pre-RC is assembled on chromatin, but its activation is inhibited after incubating G0 nuclei isolated from quiescent NIH3T3 cells in the extracts. Concomitant with the inhibition of replication, Mcm4 phosphorylation mediated by Dbf4-dependent kinase (DDK) as well as chromatin binding of DDK is suppressed in G0 nuclei without affecting the nuclear transport of DDK. We further found that the nuclear extracts of G0 but not proliferating cells inhibit the binding of recombinant DDK to pre-RC assembled plasmids. In addition, we observed rapid activation of checkpoint kinases after incubating G0 nuclei in the egg extracts. However, specific inhibitors of ATR/ATM are unable to promote DNA replication in G0 nuclei in the egg extracts. We suggest that a novel inhibitory mechanism is functional to prevent the targeting of DDK to pre-RC in G0 nuclei, thereby suppressing DNA replication in Xenopus egg extracts.

Flow cytometry may allow microscope-independent detection of holocentric chromosomes in plants.

Two chromosomal structures, known as monocentric and holocentric chromosomes, have evolved in eukaryotes. Acentric fragments of monocentric chromosomes are unequally distributed to daughter cells and/or lost, while holocentric fragments are inherited normally. In monocentric species, unequal distribution should generate chimeras of cells with different nuclear DNA content. We investigated whether such differences in monocentric species are detectable by flow cytometry (FCM) as (i) a decreased nuclear DNA content and (ii) an increased coefficient of variance (CV) of the G1 peak after gamma radiation-induced fragmentation. We compared 13 monocentric and 9 holocentric plant species. Unexpectedly, monocentrics and holocentrics did not differ with respect to parameters (i) and (ii) in their response to gamma irradiation. However, we found that the proportion of G2 nuclei was highly elevated in monocentrics after irradiation, while holocentrics were negligibly affected. Therefore, we hypothesize that DNA-damaging agents induce cell cycle arrest leading to endopolyploidy only in monocentric and not (or to much lesser extent) in holocentric plants. While current microscope-dependent methods for holocentrism detection are unreliable for small and numerous chromosomes, which are common in holocentrics, FCM can use somatic nuclei. Thus, FCM may be a rapid and reliable method of high-throughput screening for holocentric candidates across plant phylogeny.

Condensin I and II behaviour in interphase nuclei and cells undergoing premature chromosome condensation.

Condensin is an integral component of the mitotic chromosome condensation machinery, which ensures orderly segregation of chromosomes during cell division. In metazoans, condensin exists as two complexes, condensin I and II. It is not yet clear what roles these complexes may play outside mitosis, and so we have examined their behaviour both in normal interphase and in premature chromosome condensation (PCC). We find that a small fraction of condensin I is retained in interphase nuclei, and our data suggests that this interphase nuclear condensin I is active in both gene regulation and chromosome condensation. Furthermore, live cell imaging demonstrates condensin II dramatically increases on G1 nuclei following completion of mitosis. Our PCC studies show condensins I and II and topoisomerase II localise to the chromosome axis in G1-PCC and G2/M-PCC, while KIF4 binding is altered. Individually, condensins I and II are dispensable for PCC. However, when both are knocked out, G1-PCC chromatids are less well structured. Our results define new roles for the condensins during interphase and provide new information about the mechanism of PCC.

Resetting a functional G1 nucleus after mitosis.

The maintenance of the correct cellular information goes beyond the simple transmission of an intact genetic code from one generation to the next. Epigenetic changes, topological cues and correct protein-protein interactions need to be re-established after each cell division to allow the next cell cycle to resume in the correct regulated manner. This process begins with mitotic exit and re-sets all the changes that occurred during mitosis thus restoring a functional G1 nucleus in preparation for the next cell cycle. Mitotic exit is triggered by inactivation of mitotic kinases and the reversal of their phosphorylation activities on many cellular components, from nuclear lamina to transcription factors and chromatin itself. To reverse all these phosphorylations, phosphatases act during mitotic exit in a timely and spatially controlled manner directing the events that lead to a functional G1 nucleus. In this review, we will summarise the recent developments on the control of phosphatases and their known substrates during mitotic exit, and the key steps that control the restoration of chromatin status, nuclear envelope reassembly and nuclear body re-organisation. Although pivotal work has been conducted in this area in yeast, due to differences between the mitotic exit network between yeast and vertebrates, we will mainly concentrate on the vertebrate system.

