Mcl-1 dynamics influence mitotic slippage and death in mitosis.

PubMed

Sloss, Olivia; Topham, Caroline; Diez, Maria; Taylor, Stephen

2016-02-02

Microtubule-binding drugs such as taxol are frontline treatments for a variety of cancers but exactly how they yield patient benefit is unclear. In cell culture, inhibiting microtubule dynamics prevents spindle assembly, leading to mitotic arrest followed by either apoptosis in mitosis or slippage, whereby a cell returns to interphase without dividing. Myeloid cell leukaemia-1 (Mcl-1), a pro-survival member of the Bcl-2 family central to the intrinsic apoptosis pathway, is degraded during a prolonged mitotic arrest and may therefore act as a mitotic death timer. Consistently, we show that blocking proteasome-mediated degradation inhibits taxol-induced mitotic apoptosis in a Mcl-1-dependent manner. However, this degradation does not require the activity of either APC/C-Cdc20, FBW7 or MULE, three separate E3 ubiquitin ligases implicated in targeting Mcl-1 for degradation. This therefore challenges the notion that Mcl-1 undergoes regulated degradation during mitosis. We also show that Mcl-1 is continuously synthesized during mitosis and that blocking protein synthesis accelerates taxol induced death-in-mitosis. Modulating Mcl-1 levels also influences slippage; overexpressing Mcl-1 extends the time from mitotic entry to mitotic exit in the presence of taxol, while inhibiting Mcl-1 accelerates it. We suggest that Mcl-1 competes with Cyclin B1 for binding to components of the proteolysis machinery, thereby slowing down the slow degradation of Cyclin B1 responsible for slippage. Thus, modulating Mcl-1 dynamics influences both death-in-mitosis and slippage. However, because mitotic degradation of Mcl-1 appears not to be under the control of an E3 ligase, we suggest that the notion of network crosstalk is used with caution.

Search, capture and signal: games microtubules and centrosomes play.

PubMed

Schuyler, S C; Pellman, D

2001-01-01

Accurate distribution of the chromosomes in dividing cells requires coupling of cellular polarity cues with both the orientation of the mitotic spindle and cell cycle progression. Work in budding yeast has demonstrated that cytoplasmic dynein and the kinesin Kip3p define redundant pathways that ensure proper spindle orientation. Furthermore, it has been shown that the Kip3p pathway components Kar9p and Bim1p (Yeb1p) form a complex that provides a molecular link between cortical polarity cues and spindle microtubules. Recently, other studies indicated that the cortical localization of Kar9p depends upon actin cables and Myo2p, a type V myosin. In addition, a BUB2-dependent cell cycle checkpoint has been described that inhibits the mitotic exit network and cytokinesis until proper centrosome position is achieved. Combined, these studies provide molecular insight into how cells link cellular polarity, spindle position and cell cycle progression.

mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos.

PubMed

Garner, M; van Kreeveld, S; Su, T T

2001-10-16

Drosophila double park encodes a homolog of Cdt1 that functions in initiation of DNA replication in fission yeast and Xenopus. dup mutants complete the first 15 embryonic cell cycles, presumably via maternal dup products, and show defects in the 16(th) S phase (S16). Cells carrying dup(a1) allele forgo S16 altogether but enter mitosis 16 (M16). We find that the timing of entry into M16 is similar in dup(a1) and heterozygous or wild-type (wt) controls. In contrast, we find that mutant cells carrying another allele, dup(a3), undergo a partial S16 and delay the entry into M16. Thus, initiation of S16 appears necessary for delaying M16. This delay is absent in double mutants of dup(a3) and mei-41 (Drosophila ATR), indicating that a mei-41-dependent checkpoint acts to delay the entry into mitosis in response to incomplete DNA replication. dup(a3) and dup(a1) mutant cells that enter M16 become arrested in M16. We find that mitotic cyclins are stabilized and that a spindle checkpoint protein, Bub1, localizes onto chromosomes during mitotic arrest in dup mutants. These features suggest an arrest prior to metaphase-anaphase transition. dup(a3) bub1 double mutant cells exit M16, indicating that a bub1-mediated checkpoint acts to block mitotic exit in dup mutants. To our knowledge, this is the first report of (1) incomplete DNA replication affecting both the entry into and the exit from mitosis in a single cell cycle via different mechanisms and (2) the role of bub1 in regulating mitotic exit in response to incomplete DNA replication.

A novel functional domain of Cdc15 kinase is required for its interaction with Tem1 GTPase in Saccharomyces cerevisiae.

PubMed Central

Asakawa, K; Yoshida, S; Otake, F; Toh-e, A

2001-01-01

Exit from mitosis requires the inactivation of cyclin-dependent kinase (CDK) activity. In the budding yeast Saccharomyces cerevisiae, a number of gene products have been identified as components of the signal transduction network regulating inactivation of CDK (called the MEN, for the mitotic exit network). Cdc15, one of such components of the MEN, is an essential protein kinase. By the two-hybrid screening, we identified Cdc15 as a binding protein of Tem1 GTPase, another essential regulator of the MEN. Coprecipitation experiments revealed that Tem1 binds to Cdc15 in vivo. By deletion analysis, we found that the Tem1-binding domain resides near the conserved kinase domain of Cdc15. The cdc15-LF mutation, which was introduced into the Tem1-binding domain, reduced the interaction with Cdc15 and Tem1 and caused temperature-sensitive growth.The kinase activity of Cdc15 was not so much affected by the cdc15-LF mutation. However, Cdc15-LF failed to localize to the SPB at the restrictive temperature. Our data show that the interaction with Tem1 is important for the function of Cdc15 and that Cdc15 and Tem1 function in a complex to direct the exit from mitosis. PMID:11290702

The DNA damage response during mitosis.

PubMed

Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

2013-10-01

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Mutations in CENPE define a novel kinetochore-centromeric mechanism for Microcephalic Primordial Dwarfism

PubMed Central

Mirzaa, Ghayda M.; Vitre, Benjamin; Carpenter, Gillian; Abramowicz, Iga; Gleeson, Joseph G.; Paciorkowski, Alex R.; Cleveland, Don W.; Dobyns, William B.; O’Driscoll, Mark

2015-01-01

Defects in centrosome, centrosomal-associated and spindle-associated proteins are the most frequent cause of Primary Microcephaly (PM) and Microcephalic Primordial Dwarfism (MPD) syndromes in humans. Mitotic progression and segregation defects, microtubule spindle abnormalities and impaired DNA damage-induced G2-M cell cycle checkpoint proficiency have been documented in cell lines from these patients. This suggests that impaired mitotic entry, progression and exit strongly contribute to PM and MPD. Considering the vast protein networks involved in coordinating this cell cycle stage, the list of potential target genes that could underlie novel developmental disorders is large. One such complex network, with a direct microtubule-mediated physical connection to the centrosome, is the kinetochore. This centromeric-associated structure nucleates microtubule attachments onto mitotic chromosomes. Here, we described novel compound heterozygous variants in CENPE in two siblings who exhibit a profound MPD associated with developmental delay, simplified gyri and other isolated abnormalities. CENPE encodes centromere-associated protein E (CENP-E), a core kinetochore component functioning to mediate chromosome congression initially of misaligned chromosomes and in subsequent spindle microtubule capture during mitosis. Firstly, we present a comprehensive clinical description of these patients. Then, using patient cells we document abnormalities in spindle microtubule organisation, mitotic progression and segregation, before modeling the cellular pathogenicity of these variants in an independent cell system. Our cellular analysis shows that a pathogenic defect in CENP-E, a kinetochore-core protein, largely phenocopies PCNT-mutated Microcephalic Osteodysplastic Primordial Dwarfism type II patient cells. PCNT encodes a centrosome-associated protein. These results highlight a common underlying pathomechanism. Our findings provide the first evidence for a kinetochore-based route to MPD in humans. PMID:24748105

Mutations in CENPE define a novel kinetochore-centromeric mechanism for microcephalic primordial dwarfism.

PubMed

Mirzaa, Ghayda M; Vitre, Benjamin; Carpenter, Gillian; Abramowicz, Iga; Gleeson, Joseph G; Paciorkowski, Alex R; Cleveland, Don W; Dobyns, William B; O'Driscoll, Mark

2014-08-01

Defects in centrosome, centrosomal-associated and spindle-associated proteins are the most frequent cause of primary microcephaly (PM) and microcephalic primordial dwarfism (MPD) syndromes in humans. Mitotic progression and segregation defects, microtubule spindle abnormalities and impaired DNA damage-induced G2-M cell cycle checkpoint proficiency have been documented in cell lines from these patients. This suggests that impaired mitotic entry, progression and exit strongly contribute to PM and MPD. Considering the vast protein networks involved in coordinating this cell cycle stage, the list of potential target genes that could underlie novel developmental disorders is large. One such complex network, with a direct microtubule-mediated physical connection to the centrosome, is the kinetochore. This centromeric-associated structure nucleates microtubule attachments onto mitotic chromosomes. Here, we described novel compound heterozygous variants in CENPE in two siblings who exhibit a profound MPD associated with developmental delay, simplified gyri and other isolated abnormalities. CENPE encodes centromere-associated protein E (CENP-E), a core kinetochore component functioning to mediate chromosome congression initially of misaligned chromosomes and in subsequent spindle microtubule capture during mitosis. Firstly, we present a comprehensive clinical description of these patients. Then, using patient cells we document abnormalities in spindle microtubule organization, mitotic progression and segregation, before modeling the cellular pathogenicity of these variants in an independent cell system. Our cellular analysis shows that a pathogenic defect in CENP-E, a kinetochore-core protein, largely phenocopies PCNT-mutated microcephalic osteodysplastic primordial dwarfism-type II patient cells. PCNT encodes a centrosome-associated protein. These results highlight a common underlying pathomechanism. Our findings provide the first evidence for a kinetochore-based route to MPD in humans.

Spatiotemporal Regulation of the Anaphase-Promoting Complex in Mitosis

PubMed Central

Sivakumar, Sushama; Gorbsky, Gary J

2015-01-01

The appropriate timing of events that lead to chromosome segregation during mitosis and cytokinesis is essential to prevent aneuploidy, and defects in these processes can contribute to tumorigenesis. Key mitotic regulators are controlled through ubiquitylation and proteasome-mediated degradation. The Anaphase-Promoting Complex or Cyclosome (APC/C) is an E3 ubiquitin ligase that has a crucial function in the regulation of the mitotic cell cycle, particularly at the onset of anaphase and during mitotic exit. Co-activator proteins, inhibitor proteins, protein kinases and phosphatases interact with the APC/C to temporally and spatially control its activity and thus ensure accurate timing of mitotic events. PMID:25604195

Antiproliferative Fate of the Tetraploid Formed after Mitotic Slippage and Its Promotion; A Novel Target for Cancer Therapy Based on Microtubule Poisons.

PubMed

Nakayama, Yuji; Inoue, Toshiaki

2016-05-19

Microtubule poisons inhibit spindle function, leading to activation of spindle assembly checkpoint (SAC) and mitotic arrest. Cell death occurring in prolonged mitosis is the first target of microtubule poisons in cancer therapies. However, even in the presence of microtubule poisons, SAC and mitotic arrest are not permanent, and the surviving cells exit the mitosis without cytokinesis (mitotic slippage), becoming tetraploid. Another target of microtubule poisons-based cancer therapy is antiproliferative fate after mitotic slippage. The ultimate goal of both the microtubule poisons-based cancer therapies involves the induction of a mechanism defined as mitotic catastrophe, which is a bona fide intrinsic oncosuppressive mechanism that senses mitotic failure and responds by driving a cell to an irreversible antiproliferative fate of death or senescence. This mechanism of antiproliferative fate after mitotic slippage is not as well understood. We provide an overview of mitotic catastrophe, and explain new insights underscoring a causal association between basal autophagy levels and antiproliferative fate after mitotic slippage, and propose possible improved strategies. Additionally, we discuss nuclear alterations characterizing the mitotic catastrophe (micronuclei, multinuclei) after mitotic slippage, and a possible new type of nuclear alteration (clustered micronuclei).

The human protein PRR14 tethers heterochromatin to the nuclear lamina during interphase and mitotic exit.

PubMed

Poleshko, Andrey; Mansfield, Katelyn M; Burlingame, Caroline C; Andrake, Mark D; Shah, Neil R; Katz, Richard A

2013-10-31

The nuclear lamina is a protein meshwork that lies under the inner nuclear membrane of metazoan cells. One function of the nuclear lamina is to organize heterochromatin at the inner nuclear periphery. However, very little is known about how heterochromatin attaches to the nuclear lamina and how such attachments are restored at mitotic exit. Here, we show that a previously unstudied human protein, PRR14, functions to tether heterochromatin to the nuclear periphery during interphase, through associations with heterochromatin protein 1 (HP1) and the nuclear lamina. During early mitosis, PRR14 is released from the nuclear lamina and chromatin and remains soluble. Strikingly, at the onset of anaphase, PRR14 is incorporated rapidly into chromatin through HP1 binding. Finally, in telophase, PRR14 relocalizes to the reforming nuclear lamina. This stepwise reassembly of PRR14 suggests a function in the selection of HP1-bound heterochromatin for reattachment to the nuclear lamina as cells exit mitosis.

The Human Protein PRR14 Tethers Heterochromatin to the Nuclear Lamina During Interphase and Mitotic Exit

PubMed Central

Poleshko, Andrey; Mansfield, Katelyn M.; Burlingame, Caroline C.; Andrake, Mark D.; Shah, Neil R.; Katz, Richard A.

2013-01-01

SUMMARY The nuclear lamina is a protein meshwork that lies under the inner nuclear membrane of metazoan cells. One function of the nuclear lamina is to organize heterochromatin at the inner nuclear periphery. However, very little is known about how heterochromatin attaches to the nuclear lamina and how such attachments are restored at mitotic exit. Here we show that a previously unstudied human protein, PRR14, functions to tether heterochromatin to the nuclear periphery during interphase, through associations with heterochromatin protein 1 (HP1) and the nuclear lamina. During early mitosis, PRR14 is released from the nuclear lamina and chromatin, and remains soluble. Strikingly, at the onset of anaphase, PRR14 is incorporated rapidly into chromatin through HP1 binding. Finally, in telophase, PRR14 relocalizes to the reforming nuclear lamina. This stepwise reassembly of PRR14 suggests a novel function in the selection of HP1–bound heterochromatin for reattachment to the nuclear lamina as cells exit mitosis. PMID:24209742

Phosphorylation and dephosphorylation regulate APC/CCdh1 substrate degradation

PubMed Central

Simpson-Lavy, Kobi J; Zenvirth, Drora; Brandeis, Michael

2015-01-01

The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/CCdh1 mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1m11 mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/CCdh1 substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation. PMID:26252546

Structure and dimerization of the catalytic domain of the protein phosphatase Cdc14p, a key regulator of mitotic exit in Saccharomyces cerevisiae.

PubMed

Kobayashi, Junya; Matsuura, Yoshiyuki

2017-10-01

In the budding yeast Saccharomyces cerevisiae, the protein phosphatase Cdc14p orchestrates various events essential for mitotic exit. We have determined the X-ray crystal structures at 1.85 Å resolution of the catalytic domain of Cdc14p in both the apo state, and as a complex with S160-phosphorylated Swi6p peptide. Each asymmetric unit contains two Cdc14p chains arranged in an intimately associated homodimer, consistent with its oligomeric state in solution. The dimerization interface is located on the backside of the substrate-binding cleft. Structure-based mutational analyses indicate that the dimerization of Cdc14p is required for normal growth of yeast cells. © 2017 The Protein Society.

Sumoylation promotes optimal APC/C Activation and Timely Anaphase.

PubMed

Lee, Christine C; Li, Bing; Yu, Hongtao; Matunis, Michael J

2018-03-08

The Anaphase Promoting Complex/Cyclosome (APC/C) is a ubiquitin E3 ligase that functions as the gatekeeper to mitotic exit. APC/C activity is controlled by an interplay of multiple pathways during mitosis, including the spindle assembly checkpoint (SAC), that are not yet fully understood. Here, we show that sumoylation of the APC4 subunit of the APC/C peaks during mitosis and is critical for timely APC/C activation and anaphase onset. We have also identified a functionally important SUMO interacting motif in the cullin-homology domain of APC2 located near the APC4 sumoylation sites and APC/C catalytic core. Our findings provide evidence of an important regulatory role for SUMO modification and binding in affecting APC/C activation and mitotic exit. © 2018, Lee et al.

Genome co-amplification upregulates a mitotic gene network activity that predicts outcome and response to mitotic protein inhibitors in breast cancer

DOE PAGES

Hu, Zhi; Mao, Jian-Hua; Curtis, Christina; ...

2016-07-01

Background: High mitotic activity is associated with the genesis and progression of many cancers. Small molecule inhibitors of mitotic apparatus proteins are now being developed and evaluated clinically as anticancer agents. With clinical trials of several of these experimental compounds underway, it is important to understand the molecular mechanisms that determine high mitotic activity, identify tumor subtypes that carry molecular aberrations that confer high mitotic activity, and to develop molecular markers that distinguish which tumors will be most responsive to mitotic apparatus inhibitors. Methods: We identified a coordinately regulated mitotic apparatus network by analyzing gene expression profiles for 53 malignantmore » and non-malignant human breast cancer cell lines and two separate primary breast tumor datasets. We defined the mitotic network activity index (MNAI) as the sum of the transcriptional levels of the 54 coordinately regulated mitotic apparatus genes. The effect of those genes on cell growth was evaluated by small interfering RNA (siRNA). Results: High MNAI was enriched in basal-like breast tumors and was associated with reduced survival duration and preferential sensitivity to i nhibitors of the mitotic apparatus proteins, polo-like kinase, centromere associated protein E and aurora kinase designated GSK462364, GSK923295 and GSK1070916, respectively. Co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25 was associated with the transcriptional upregulation of this network of 54 mitotic apparatus genes, and we identify transcription factors that localize to these regions and putatively regulate mitotic activity. Knockdown of the mitotic network by siRNA identified 22 genes that might be considered as additional therapeutic targets for this clinically relevant patient subgroup. Conclusions: We define a molecular signature which may guide therapeutic approaches for tumors with high mitotic network activity.« less

Genome co-amplification upregulates a mitotic gene network activity that predicts outcome and response to mitotic protein inhibitors in breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Zhi; Mao, Jian-Hua; Curtis, Christina

Background: High mitotic activity is associated with the genesis and progression of many cancers. Small molecule inhibitors of mitotic apparatus proteins are now being developed and evaluated clinically as anticancer agents. With clinical trials of several of these experimental compounds underway, it is important to understand the molecular mechanisms that determine high mitotic activity, identify tumor subtypes that carry molecular aberrations that confer high mitotic activity, and to develop molecular markers that distinguish which tumors will be most responsive to mitotic apparatus inhibitors. Methods: We identified a coordinately regulated mitotic apparatus network by analyzing gene expression profiles for 53 malignantmore » and non-malignant human breast cancer cell lines and two separate primary breast tumor datasets. We defined the mitotic network activity index (MNAI) as the sum of the transcriptional levels of the 54 coordinately regulated mitotic apparatus genes. The effect of those genes on cell growth was evaluated by small interfering RNA (siRNA). Results: High MNAI was enriched in basal-like breast tumors and was associated with reduced survival duration and preferential sensitivity to i nhibitors of the mitotic apparatus proteins, polo-like kinase, centromere associated protein E and aurora kinase designated GSK462364, GSK923295 and GSK1070916, respectively. Co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25 was associated with the transcriptional upregulation of this network of 54 mitotic apparatus genes, and we identify transcription factors that localize to these regions and putatively regulate mitotic activity. Knockdown of the mitotic network by siRNA identified 22 genes that might be considered as additional therapeutic targets for this clinically relevant patient subgroup. Conclusions: We define a molecular signature which may guide therapeutic approaches for tumors with high mitotic network activity.« less

Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis.

PubMed

Saurin, Adrian T; van der Waal, Maike S; Medema, René H; Lens, Susanne M A; Kops, Geert J P L

2011-01-01

The mitotic checkpoint prevents mitotic exit until all chromosomes are attached to spindle microtubules. Aurora B kinase indirectly invokes this checkpoint by destabilizing incorrect attachments; however, a more direct role remains controversial. In contrast, activity of the kinase Mps1 is indispensible for the mitotic checkpoint. Here we show that Aurora B and Hec1 are needed for efficient Mps1 recruitment to unattached kinetochores, allowing rapid Mps1 activation at the onset of mitosis. Live monitoring of cyclin B degradation reveals that this is essential to establish the mitotic checkpoint quickly at the start of mitosis. Delayed Mps1 activation and checkpoint establishment upon Aurora B inhibition or Hec1 depletion are rescued by tethering Mps1 to kinetochores, demonstrating that Mps1 recruitment is the primary role of Aurora B and Hec1 in mitotic checkpoint signalling. These data demonstrate a direct role for Aurora B in initiating the mitotic checkpoint rapidly at the onset of mitosis.

The tumor suppressor CDKN3 controls mitosis

PubMed Central

Nalepa, Grzegorz; Barnholtz-Sloan, Jill; Enzor, Rikki; Dey, Dilip; He, Ying; Gehlhausen, Jeff R.; Lehmann, Amalia S.; Park, Su-Jung; Yang, Yanzhu; Yang, Xianlin; Chen, Shi; Guan, Xiaowei; Chen, Yanwen; Renbarger, Jamie; Yang, Feng-Chun; Parada, Luis F.

2013-01-01

Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2pThr-161 at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CKβ phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics. PMID:23775190

An APC/C-Cdh1 Biosensor Reveals the Dynamics of Cdh1 Inactivation at the G1/S Transition.

PubMed

Ondracka, Andrej; Robbins, Jonathan A; Cross, Frederick R

2016-01-01

B-type cyclin-dependent kinase activity must be turned off for mitotic exit and G1 stabilization. B-type cyclin degradation is mediated by the anaphase-promoting complex/cyclosome (APC/C); during and after mitotic exit, APC/C is dependent on Cdh1. Cdh1 is in turn phosphorylated and inactivated by cyclin-CDK at the Start transition of the new cell cycle. We developed a biosensor to assess the cell cycle dynamics of APC/C-Cdh1. Nuclear exit of the G1 transcriptional repressor Whi5 is a known marker of Start; APC/C-Cdh1 is inactivated 12 min after Whi5 nuclear exit with little measurable cell-to-cell timing variability. Multiple phosphorylation sites on Cdh1 act in a redundant manner to repress its activity. Reducing the number of phosphorylation sites on Cdh1 can to some extent be tolerated for cell viability, but it increases variability in timing of APC/C-Cdh1 inactivation. Mutants with minimal subsets of phosphorylation sites required for viability exhibit striking stochasticity in multiple responses including budding, nuclear division, and APC/C-Cdh1 activity itself. Multiple cyclin-CDK complexes, as well as the stoichiometric inhibitor Acm1, contribute to APC/C-Cdh1 inactivation; this redundant control is likely to promote rapid and reliable APC/C-Cdh1 inactivation immediately following the Start transition.

The NIMA Kinase Is Required To Execute Stage-Specific Mitotic Functions after Initiation of Mitosis

PubMed Central

Govindaraghavan, Meera; Lad, Alisha A.

2014-01-01

The G2-M transition in Aspergillus nidulans requires the NIMA kinase, the founding member of the Nek kinase family. Inactivation of NIMA results in a late G2 arrest, while overexpression of NIMA is sufficient to promote mitotic events independently of cell cycle phase. Endogenously tagged NIMA-GFP has dynamic mitotic localizations appearing first at the spindle pole body and then at nuclear pore complexes before transitioning to within nuclei and the mitotic spindle and back at the spindle pole bodies at mitotic exit, suggesting that it functions sequentially at these locations. Since NIMA is indispensable for mitotic entry, it has been difficult to determine the requirement of NIMA for subaspects of mitosis. We show here that when NIMA is partially inactivated, although mitosis can be initiated, a proportion of cells fail to successfully generate two daughter nuclei. We further define the mitotic defects to show that normal NIMA function is required for the formation of a bipolar spindle, nuclear pore complex disassembly, completion of chromatin segregation, and the normal structural rearrangements of the nuclear envelope required to generate two nuclei from one. In the remaining population of cells that enter mitosis with inadequate NIMA, two daughter nuclei are generated in a manner dependent on the spindle assembly checkpoint, indicating highly penetrant defects in mitotic progression without sufficient NIMA activity. This study shows that NIMA is required not only for mitotic entry but also sequentially for successful completion of stage-specific mitotic events. PMID:24186954

Perturbation of Incenp function impedes anaphase chromatid movements and chromosomal passenger protein flux at centromeres

PubMed Central

Ahonen, Leena J.; Kukkonen, Anu M.; Pouwels, Jeroen; Bolton, Margaret A.; Jingle, Christopher D.; Stukenberg, P. Todd; Kallio, Marko J.

2012-01-01

Incenp is an essential mitotic protein that, together with Aurora B, Survivin, and Borealin, forms the core of the chromosomal passenger protein complex (CPC). The CPC regulates various mitotic processes and functions to maintain genomic stability. The proper subcellular localization of the CPC and its full catalytic activity require the presence of each core subunit in the complex. We have investigated the mitotic tasks of the CPC using a function blocking antibody against Incenp microinjected into cells at different mitotic phases. This method allowed temporal analysis of CPC functions without perturbation of complex assembly or activity prior to injection. We have also studied the dynamic properties of Incenp and Aurora B using fusion protein photobleaching. We found that in early mitotic cells, Incenp and Aurora B exhibit dynamic turnover at centromeres, which is prevented by the anti-Incenp antibody. In these cells, the loss of centromeric CPC turnover is accompanied by forced mitotic exit without the execution of cytokinesis. Introduction of anti-Incenp antibody into early anaphase cells causes abnormalities in sister chromatid separation through defects in anaphase spindle functions. In summary, our data uncovers new mitotic roles for the CPC in anaphase and proposes that CPC turnover at centromeres modulates spindle assembly checkpoint signaling. PMID:18784935

Perturbation of Incenp function impedes anaphase chromatid movements and chromosomal passenger protein flux at centromeres.

PubMed

Ahonen, Leena J; Kukkonen, Anu M; Pouwels, Jeroen; Bolton, Margaret A; Jingle, Christopher D; Stukenberg, P Todd; Kallio, Marko J

2009-02-01

Incenp is an essential mitotic protein that, together with Aurora B, Survivin, and Borealin, forms the core of the chromosomal passenger protein complex (CPC). The CPC regulates various mitotic processes and functions to maintain genomic stability. The proper subcellular localization of the CPC and its full catalytic activity require the presence of each core subunit in the complex. We have investigated the mitotic tasks of the CPC using a function blocking antibody against Incenp microinjected into cells at different mitotic phases. This method allowed temporal analysis of CPC functions without perturbation of complex assembly or activity prior to injection. We have also studied the dynamic properties of Incenp and Aurora B using fusion protein photobleaching. We found that in early mitotic cells, Incenp and Aurora B exhibit dynamic turnover at centromeres, which is prevented by the anti-Incenp antibody. In these cells, the loss of centromeric CPC turnover is accompanied by forced mitotic exit without the execution of cytokinesis. Introduction of anti-Incenp antibody into early anaphase cells causes abnormalities in sister chromatid separation through defects in anaphase spindle functions. In summary, our data uncovers new mitotic roles for the CPC in anaphase and proposes that CPC turnover at centromeres modulates spindle assembly checkpoint signaling.

ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores

PubMed Central

Isokane, Mayumi; Walter, Thomas; Mahen, Robert; Nijmeijer, Bianca; Hériché, Jean-Karim; Miura, Kota; Maffini, Stefano; Ivanov, Miroslav Penchev; Kitajima, Tomoya S.; Peters, Jan-Michael

2016-01-01

To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17’s RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment. PMID:26953350

ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores.

PubMed

Isokane, Mayumi; Walter, Thomas; Mahen, Robert; Nijmeijer, Bianca; Hériché, Jean-Karim; Miura, Kota; Maffini, