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In favorable conditions, eukaryotic cells proceed irreversibly through the cell division cycle (G1-S-G2-M) in order to produce two daughter cells with the same number and identity of chromosomes of their progenitor. The integrity of this process is maintained by "checkpoints" that hold a cell at particular transition points of the cycle until all requisite events are completed. The crucial functions of these checkpoints seem to depend on irreversible bistability of the underlying checkpoint control systems. Bistability of cell cycle transitions has been confirmed experimentally in frog egg extracts, budding yeast cells and mammalian cells. For fission yeast cells, a recent paper by Patterson et al. (2021) provides experimental evidence for an abrupt transition from G2 phase into mitosis, and we show that these data are consistent with a stochastic model of a bistable switch governing the G2/M checkpoint. Interestingly, our model suggests that their experimental data could also be explained by a reversible/sigmoidal switch, and stochastic simulations confirm this supposition. We propose a simple modification of their experimental protocol that could provide convincing evidence for (or against) bistability of the G2/M transition in fission yeast.

Atherosclerosis is a multicausal disease characterized by the formation of cholesterol-containing plaque in the pronounced intima nearest to the heart's elastic-type arteries that have high levels of blood circulation. Plaques are formed due to arterial pressure-induced damage to the endothelium in areas of turbulent blood flow. It is found in the majority of the Western population, including young people. This denies the monogenic mechanism of atherogenesis. In 1988, Orekhov et al. and Kawai et al. discovered that the presence of atherogenic (modified, including oxidized ones) LDLs is necessary for atherogenesis. On the basis of our discovery, suggesting that the overloading of enterocytes with lipids could lead to the formation of modified LDLs, we proposed a new hypothesis explaining the main factors of atherogenesis. Indeed, when endothelial cells are damaged and then pass through the G2 phase of their cell cycle they secrete proteins into their basement membrane. This leads to thickening of the basement membrane and increases its affinity to LDL especially for modified ones. When the enterocyte transcytosis pathway is overloaded with fat, very large chylomicrons are formed, which have few sialic acids, circulate in the blood for a long time, undergo oxidation, and can induce the production of autoantibodies. It is the sialic acids that shield the short forks of the polysaccharide chains to which autoantibodies are produced. Here, these data are evaluated from the point of view of our new model.

Heterotrimeric G proteins (αβγ) function at the cytoplasmic surface of a cell's plasma membrane to transduce extracellular signals into cellular responses. However, numerous studies indicate that G proteins also play noncanonical roles at unique intracellular locations. Previous work has established that G protein βγ subunits (Gβγ) regulate a signaling pathway on the cytoplasmic surface of Golgi membranes that controls the exit of select protein cargo. Now, we demonstrate a novel role for Gβγ in regulating mitotic Golgi fragmentation, a key checkpoint of the cell cycle that occurs in the late G2 phase. We show that small interfering RNA-mediated depletion of Gβ1 and Gβ2 in synchronized cells causes a decrease in the number of cells with fragmented Golgi in late G2 and a delay of entry into mitosis and progression through G2/M. We also demonstrate that during G2/M Gβγ acts upstream of protein kinase D and regulates the phosphorylation of the Golgi structural protein GRASP55. Expression of Golgi-targeted GRK2ct, a Gβγ-sequestering protein used to inhibit Gβγ signaling, also causes a decrease in Golgi fragmentation and a delay in mitotic progression. These results highlight a novel role for Gβγ in regulation of Golgi structure.

Drosophila female germline stem cells (GSCs) are found inside the cellular niche at the tip of the ovary. They undergo asymmetric divisions to renew the stem cell lineage and to produce sibling cystoblasts that will in turn enter differentiation. GSCs and cystoblasts contain spectrosomes, membranous structures essential for orientation of the mitotic spindle and that, particularly in GSCs, change shape depending on the cell cycle phase. Using live imaging and a fusion protein of GFP and the spectrosome component Par-1, we follow the complete spectrosome cycle throughout GSC division and quantify the relative duration of the different spectrosome shapes. We also determine that the Par-1 kinase shuttles between the spectrosome and the cytoplasm during mitosis and observe the continuous addition of new material to the GSC and cystoblast spectrosomes. Next, we use the Fly-FUCCI tool to define, in live and fixed tissues, that GSCs have a shorter G1 compared with the G2 phase. The observation of centrosomes in dividing GSCs allowed us to determine that centrosomes separate very early in G1, before centriole duplication. Furthermore, we show that the anterior centrosome associates with the spectrosome only during mitosis and that, upon mitotic spindle assembly, it translocates to the cell cortex, where it remains anchored until centrosome separation. Finally, we demonstrate that the asymmetric division of GSCs is not an intrinsic property of these cells, as the spectrosome of GSC-like cells located outside of the niche can divide symmetrically. Thus, GSCs display unique properties during division, a behaviour influenced by the surrounding niche., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Ethnopharmacological Relevance: The Menispermaceae plant Tiliacora racemosa is immensely popular in Indian traditional Ayurvedic medicine as "Krishnavetra" for its remarkable anti-cancerous property, and is commonly used by tribal population for the treatment of skin infections, snake bites and filariasis., Aim of the Study: This present study intends to identify the modus operandi behind the cytotoxic activity of Tiliacora racemosa leaves in cervical cancer cells SiHa. Focus has been instilled in the ability of the plant extract to target multiple signaling pathways leading to cell cycle arrest and cell death in SiHa cells, followed by a pharmacological characterization to identify the bioactive principle., Materials and Methods: T. racemosa leaves extracted in methanol, ethyl acetate, hexane and aqueous solvent were screened for cytotoxicity in HeLa, SiHa, C33A (cervical cancer cells) and HEK cells by MTT assay. SiHa cells were treated with the most potent extract (TRM). Cellular morphology, clonogenic and wound healing potential, presence of intracellular ROS and NO, lipid peroxidation, activity of cellular antioxidants (SOD, CAT, GSH), DNA damage detection by comet assay and localisation of γ-H2AX foci, intracellular expression of PARP-1, Bax/Bcl2 and caspase-3, loss in mitochondrial membrane potential by JC1 (flow cytometry) and Rh123 (microscopy), cell cycle analysis, Annexin-FITC assay, AO/EtBr microscopy and apoptotic proteome profiling were undertaken in the treated cells. All the related proteins were studied by immunoblots. Effect of NAC (ROS-scavenger) on cell viability, DNA damage and apoptosis were studied. Phytochemical characterization of all TR extracts was followed by LC-MS analysis of TRM and isolated alkaloid of TR was assessed for cytotoxicity., Results: The methanol extract of T. racemosa (TRM) rich in bisbenzylisoquinoline and other alkaloids impeded the proliferation of cervical cancer cells SiHa in vitro through disruption of cellular redox homeostasis caused by increase in cellular ROS and NO with concomitant decrease in the cellular antioxidants. Double-stranded DNA damage was noted from γH2AX foci accumulation and Parp-1 activation leading to ATM-Chk2-p53 pathway arresting the cells at G2/M-phase through cyclin B1 inhibition. The mitochondrial membrane potential was also disturbed leading to caspase-3 dependent apoptotic induction by both extrinsic and intrinsic pathway. Immunoblots show TRM also inhibited PI3K/Akt and NFκB pathway. NAC pre-treatment rescued the cell viability proving DNA damage and apoptosis to be direct consequences of ROS overproduction. Lastly, the therapeutic potential of T. racemosa is was hypothesized to be possibly derived from its alkaloid content., Conclusion: This study proves the age old ethnnopharmacological anticancer role of T. racemosa. The leaf extracts inhibited the anomalous proliferation of SiHa cells by virtue of G2/M-phase cell cycle arrest and apoptotic cell death. Oxidative stress mediated double stranded DNA damage paved the way towards apoptotic cell death through multiple routes, including PI3K/Akt/NFκB pathway. The abundant alkaloid content of T. racemosa was denoted as the probable responsible cytotoxic principle., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The precise control of the cell cycle G2 phase to Mitosis (M phase) transition is central for cell fate determination. The commonly used methods for assessing G2 to M phase progression are based on synchronizing cells and involve perturbation of the natural cell cycle progression. Additionally, these methods are often time-consuming and labor-intensive. Here, we report a flow cytometry-based method that offers a kinetic analysis of G2 to M phase progression in asynchronous cells using nocodazole, 5-Ethynyl-2´-deoxyuridine staining, and histone H3 serine 28 phosphorylation (pH3) staining. Nocodazole is used to collect mitotic cells and prevent their progression into G1, at the same time EdU is added for use as a dump channel during analysis. The remaining cells can then be identified as either G1 or G2/M based on their DNA content. Finally, G2 and M phase cells can be separated based on a mitotic marker, phosphorylation of ser28 on histone H3. While developed to assay G2/M phase progression, this method also resolves G1/S phase progression with no additional steps other than analysis. Compared to double thymidine block, this method does not require extended pre-treatments and is compatible with a greater variety of cell lines, while at the same time offering enhanced consistency and temporal resolution.

Pinostrobin, a flavonoid compound known for its diverse pharmacological actions, including anti-leukemic and anti-inflammatory activities, has been repeatedly isolated by various screenings, but its action mechanism is still obscure. Previously, pinostrobin was rediscovered in our laboratory using a yeast-based assay procedure devised specifically for the inhibitory effect on the activated Ca2+ signaling that leads the cells to severe growth retardation in the G2 phase. Here, we attempted to identify target of pinostrobin employing the genetic techniques available in the yeast. Using various genetically engineered yeast strains in which the Ca2+-signaling cascade can be activated by the controlled expression of the various signaling molecules of the cascade, its target was narrowed down to Swe1, the cell-cycle regulatory protein kinase. The Swe1 kinase is situated at the downstream of the Ca2+-signaling cascade and downregulates the Cdc28/Clb complex by phosphorylating the Cdc28 moiety of the complex in the G2 phase. We further demonstrated that pinostrobin inhibits the protein kinase activity of Swe1 in vivo as estimated by the decreased level of Cdc28 phosphorylation at Tyr-19. Since the yeast SWE1 gene is an ortholog for the human WEE1 gene, our finding implied a potentiality of pinostrobin as the G2 checkpoint abrogator in cancer chemotherapy., (© FEMS 2020.)

E2F8 is a transcriptional repressor that antagonizes E2F1 at the crossroads of the cell cycle, apoptosis, and cancer. Previously, we discovered that E2F8 is a direct target of the APC/C ubiquitin ligase. Nevertheless, it remains unknown how E2F8 is dynamically controlled throughout the entirety of the cell cycle. Here, using newly developed human cell-free systems that recapitulate distinct inter-mitotic and G1 phases and a continuous transition from prometaphase to G1, we reveal an interlocking dephosphorylation switch coordinating E2F8 degradation with mitotic exit and the activation of APC/C Cdh1 . Further, we uncover differential proteolysis rates for E2F8 at different points within G1 phase, accounting for its accumulation in late G1 while APC/C Cdh1 is still active. Finally, we demonstrate that the F-box protein Cyclin F regulates E2F8 in G2-phase. Altogether, our data define E2F8 regulation throughout the cell cycle, illuminating an extensive coordination between phosphorylation, ubiquitination and transcription in mammalian cell cycle.

Cdk2-dependent TopBP1-treslin interaction is critical for DNA replication initiation. However, it remains unclear how this association is terminated after replication initiation is finished. Here, we demonstrate that phosphorylation of TopBP1 by Akt coincides with cyclin A activation during S and G 2 phases and switches the TopBP1-interacting partner from treslin to E2F1, which results in the termination of replication initiation. Premature activation of Akt in G 1 phase causes an early switch and inhibits DNA replication. TopBP1 is often overexpressed in cancer and can bypass control by Cdk2 to interact with treslin, leading to enhanced DNA replication. Consistent with this notion, reducing the levels of TopBP1 in cancer cells restores sensitivity to a Cdk2 inhibitor. Together, our study links Cdk2 and Akt pathways to the control of DNA replication through the regulation of TopBP1-treslin interaction. These data also suggest an important role for TopBP1 in driving abnormal DNA replication in cancer., (Copyright © 2020 American Society for Microbiology.)

Aim: We aimed to characterize the tumor-targeting and radiosensitization properties of the photo-responsive gold nanoparticles (AuNPs) decorated photolabile diazirine group and folic acid for improved radiotherapy and computed tomography imaging of tumors. Methods: Folic acid and photolabile diazirine group were covalently conjugated on the surface of AuNPs to afford the desired photo-responsive dAuNP-FA (AuNPs capped with poly(ethylene) glycol ligands bearing photolabile diazirine group and folic acid). The probes were intravenously injected into tumor-bearing mice followed by photocrosslinking upon 405 nm laser irradiation for radiotherapy and computed tomography imaging of tumors in vivo . Results: Light-triggered crosslinking of AuNPs in vivo remarkably enhanced the accumulation and retention of AuNPs within tumors. Conclusion: We have successfully developed a novel photo-responsive Au particle-based tumor theranostic probe showing remarkably improved tumor targeting ability and radiosensitization effect.

E2F7 and E2F8 act as tumor suppressors via transcriptional repression of genes involved in S-phase entry and progression. Previously, we demonstrated that these atypical E2Fs are degraded by APC/C C dh1 during G1 phase of the cell cycle. However, the mechanism driving the downregulation of atypical E2Fs during G2 phase is unknown. Here, we show that E2F7 is targeted for degradation by the E3 ubiquitin ligase SCF cyclin F during G2. Cyclin F binds via its cyclin domain to a conserved C-terminal CY motif on E2F7. An E2F7 mutant unable to interact with SCF cyclin F remains stable during G2. Furthermore, SCF cyclin F can also interact and induce degradation of E2F8. However, this does not require the cyclin domain of SCF cyclin F nor the CY motifs in the C-terminus of E2F8, implying a different regulatory mechanism than for E2F7. Importantly, depletion of cyclin F causes an atypical-E2F-dependent delay of the G2/M transition, accompanied by reduced expression of E2F target genes involved in DNA repair. Live cell imaging of DNA damage revealed that cyclin F-dependent regulation of atypical E2Fs is critical for efficient DNA repair and cell cycle progression., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Primordial germ cells (PGCs) are reprogrammed into pluripotent embryonic germ cells (EGCs) under specific culture conditions, but the detailed mechanisms of PGC reprogramming have not yet been fully clarified. Previous studies have demonstrated that AKT, an important intracellular signaling molecule, promotes reprogramming of PGCs into EGCs. Because AKT likely inhibits p53 functions to enhance PGC reprogramming, and p53 negatively regulates cell cycle progression, we analyzed cell cycle changes in PGCs following AKT activation and found that the ratio of PGCs in the G1/G0 phase was decreased while that of PGCs in the G2/M phase was increased after AKT activation. We also showed that the expression of the CDK inhibitor p27kip1, which prevents the G1‐S transition and is transcriptionally activated by p53, was significantly downregulated by AKT activation. The results suggested that the characteristic cell cycle changes of PGCs by AKT activation are, at least in part, due to decreased expression of p27 kip1 . We also investigated changes in histone H3K27 tri-methylation (H3K27me3) by AKT activation in PGCs, because we previously found that decreased H3K27me3 was involved in PGC reprogramming via upregulation of cyclin D1. We observed that AKT activation in PGCs resulted in H3K27 hypomethylation. In addition, DZNeP, an inhibitor of the H3K27 trimethyl transferase Ezh2, stimulated EGC formation. These results together suggested that AKT activation promotes G1-S transition and downregulates H3K27me3 to enhance PGC reprogramming., (© 2019 Japanese Society of Developmental Biologists.)

Quiescent neural stem cells (NSCs) in the adult brain are regenerative cells that could be activated therapeutically to repair damage. It is becoming apparent that quiescent NSCs exhibit heterogeneity in their propensity for activation and in the progeny that they generate. We discovered recently that NSCs undergo quiescence in either G 0 or G 2 in the Drosophila brain, challenging the notion that all quiescent stem cells are G 0 arrested. We found that G 2 -quiescent NSCs become activated prior to G 0 NSCs. Here, we show that the cyclin-dependent kinase inhibitor Dacapo (Dap; ortholog of p57 KIP2 ) determines whether NSCs enter G 0 or G 2 quiescence during embryogenesis. We demonstrate that the dorsal patterning factor, Muscle segment homeobox (Msh; ortholog of MSX1/2/3) binds directly to the Dap locus and induces Dap expression in dorsal NSCs, resulting in G 0 arrest, while more ventral NSCs undergo G 2 quiescence. Our results reveal region-specific regulation of stem cell quiescence., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Hook proteins are evolutionarily conserved dynein adaptors that promote assembly of highly processive dynein-dynactin motor complexes. Mammals express three Hook paralogs, namely Hook1, Hook2, and Hook3, that have distinct subcellular localizations and expectedly, distinct cellular functions. Here we demonstrate that Hook2 binds to and promotes dynein-dynactin assembly specifically during mitosis. During the late G2 phase, Hook2 mediates dynein-dynactin localization at the nuclear envelope (NE), which is required for centrosome anchoring to the NE. Independent of its binding to dynein, Hook2 regulates microtubule nucleation at the centrosome; accordingly, Hook2-depleted cells have reduced astral microtubules and spindle positioning defects. Besides the centrosome, Hook2 localizes to and recruits dynactin and dynein to the central spindle. Dynactin-dependent targeting of centralspindlin complex to the midzone is abrogated upon Hook2 depletion; accordingly, Hook2 depletion results in cytokinesis failure. We find that the zebrafish Hook2 homologue promotes dynein-dynactin association and was essential for zebrafish early development. Together, these results suggest that Hook2 mediates assembly of the dynein-dynactin complex and regulates mitotic progression and cytokinesis., (© 2019 Dwivedi et al.)

Vesicular stomatitis virus (VSV) (a rhabdovirus) and its variant VSV-ΔM51 are widely used model systems to study mechanisms of virus-host interactions. Here, we investigated how the cell cycle affects replication of these viruses using an array of cell lines with different levels of impairment of antiviral signaling and a panel of chemical compounds arresting the cell cycle at different phases. We observed that all compounds inducing cell cycle arrest in G 2 /M phase strongly enhanced the replication of VSV-ΔM51 in cells with functional antiviral signaling. G 2 /M arrest strongly inhibited type I and type III interferon (IFN) production as well as expression of IFN-stimulated genes in response to exogenously added IFN. Moreover, G 2 /M arrest enhanced the replication of Sendai virus (a paramyxovirus), which is also highly sensitive to the type I IFN response but did not stimulate the replication of a wild-type VSV that is more effective at evading antiviral responses. In contrast, the positive effect of G 2 /M arrest on virus replication was not observed in cells defective in IFN signaling. Altogether, our data show that replication of IFN-sensitive cytoplasmic viruses can be strongly stimulated during G 2 /M phase as a result of inhibition of antiviral gene expression, likely due to mitotic inhibition of transcription, a global repression of cellular transcription during G 2 /M phase. The G 2 /M phase thus could represent an "Achilles' heel" of the infected cell, a phase when the cell is inadequately protected. This model could explain at least one of the reasons why many viruses have been shown to induce G 2 /M arrest. IMPORTANCE Vesicular stomatitis virus (VSV) (a rhabdovirus) and its variant VSV-ΔM51 are widely used model systems to study mechanisms of virus-host interactions. Here, we investigated how the cell cycle affects replication of VSV and VSV-ΔM51. We show that G 2 /M cell cycle arrest strongly enhances the replication of VSV-ΔM51 (but not of wild-type VSV) and Sendai virus (a paramyxovirus) via inhibition of antiviral gene expression, likely due to mitotic inhibition of transcription, a global repression of cellular transcription during G 2 /M phase. Our data suggest that the G 2 /M phase could represent an "Achilles' heel" of the infected cell, a phase when the cell is inadequately protected. This model could explain at least one of the reasons why many viruses have been shown to induce G 2 /M arrest, and it has important implications for oncolytic virotherapy, suggesting that frequent cell cycle progression in cancer cells could make them more permissive to viruses., (Copyright © 2019 Bressy et al.)

The centromere is an evolutionarily conserved eukaryotic protein machinery essential for precision segregation of the parental genome into two daughter cells during mitosis. Centromere protein A (CENP-A) organizes the functional centromere via a constitutive centromere-associated network composing the CENP-T complex. However, how CENP-T assembles onto the centromere remains elusive. Here we show that CENP-T binds directly to Holliday junction recognition protein (HJURP), an evolutionarily conserved chaperone involved in loading CENP-A. The binding interface of HJURP was mapped to the C terminus of CENP-T. Depletion of HJURP by CRISPR-elicited knockout minimized recruitment of CENP-T to the centromere, indicating the importance of HJURP in CEPN-T loading. Our immunofluorescence analyses indicate that HJURP recruits CENP-T to the centromere in S/G 2 phase during the cell division cycle. Significantly, the HJURP binding-deficient mutant CENP-T 6L failed to locate to the centromere. Importantly, CENP-T insufficiency resulted in chromosome misalignment, in particular chromosomes 15 and 18. Taken together, these data define a novel molecular mechanism underlying the assembly of CENP-T onto the centromere by a temporally regulated HJURP-CENP-T interaction., (© 2019 Ding et al.)

Protein posttranslational modifications (PTMs) play a central role in the DNA damage response. In particular, protein phosphorylation and ubiquitination have been shown to be essential in the signaling cascade that coordinates break repair with cell cycle progression. Here, we performed whole-cell quantitative proteomics to identify global changes in protein ubiquitination that are induced by DNA double-strand breaks. In total, we quantified more than 9,400 ubiquitin sites and found that the relative abundance of ∼10% of these sites was altered in response to DNA double-strand breaks. Interestingly, a large proportion of ribosomal proteins, including those from the 40S as well as the 60S subunit, were ubiquitinated in response to DNA damage. In parallel, we discovered that DNA damage leads to the inhibition of ribosome function. Taken together, these data uncover the ribosome as a major target of the DNA damage response., (© 2018 Halim et al.)

Aurora A kinase (AURKA) is a master cell-cycle regulator that is often dysregulated in human cancers. Its overexpression has been associated with genome instability and oncogenic transformation. The protein kinase D (PKD) family is an emerging therapeutic target of cancer. Aberrant PKD activation has been implicated in tumor growth and survival, yet the underlying mechanisms remain to be elucidated. This study identified, for the first time, a functional crosstalk between PKD2 and Aurora A kinase in cancer cells. The data demonstrate that PKD2 is catalytically active during the G 2 -M phases of the cell cycle, and inactivation or depletion of PKD2 causes delay in mitotic entry due to downregulation of Aurora A, an effect that can be rescued by overexpression of Aurora A. Moreover, PKD2 localizes in the centrosome with Aurora A by binding to γ-tubulin. Knockdown of PKD2 caused defects in centrosome separation, elongated G 2 phase, mitotic catastrophe, and eventually cell death via apoptosis. Mechanistically, PKD2 interferes with Fbxw7 function to protect Aurora A from ubiquitin- and proteasome-dependent degradation. Taken together, these results identify PKD as a cell-cycle checkpoint kinase that positively modulates G 2 -M transition through Aurora A kinase in mammalian cells. Implications: PKD2 is a novel cell-cycle regulator that promotes G 2 -M transition by modulating Aurora A kinase stability in cancer cells and suggests the PKD2/Aurora A kinase regulatory axis as new therapeutic targets for cancer treatment. Mol Cancer Res; 16(11); 1785-97. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Cytoplasmic dynein is involved in diverse cell cycle-dependent functions regulated by several accessory factors, including Nde1 and Ndel1. Little is known about the role of these proteins in dynein cargo binding, and less is known about their cell cycle--dependent dynein regulation. Using Nde1 RNAi, mutant cDNAs, and a phosphorylation site-specific antibody, we found a specific association of phospho-Nde1 with the late G2-M nuclear envelope and prophase to anaphase kinetochores, comparable to the pattern for the Nde1 interactor CENP-F. Phosphomutant-Nde1 associated only with prometaphase kinetochores and showed weaker CENP-F binding in in vitro assays. Nde1 RNAi caused severe delays in mitotic progression, which were substantially rescued by both phosphomimetic and phosphomutant Nde1. Expression of a dynein-binding-deficient Nde1 mutant reduced kinetochore dynein by half, indicating a major role for Nde1 in kinetochore dynein recruitment. These results establish CENP-F as the first well-characterized Nde1 cargo protein, and reveal phosphorylation control of Nde1 cargo binding throughout a substantial fraction of the cell cycle., (© 2018 Wynne and Vallee.)

Ostreococcus tauri is the smallest free-living unicellular organism with one copy of each core cell cycle genes in its genome. There is a growing interest in this green algae due to its evolutionary origin. Since O. tauri is diverged early in the green lineage, relatively close to the ancestral eukaryotic cell, it might hold a key phylogenetic position in the eukaryotic tree of life. In this study, we focus on the regulatory network of its cell division cycle. We propose a mathematical modelling framework to integrate the existing knowledge of cell cycle network of O. tauri. We observe that feedback loop regulation of both G1/S and G2/M transitions in O. tauri is conserved, which can make the transition bistable. This is essential to make the transition irreversible as shown in other eukaryotic organisms. By performing sequence analysis, we also predict the presence of the Greatwall/PP2A pathway in the cell cycle of O. tauri. Since O. tauri cell cycle machinery is conserved, the exploration of the dynamical characteristic of the cell division cycle will help in further understanding the regulation of cell cycle in higher eukaryotes., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

From unicellular to multicellular organisms, cell-cycle progression is tightly coupled to biosynthetic and bioenergetic demands. Accumulating evidence has demonstrated the G 1 /S-phase transition as a key checkpoint where cells respond to their metabolic status and commit to replicating the genome. However, the mechanism underlying the coordination of metabolism and the G 2 /M-phase transition in mammalian cells remains unclear. Here, we show that the activation of AMP-activated protein kinase (AMPK), a highly conserved cellular energy sensor, significantly delays mitosis entry. The cell-cycle G 2 /M-phase transition is controlled by mitotic cyclin-dependent kinase complex (CDC2-cyclin B), which is inactivated by WEE1 family protein kinases and activated by the opposing phosphatase CDC25C. AMPK directly phosphorylates CDC25C on serine 216, a well-conserved inhibitory phosphorylation event, which has been shown to mediate DNA damage-induced G 2 -phase arrest. The acute induction of CDC25C or suppression of WEE1 partially restores mitosis entry in the context of AMPK activation. These findings suggest that AMPK-dependent phosphorylation of CDC25C orchestrates a metabolic checkpoint for the cell-cycle G 2 /M-phase transition., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Prohibitin 2(PHB2) is a member of the SFPH trans-membrane family proteins. It is a highly conserved and functionally diverse protein that plays an important role in preserving the structure and function of the mitochondria. In this study, the lamprey PHB2 gene was expressed in HeLa cells to investigate its effect on cell proliferation. The effect of Lm-PHB2 on the proliferation of HeLa cells was determined by treating the cells with pure Lm-PHB2 protein followed by MTT assay. Using the synchronization method with APC-BrdU and PI double staining revealed rLm-PHB2 treatment induced the decrease of both S phase and G0/G1 phase and then increase of G2/M phase. Similarly, cells transfected with pEGFP-N1-Lm-PHB2 also exhibited remarkable reduction in proliferation. Western blot and quantitative real-time PCR(qRT-PCR) assays suggested that Lm-PHB2 caused cell cycle arrest in HeLa cells through inhibition of CDC25C and CCNB1 expression. According to our western blot analysis, Lm-PHB2 was also found to reduce the expression level of Wee1 and PLK1 and the phosphorylation level of CCNB1, CDC25C and CDK1 in HeLa cells. Lamprey prohibitin 2 could arrest G2/M phase transition of HeLa cells through down-regulating expression and phosphorylation level of cell cycle proteins.

Transcriptional silencing is a conserved process used by embryonic germ cells to repress somatic fate and maintain totipotency and immortality. In Drosophila, this transcriptional silencing is mediated by polar granule component (pgc). Here, we show that in the adult ovary, pgc is required for timely germline stem cell (GSC) differentiation. Pgc is expressed transiently in the immediate GSC daughter (pre-cystoblast), where it mediates a pulse of transcriptional silencing. This transcriptional silencing mediated by pgc indirectly promotes the accumulation of Cyclin B (CycB) and cell cycle progression into late-G2 phase, when the differentiation factor bag of marbles (bam) is expressed. Pgc mediated accumulation of CycB is also required for heterochromatin deposition, which protects the germ line genome against selfish DNA elements. Our results suggest that transient transcriptional silencing in the pre-cystoblast "re-programs" it away from self-renewal and toward the gamete differentiation program., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Participation and relative importance of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling, either alone or in combination, have been investigated during 17α,20β-dihydroxy-4-pregnen-3-one (DHP)-induced meiotic G2-M1 transition in denuded zebrafish oocyte. Results demonstrate that concomitant with rapid phosphorylation (activation) of Akt (Ser473) and MAPK (ERK1/2) at as early as 15 min of incubation, DHP stimulation promotes enhanced an GVBD response and histone H1 kinase activation between 1 and 5 h in full-grown oocytes in vitro. While p-Akt reaches its peak at 60 to 90 min and undergoes downregulation to the basal level by 240 min, ERK1/2 phosphorylation (activation) increases gradually until 120 min and remains high thereafter. Although, priming with MEK1/2 inhibitor U0126 is without effect, PI3K inhibitors, wortmannin or LY294002, delay the GVBD response significantly (P < 0.001) until 3 h but not at 5 h of incubation. Interestingly, blocking PI3K and MEK function together could abrogate steroid-induced oocyte maturation at all time points tested. While DHP stimulation promotes phospho-PKA catalytic (p-PKAc) dephosphorylation (inactivation) between 30-120 min of incubation, simultaneous inhibition of PI3K and MEK1/2 kinases abrogates DHP action. Conversely, elevated intra-oocyte cAMP, through priming with either adenylyl cyclase (AC) activator forskolin (FK) or dibutyryl cAMP (db-cAMP), abrogates steroid-induced Akt and ERK1/2 phosphorylation. Taken together, these results suggest that DHP-induced Akt and ERK activation precedes the onset of meiosis (GVBD response) in a cAMP-sensitive manner and PI3K/Akt and MEK/MAPK pathways together have a pivotal influence in the downregulation of PKA and resumption of meiotic maturation in zebrafish oocytes in vitro.

A chromosome is composed of structurally and functionally distinct domains. Telomeres, which are located at the ends of linear chromosomes, play crucial roles in genome stability. Although substantial knowledge of telomeres has been accumulated, the regulation and function of subtelomeres, which are the domains adjacent to telomeres, remain largely unknown. In this review, I describe recent discoveries about the multiple roles of a shugoshin family protein, Sgo2, which is localized at centromeres in mitosis and contributes to precise chromosome segregation, in defining chromatin structure and functions of the subtelomeres in fission yeast. Sgo2 becomes enriched at the subtelomeres, particularly during G 2 phase, and is essential for the formation of a highly condensed subtelomeric chromatin body called the knob. Furthermore, Sgo2 maintains the expression levels of subtelomeric genes and the timing of DNA replication at subtelomeric late origins.

This chapter describes a microfluidic device that enables immobilization and culturing of single rod-shaped S. pombe cells in a stand-up mode. The wide-band electrical impedance spectroscopy (EIS) has been integrated in the microfluidic device to continuously measure cell growth of single S. pombe cells. Cell growth curves showing cellular and intracellular features at high spatiotemporal resolution can be obtained from EIS signals. The features include longitudinal cell elongation in the G2 phase, mitosis, and cell division during an entire cell cycle of S. pombe cells. Microfluidics-based EIS systems provide, hence, a tool for dynamic single-cell studies.

DNA repair is a complex process that prevents genomic instability. Many proteins play fundamental roles in regulating the optimal repair of DNA lesions. Proliferating cell nuclear antigen (PCNA) is a key factor that initiates recombination-associated DNA synthesis after injury. Here, in very early S-phase, we show that the fluorescence intensity of mCherry-tagged PCNA after local micro-irradiation was less than the fluorescence intensity of non-irradiated mCherry-PCNA-positive replication foci. However, PCNA protein accumulated at locally irradiated chromatin in very late S-phase of the cell cycle, and this effect was more pronounced in the following G2 phase. In comparison to the dispersed form of PCNA, a reduced mobile fraction appeared in PCNA-positive replication foci during S-phase, and we observed similar recovery time after photobleaching at locally induced DNA lesions. This diffusion of mCherry-PCNA in micro-irradiated regions was not affected by cell cycle phases. We also studied the link between function of PCNA and A-type lamins in late S-phase. We found that the accumulation of PCNA at micro-irradiated chromatin is identical in wild-type and A-type lamin-deficient cells. Only micro-irradiation of the nuclear interior, and thus the irradiation of internal A-type lamins, caused the fluorescence intensity of mCherry-tagged PCNA to increase. In summary, we showed that PCNA begins to play a role in DNA repair in late S-phase and that PCNA function in repair is maintained during the G2 phase of the cell cycle. However, PCNA mobility is reduced after local micro-irradiation regardless of the cell cycle phase.

Centromere protein F (CENP-F) is a component of the kinetochore and a regulator of cell cycle progression. CENP-F recruits the dynein transport machinery and orchestrates several cell cycle-specific transport events, including transport of the nucleus, mitochondria and chromosomes. A key regulatory step for several of these functions is likely the G2 phase-specific export of CENP-F from the nucleus to the cytosol, where the cytoplasmic dynein transport machinery resides; however, the molecular mechanism of this process is elusive. Here, we have identified 3 phosphorylation sites within the bipartite classical nuclear localization signal (cNLS) of CENP-F. These sites are specific for cyclin-dependent kinase 1 (Cdk1), which is active in G2 phase. Phosphomimetic mutations of these residues strongly diminish the interaction of the CENP-F cNLS with its nuclear transport receptor karyopherin α. These mutations also diminish nuclear localization of the CENP-F cNLS in cells. Notably, the cNLS is phosphorylated in the -1 position, which is important to orient the adjacent major motif for binding into its pocket on karyopherin α. We propose that localization of CENP-F is regulated by a cNLS, and a nuclear export pathway, resulting in nuclear localization during most of interphase. In G2 phase, the cNLS is weakened by phosphorylation through Cdk1, likely resulting in nuclear export of CENP-F via the still active nuclear export pathway. Once CENP-F resides in the cytosol, it can engage in pathways that are important for cell cycle progression, kinetochore assembly and the faithful segregation of chromosomes into daughter cells.

Commitment to mitosis must be tightly coordinated with DNA replication to preserve genome integrity. While we have previously established that the timely activation of CyclinB1-Cdk1 in late G2 triggers mitotic entry, the upstream regulatory mechanisms remain unclear. Here, we report that Polo-like kinase 1 (Plk1) is required for entry into mitosis during an unperturbed cell cycle and is rapidly activated shortly before CyclinB1-Cdk1. We determine that Plk1 associates with the Cdc25C1 phosphatase and induces its phosphorylation before mitotic entry. Plk1-dependent Cdc25C1 phosphosites are sufficient to promote mitotic entry, even when Plk1 activity is inhibited. Furthermore, we find that activation of Plk1 during G2 relies on CyclinA2-Cdk activity levels. Our findings thus elucidate a critical role for Plk1 in CyclinB1-Cdk1 activation and mitotic entry and outline how CyclinA2-Cdk, an S-promoting factor, poises cells for commitment to mitosis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: A new method was presented to prepare clinical-grade human adipose-derived stromal stem cells (ASCs) and its safety in vitro, such as biological characteristics and genetic features alteration were investigated., Methods: The morphology of the ASCs which were cultured in vitro using serum-free medium was observed. Cell cycle and CD markers profile were tested by flow cytometry, while karyotype was analyzed by the chromosome G-banding technology. Growth factors expression was tested by ELISA and tumor-related genes were analyzed by the real-time PCR, respectively., Results: ASCs were adult stem cells with spindle shape. The proliferation ratio of ASCs began to slow down after 10 passages, and was significant after 15 passages. Cell cycle analysis revealed that the percentage of G2 phase and S phase cells was stable. There was no obvious missing, translocation or dislocation in terms of karyotype. Expression level of tumor relevant genes and cytokines at different passages had no significant difference., Conclusions: The clinical-grade ASCs prepared with this new method, less than ten passages, was safe for clinical trials., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

To maintain size homeostasis in a unicellular culture, cells should coordinate growth to the division cycle. This is achieved via size control mechanisms (also known as size checkpoints), i.e. some events during the mitotic cycle supervene only if the cell has reached a critical size. Rod-shaped cells like those of fission yeast are ideal model organisms to study these checkpoints via time-lapse microphotography. By applying this method, once we can analyse the growth process between two consecutive divisions at a single (or even at an 'average') cellular level, moreover, we can also position the size checkpoint(s) at the population level. Finally, any of these controls can be abolished in appropriate cell cycle mutants, either in steady-state or in induction synchronised cultures. In the latter case, we produce abnormally oversized cells, and microscopic experiments with them clearly show the existence of a critical size above which the size checkpoint ceases (becomes cryptic). In this review, we delineate the development of our knowledge both on the growth mode of fission yeast and on the operating size control(s) during its mitotic cycle. We finish these historical stories with our recent findings, arguing that three different size checkpoints exist in the fission yeast cell cycle, namely in late G1, in mid G2 and in late G2, which has been concluded by analysing these controls in several cell cycle mutants.

Background/aim: The transcription factor Y-box-binding protein 1 (YB1) is overexpressed in many types of human cancers. YB1 regulates the G 1 phase of the cell cycle by controlling transcription of G 1 regulators. Here, we report that YB1 is also involved in regulating G 2 /M phase., Materials and Methods: YB1-depleted TKO cells were subjected to quantitative reverse transcription-polymerase chain reaction and cell-cycle analysis. RNA immunoprecipitation (RIP)-chip assay was performed using anti-YB1 antibodies. Precipitated RNAs were subjected to microarray analysis., Results: Silencing YB1 inhibited the proliferation of TKO cells, which lost the machinery required for G 1 phase arrest. Cell-cycle analysis showed that silencing YB1 caused G 2 /M phase cell-cycle arrest. RIP-chip assay showed that YB1 associated with mRNA of multiple cell-cycle-related genes, including G 2 /M phase regulators., Conclusion: YB1 positively regulates not only the G 1 phase but also G 2 /M phase by regulating multiple cell-cycle-related genes., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The function of the primary cilia, which is assembled in most vertebrate cells, is achieved by transport in and out of kinds of signaling receptors. The BBSome protein complex could recognize and target membrane proteins to the cilia, but how the BBSome itself is transported into the cilia is poorly understood. Here we demonstrate that the centrosome protein Dzip1 mediates the assembly of the BBSome-Dzip1-PCM1 complex in the centriolar satellites (CS) at the G 0 phase for ciliary translocation of the BBSome. Phosphorylation of Dzip1 at Ser-210 by Plk1 (polo-like kinase 1) during the G 2 phase promotes disassembly of this complex, resulting in removal of Dzip1 and the BBSome from the CS. Inhibiting the kinase activity of Plk1 maintains the CS localization of the BBSome and Dzip1 at the G 2 phase. Collectively, our findings reveal the cell cycle-dependent regulation of BBSome transport to the CS and highlight a potential mechanism that the BBSome-mediated signaling pathways are accordingly regulated during the cell cycle., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) are co-receptors for Wnt ligands. Upon ligand binding, LRP5/6 undergo glycogen synthase kinase 3 (GSK3)/casein kinase I (CKI)-mediated phosphorylation at multiple PPP(S/T)P motifs in the intracellular domain, which is essential for canonical Wnt signal transduction. On the other hand, in the Wnt-off state, the mitosis-specific CDK14-Cyclin Y kinase complex phosphorylates Ser-1490 of LRP5/6 at G 2 /M, thereby priming the receptor for Wnt-induced phosphorylation. However, it remains unclear how CDK14/Cyclin Y is recruited to LRP5/6 and whether there are other cofactors involved in this process. Previously, we identified Caprin-2 as a positive regulator of canonical Wnt signaling by promoting GSK3-depedent LRP5/6 phosphorylation upon Wnt stimulation. Here we uncovered that Caprin-2 positively regulates constitutive LRP5/6 Ser-1490 phosphorylation by complexing with CDK14/Cyclin Y. Caprin-2-mediated LRP5/6 phosphorylation is cell cycle-dependent in a pattern similar to that of CDK14/Cyclin Y-dependent LRP5/6 phosphorylation. Moreover, knockdown of Caprin-2 disrupts not only the interaction between CDK14 and Cyclin Y but also the interaction between CDK14/Cyclin Y and LRP6. Overall, our findings revealed an unrecognized role of Caprin-2 in facilitating LRP5/6 constitutive phosphorylation at G 2 /M through forming a quaternary complex with CDK14, Cyclin Y, and LRP5/6., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

A robust method for simultaneous visualization of all four cell cycle phases in living cells is highly desirable. We developed an intensiometric reporter of the transition from S to G2 phase and engineered a far-red fluorescent protein, mMaroon1, to visualize chromatin condensation in mitosis. We combined these new reporters with the previously described Fucci system to create Fucci4, a set of four orthogonal fluorescent indicators that together resolve all cell cycle phases.

The duration of the DNA synthesis stage (S phase) of the cell cycle is fundamental in our understanding of cell cycle kinetics, cell proliferation, and DNA replication timing programs. Most S-phase duration estimates that exist for plants are based on indirect measurements. We present a method for directly estimating S-phase duration by pulse-labeling root tips or actively dividing suspension cells with the halogenated thymidine analog 5-ethynl-2'-deoxyuridine (EdU) and analyzing the time course of replication with bivariate flow cytometry. The transition between G 1 and G 2 DNA contents can be followed by measuring the mean DNA content of EdU-labeled S-phase nuclei as a function of time after the labeling pulse. We applied this technique to intact root tips of maize (Zea mays L.), rice (Oryza sativa L.), barley (Hordeum vulgare L.), and wheat (Triticum aestivum L.), and to actively dividing cell cultures of Arabidopsis (Arabidopsis thaliana (L.) Heynh.) and rice. Estimates of S-phase duration in root tips were remarkably consistent, varying only by ~3-fold, although the genome sizes of the species analyzed varied >40-fold., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Kinesins comprise a superfamily of microtubule-based motor proteins involved in essential processes in plant development, but few kinesins have been functionally identified during seed development. Especially, few kinesins that regulate cell division during embryogenesis have been identified. Here we report the functional characterization of NtKRP, a motor protein of the kinesin-12 family. NtKRP is predominantly expressed in embryos and embryonic roots. NtKRP RNAi lines displayed reductions in cell numbers in the meristematic zone, in embryonic root length, and in mature embryo and seed sizes. Furthermore, we also show that CDKA;1 binds to NtKRP at the consensus phosphorylation sites and that the decreased cell numbers in NtKRP-silenced embryos are due to a delay in cell division cycle at the G2/M transition. In addition, binding between the cargo-binding tail domain of NtKRP and CDKA; 1 was also determined. Our results reveal a novel molecular pathway that regulates embryo/seed development and critical role of kinesin in temporal and spatial regulation of a specific issue of embryo developmental.

The Golgi apparatus is composed of stacks of cisternae laterally connected by tubules to form a ribbon-like structure. At the onset of mitosis, the Golgi ribbon is broken down into discrete stacks, which then undergo further fragmentation. This ribbon cleavage is required for G2/M transition, which thus indicates that a 'Golgi mitotic checkpoint' couples Golgi inheritance with cell cycle transition. We previously showed that the Golgi-checkpoint regulates the centrosomal recruitment of the mitotic kinase Aurora-A; however, how the Golgi unlinking regulates this recruitment was unknown. Here we show that, in G2, Aurora-A recruitment is promoted by activated Src at the Golgi. Our data provide evidence that Src and Aurora-A interact upon Golgi ribbon fragmentation; Src phosphorylates Aurora-A at tyrosine 148 and this specific phosphorylation is required for Aurora-A localization at the centrosomes. This process, pivotal for centrosome maturation, is a fundamental prerequisite for proper spindle formation and chromosome segregation.

During neurulation of chordate ascidians, the 11th mitotic division within the epidermal layer shows a posterior-to-anterior wave that is precisely coordinated with the unidirectional progression of the morphogenetic movement. Here we show that the first sign of this patterned mitosis is an asynchronous anterior-to-posterior S-phase length and that mitotic synchrony is reestablished by a compensatory asynchronous G2-phase length. Live imaging combined with genetic experiments demonstrated that compensatory G2-phase regulation requires transcriptional activation of the G2/M regulator cdc25 by the patterning genes GATA and AP-2. The downregulation of GATA and AP-2 at the onset of neurulation leads to loss of compensatory G2-phase regulation and promotes the transition to patterned mitosis. We propose that such developmentally regulated cell-cycle compensation provides an abrupt switch to spatially patterned mitosis in order to achieve the coordination between mitotic timing and morphogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Golgi complex of mammalian cells is composed of stacks of flattened cisternae that are connected by tubules to form a continuous membrane system, also known as the Golgi ribbon. At the onset of mitosis, the Golgi ribbon is progressively fragmented into small tubular-vesicular clusters and it is reconstituted before completion of cytokinesis. The investigation of the mechanisms behind this reversible cycle of disassembly and reassembly has led to the identification of structural Golgi proteins and regulators. Moreover, these studies allowed to discover that disassembly of the ribbon is necessary for cell entry into mitosis. Here, we describe an in vitro assay that reproduces the mitotic Golgi fragmentation and that has been successfully employed to identify many important mechanisms and proteins involved in the mitotic Golgi reorganization.

Oocyte meiosis is a transcription quiescence process and the cell-cycle progression is coordinated by multiple post-translational modifications, including SUMOylation. SENP3 an important deSUMOylation protease has been intensively studied in ribosome biogenesis and oxidative stress. However, the roles of SENP3 in cell-cycle regulation remain enigmatic, particularly for oocyte meiotic maturation. Here, we found that SENP3 co-localized with spindles during oocyte meiosis and silencing of SENP3 severely compromised the M phase entry (germinal vesicle breakdown, GVBD) and first polar body extrusion (PBI). The failure in polar body extrusion was due to the dysfunction of γ-tubulin that caused defective spindle morphogenesis. SENP3 depletion led to mislocalization and a substantial loss of Aurora A (an essential protein for MTOCs localization and spindle dynamics) while irregularly dispersed distribution of Bora (a binding partner and activator of Aurora A) in cytoplasm instead of concentrating at spindles. The SUMO-2/3 but not SUMO-1 conjugates were globally decreased by SENP3 RNAi. Additionally, the spindle assembly checkpoint remained functional upon SENP3 RNAi. Our findings renew the picture of SENP3 function by exploring its role in meiosis resumption, spindle assembly and following polar body emission during mouse oocyte meiotic maturation, which is potentially due to its proteolytic activity that facilitate SUMO-2/3 maturation.

Background: Metabolic syndrome (MetS), a complex cluster of risk factors for chronic diseases such as cardiovascular disease, is observed to be increasingly associated with periodontal disease. However, the fundamental contribution of periodontal bacteria to periodontal bone loss in patients with MetS remains unclear. The aim of the present study is to analyze the effect of Porphyromonas gingivalis on differentiation of primary osteoblasts from New Zealand obese (NZO) mice, a model for MetS, compared with C57 Black 6 JAX (C57BL/6J) mice osteoblasts., Methods: Primary calvarial osteoblasts, isolated from 3-day-old NZO and control C57BL/6J mice, were stimulated with P. gingivalis. Proliferation was quantified by 5-bromo-2'-deoxyuridine incorporation. Cell cycle and early and late apoptosis were measured by flow cytometry. Gene expression was determined by real-time polymerase chain reaction (RT-PCR)., Results: Twelve hours after P. gingivalis stimulation, NZO osteoblasts showed significantly decreased proliferation (P ≤0.01) with increased G2 cell cycle phase compared with normal osteoblasts. Flow cytometry analysis demonstrated significant (P ≤0.01) increase of early apoptotic cells (annexin V positive) and late apoptosis (caspase 3 activity) in NZO cells compared with control cells at 3 and 6 hours after stimulation. No significant lactate dehydrogenase release was found after P. gingivalis stimulation. RT-PCR data showed significantly suppressed expression (P ≤0.01) of collagen 1, osteocalcin, and Runt-related transcription factor 2 in NZO cells compared with normal osteoblasts., Conclusions: The present data demonstrate that P. gingivalis downregulates proliferation and promotes apoptosis in primary NZO osteoblasts, unlike C57BL/6J osteoblasts. Also, suppressed osteoblastic marker expression in NZO cells may contribute to pathogenesis of periodontitis, suggesting a similar process in patients with MetS.

GNL3L is an evolutionarily conserved high molecular weight GTP binding nucleolar protein belonging to HSR1-MMR1 subfamily of GTPases. The present investigation reveals that GNL3L is a nucleo-cytoplasmic shuttling protein and its export from the nucleus is sensitive to Leptomycin B. Deletion mutagenesis reveals that the C-terminal domain (amino acids 501-582) is necessary and sufficient for the export of GNL3L from the nucleus and the exchange of hydrophobic residues (M567, L570 and 572) within the C-terminal domain impairs this process. Results from the protein-protein interaction analysis indicate that GNL3L interaction with CRM1 is critical for its export from the nucleus. Ectopic expression of GNL3L leads to lesser accumulation of cells in the 'G2/M' phase of cell cycle whereas depletion of endogenous GNL3L results in 'G2/M' arrest. Interestingly, cell cycle analysis followed by BrdU labeling assay indicates that significantly increased DNA synthesis occurs in cells expressing nuclear export defective mutant (GNL3L∆NES) compared to the wild type or nuclear import defective GNL3L. Furthermore, increased hyperphosphorylation of Rb at Serine 780 and the upregulation of E2F1, cyclins A2 and E1 upon ectopic expression of GNL3L∆NES results in faster 'S' phase progression. Collectively, the present study provides evidence that GNL3L is exported from the nucleus in CRM1 dependent manner and the nuclear localization of GNL3L is important to promote 'S' phase progression during cell proliferation.

In mammalian cells, the Golgi complex is composed of stacks that are connected by membranous tubules. During G2, the Golgi complex is disassembled into isolated stacks. This process is required for entry into mitosis, indicating that the correct inheritance of the organelle is monitored by a 'Golgi mitotic checkpoint'. However, the regulation and the molecular mechanisms underlying this Golgi disassembly are still poorly understood. Here, we show that JNK2 has a crucial role in the G2-specific separation of the Golgi stacks through phosphorylation of Ser277 of the Golgi-stacking protein GRASP65 (also known as GORASP1). Inhibition of JNK2 by RNA interference or by treatment with three unrelated JNK inhibitors causes a potent and persistent cell cycle block in G2. JNK activity becomes dispensable for mitotic entry if the Golgi complex is disassembled by brefeldin A treatment or by GRASP65 depletion. Finally, measurement of the Golgi fluorescence recovery after photobleaching demonstrates that JNK is required for the cleavage of the tubules connecting Golgi stacks. Our findings reveal that a JNK2-GRASP65 signalling axis has a crucial role in coupling Golgi inheritance and G2/M transition., (© 2015. Published by The Company of Biologists Ltd.)

Purpose: Cell cycle analysis by flow cytometry has not been adequately studied in pediatric brain tumors. We investigated the value of cell cycle analysis by propidium-iodine (PI) staining of CD56+ (gated) cells for the characterization of pediatric brain tumor's malignancy., Methods: Patients that underwent surgery for an intracranial lesion and tissue sample was available were included in the study. All tumor samples were histopathologically verified before flow cytometric analysis., Results: There were 46 pediatric brain tumor cases. As control we used samples from three cases of brain tissue obtained during surgery for epilepsy. All tumors had significant lower G0/G1 and significant higher S-phase, G2/M fraction, S+G2/M and S+G2/M/G0/G1-phase fraction than normal brain tissue. Low-grade tumors had significant lower S-phase than high grade tumors. Grade IV tumors had significant lower G0/G1 fraction and higher S+G2/M/G0/G1 index than grade III tumors. DNA diploidy was detected in 24 tumors and DNA aneuploidy was detected in 23 tumors. There was a significant correlation between Ki-67 index and both S+G2/M and S+G2/M/G0/G1 phase fraction., Conclusions: Cell cycle analysis by PI staining of CD56+ cells is a promising method for the assessment of pediatric brain tumors malignancy and could be a valuable adjunct to histopathological evaluation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AX (γH2AX) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of γH2AX in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable γH2AX and no γH2AX-foci in G1 cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of γH2AX increased immediately upon S phase entry, and about 10 and 30 γH2AX foci were found in mid-S phase control and PARPi-treated cells, respectively. The γH2AX-labeled damage caused by DNA replication was not fully repaired before entry into G2. Intriguingly, G2 cells populated a continuous distribution of γH2AX levels, from cells with a high content of γH2AX and the same number of foci as S phase cells (termed "G2H" compartment), to cells that there were almost negative and had about 2 foci (termed "G2L" compartment). EdU-labeling of S phase cells revealed that G2H was directly populated from S phase, while G2L was populated from G2H, but in control cells also directly from S phase. The length of G2H in particular increased after PARPi treatment, compatible with longer DNA-repair times. Our results show that cells repair replication-induced damage in G2H, and enter mitosis after a 2-3 h delay in G2L.

PARP inhibitors have been approved for treatment of tumors with mutations in or loss of BRCA1/2. The molecular mechanisms and particularly the cellular phenotypes resulting in synthetic lethality are not well understood and varying clinical responses have been observed. We have investigated the dose- and time-dependency of cell growth, cell death and cell cycle traverse of 4 malignant lymphocyte cell lines treated with the PARP inhibitor Olaparib. PARP inhibition induced a severe growth inhibition in this cell line panel and increased the levels of phosphorylated H2AX-associated DNA damage in S phase. Repair of the remaining replication related damage caused a G2 phase delay before entry into mitosis. The G2 delay, and the growth inhibition, was more pronounced in the absence of functional ATM. Further, Olaparib treated Reh and Granta-519 cells died by apoptosis, while U698 and JVM-2 cells proceeded through mitosis with aberrant chromosomes, skipped cytokinesis, and eventually died by necrosis. The TP53-deficient U698 cells went through several rounds of DNA replication and mitosis without cytokinesis, ending up as multinucleated cells with DNA contents of up to 16c before dying. In summary, we report here for the first time cell cycle-resolved DNA damage induction, and cell line-dependent differences in the mode of cell death caused by PARP inhibition.

Wnt-β-catenin (β-catenin is also known as CTNNB1 in human) signaling through the β-catenin-TCF complex plays crucial roles in tissue homeostasis. Wnt-stimulated β-catenin-TCF complex accumulation in the nucleus regulates cell survival, proliferation and differentiation through the transcription of target genes. Compared with their levels in G1, activation of the receptor LRP6 and cytosolic β-catenin are both upregulated in G2 cells. However, accumulation of the Wnt pathway negative regulator AXIN2 also occurs in this phase. Therefore, it is unclear whether Wnt signaling is active in G2 phase cells. Here, we established a bimolecular fluorescence complementation (BiFC) biosensor system for the direct visualization of the β-catenin-TCF interaction in living cells. Using the BiFC biosensor and co-immunoprecipitation experiments, we demonstrate that levels of the nucleus-localized β-catenin-TCF complex increase during the S and G2 phases, and declines in the next G1 phase. Accordingly, a subset of Wnt target genes is transcribed by the β-catenin-TCF complex during both the S and G2 phases. By contrast, transient inhibition of this complex disturbs both cell survival and G2/M progression. Our results suggest that in S and G2 phase cells, Wnt-β-catenin signaling is highly active and functions to ensure cell survival and cell cycle progression., (© 2014. Published by The Company of Biologists Ltd.)