Endoreduplication in floral structure, vegetative and fruits of red pitaya with white pulp

Endoreduplication is the change of cellular cycle that result DNA duplication without cell division and could result endopolyploid cells. This phenomenon is common in plants and animals and considered as evaluative strategy. Although endoreduplication reported in various plant species, the information about these phenomena in red pitaya is rare. Therefore, this work was done with the objective of studying the endoreduplication in Hylocereus undatus Haw. using flow cytometry analysis. In this study were used the tissue from the flower structure, fruits, roots, cladode, and thorns of the pitaya plant.To determine the DNA content approximately 50 mg the sample of each treatment with Pisum sativum (the internal standard reference) were grind in plate of petri dishes contained 1 mL of cold Marie buffer to release the nucleus.The nuclear suspension was filtered through 50 μm mesh. The nucleuses were colored with 25 μL of 1 mg/L mL of propidium iodide. For each treatment, three samples of 10 thousand nucleuses were analyzed in cytometry Facscalibur (Becton Dickinson). The content of nuclear DNA (pg) was estimated as the ratio between fluorescence intensity of the G1 nucleus of the standard and the G1nucleus of the sample multiplied by the quantity of DNA of the internal reference. In conclusion, the endoreduplication occurred in all part of the plants analyzed except in the aculeus and the roots. The analysis evidenced different index of DNA content in the tissues analyzed being observed up to four different ploidy levels. The phenomena of endoreduplication occurs in all parts of the plant analyzed except in aculeus and roots.

The Inner Nuclear Membrane Protein Src1 Is Required for Stable Post-Mitotic Progression into G1 in Aspergillus nidulans.

How membranes and associated proteins of the nuclear envelope (NE) are assembled specifically and inclusively around segregated genomes during exit from mitosis is incompletely understood. Inner nuclear membrane (INM) proteins play key roles by providing links between DNA and the NE. In this study we have investigated the highly conserved INM protein Src1 in Aspergillus nidulans and have uncovered a novel cell cycle response during post mitotic formation of G1 nuclei. Live cell imaging indicates Src1 could have roles during mitotic exit as it preferentially locates to the NE abscission points during nucleokinesis and to the NE surrounding forming daughter G1 nuclei. Deletion analysis further supported this idea revealing that although Src1 is not required for interphase progression or mitosis it is required for stable post-mitotic G1 nuclear formation. This conclusion is based upon the observation that in the absence of Src1 newly formed G1 nuclei are structurally unstable and immediately undergo architectural modifications typical of mitosis. These changes include NPC modifications that stop nuclear transport as well as disassembly of nucleoli. More intriguingly, the newly generated G1 nuclei then cycle between mitotic- and interphase-like states. The findings indicate that defects in post-mitotic G1 nuclear formation caused by lack of Src1 promote repeated failed attempts to generate stable G1 nuclei. To explain this unexpected phenotype we suggest a type of regulation that promotes repetition of defective cell cycle transitions rather than preventing progression past the defective cell cycle transition. We suggest the term “reboot regulation” to define this mode of cell cycle regulation. The findings are discussed in relationship to recent studies showing the Cdk1 master oscillator can entrain subservient oscillators that when uncoupled cause cell cycle transitions to be repeated.

Endopolyploidy changes with age-related polyethism in the honey bee, Apis mellifera.

Honey bees (Apis mellifera) exhibit age polyethism, whereby female workers assume increasingly complex colony tasks as they age. While changes in DNA methylation accompany age polyethism, other DNA modifications accompanying age polyethism are less known. Changes in endopolyploidy (DNA amplification in the absence of cell division) with increased larval age are typical in many insect cells and are essential in adults for creating larger cells, more copies of essential loci, or greater storage capacity in secretory cells. However, changes in endopolyploidy with increased adult worker age and polyethism are unstudied. In this study, we examined endopolyploidy in honey bee workers ranging in age from newly emerged up to 55 days old. We found a nonsignificant increase in ploidy levels with age (P < 0.1) in the most highly endopolyploid secretory cells, the Malpighian tubules. All other cell types decreased ploidy levels with age. Endopolyploidy decreased the least amount (nonsignificant) in neural (brain) cells and the stinger (P < 0.1). There was a significant reduction of endopolyploidy with age in leg (P < 0.05) and thoracic (P < 0.001) muscles. Ploidy in thoracic muscle dropped from an average of 0.5 rounds of replication in newly emerged workers to essentially no rounds of replication (0.125) in the oldest workers. Ploidy reduction in flight muscle cells is likely due to the production of G1 (2C) nuclei by amitotic division in the multinucleate striated flight muscles that are essential to foragers, the oldest workers. We suggest that ploidy is constrained by the shape, size and makeup of the multinucleate striated muscle cells. Furthermore, the presence of multiple 2C nuclei might be optimal for cell function, while higher ploidy levels might be a dead-end strategy of some aging adult tissues, likely used to increase cell size and storage capacity in secretory cells.

Proteomic Analysis of Barley Cell Nuclei Purified by Flow Sorting

Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.

Cell cycle synchronization of leukemia inhibitory factor (LIF)-dependent porcine-induced pluripotent stem cells and the generation of cloned embryos

Nuclear transfer (NT) from porcine iPSC to create cloned piglets is unusually inefficient. Here we examined whether such failure might be related to the cell cycle stage of donor nuclei. Porcine iPSC, derived here from the inner cell mass of blastocysts, have a prolonged S phase and are highly sensitive to drugs normally used for synchronization. However, a double-blocking procedure with 0.3 μM aphidicolin for 10 h followed by 20 ng/ml nocodazole for 4 h arrested 94.3% of the cells at G2/M and, after release from the block, provided 70.1% cells in the subsequent G1 phase without causing any significant loss of cell viability or pluripotent phenotype. Nuclei from different cell cycle stages were used as donors for NT to in vitro-matured metaphase II oocytes. G2/M nuclei were more efficient than either G1 and S stage nuclei in undergoing first cleavage and in producing blastocysts, but all groups had a high incidence of chromosomal/nuclear abnormalities at 2 h and 6 h compared with non-synchronized NT controls from fetal fibroblasts. Many G2 embryos extruded a pseudo-second polar body soon after NT and, at blastocyst, tended to be either polyploid or diploid. By contrast, the few G1 blastocysts that developed were usually mosaic or aneuploid. The poor developmental potential of G1 nuclei may relate to lack of a G1/S check point, as the cells become active in DNA synthesis shortly after exit from mitosis. Together, these data provide at least a partial explanation for the almost complete failure to produce cloned piglets from piPSC.

Effect of Statins and Aerobic Physical Exercise on Liver Function in Dyslipidemic Rats: Morphometric Study

^len^aThe present study was carried out to evaluate the effect of statins associated with physical exercise (PE) in liver cells in dyslipidemic rats through cariometry. The animals were divided into six groups: animals subjected to a hypercholesterolemic diet (HD), simvastatin, with (G1) and without (G2) physical exercise (PE); HD submitted (G3) or not (G4) to PE, and commercial food diet (F) with (G5) and without (G6) PE. Histological analysis of the liver was performed by staining the slides with hematoxylin and eosin. The cariometric study included measuring the major and minor diameters of the hepatocytes nuclei. The Shapiro-Wilk test was also performed. To determine the differences among the groups, the Kruskal-Wallis Test with Dunn's post-test were conducted. The significance level was set at 5%. No difference was found in the hepatocytes nuclei between G5 and G6. When these groups were related with G3 and G4, reduced nuclei were observed. There was no difference between G1 and G6. The comparison between G6 and G2 showed that the nuclei in G2 were smaller. No difference was detected between G5 and G1. Changes were observed in the nuclei shape in G2 in comparison to G1. Considering G2 and G3, a decrease in the size of nuclei was observed in G3. On the other hand, G2 showed changes in shape in the comparative analysis with G4. The size and shape of G1 nuclei were larger than G3 as well as changes in shape were observed when compared to G4. G4 showed smaller nuclei than G3. Therefore, F, associated or not with the practice of PE, does not alter the size and shape of the hepatocytes nuclei; HD combined with sedentarism influences changes in the morphometric parameters of hepatocytes; and the association of simvastatin and PE seems to protect the hepatocytes nuclei with regard to HD.^les

Alternariol induces abnormal nuclear morphology and cell cycle arrest in murine RAW 264.7 macrophages

The mycotoxin alternariol (AOH), a frequent contaminant in fruit and cereal products, is known to induce DNA damage with subsequent cell cycle arrest. Here we elucidated the effects of AOH on stages of cell cycle progression using the RAW 264.7 macrophage model. AOH resulted in an accumulation of cells in the G2/M-phase (4N). Most cells exhibited a large G2 nucleus whereas numbers of true mitotic cells were reduced relative to control. Both cyclin B1 and p-cdc2 levels increased, while cyclin B1 remained in the cytoplasm; suggesting arrest in the G2/M transition point. Remarkably, after exposure to AOH for 24h, most of the cells exhibited abnormally shaped nuclei, as evidenced by partly divided nuclei, nuclear blebs, polyploidy and micronuclei (MN). AOH treatment also induced abnormal Aurora B bridges, suggesting that cytokinesis was interfered within cells undergoing karyokinesis. A minor part of the resultant G1 tetraploid (4N) cells re-entered the S-phase and progressed to 8N cells.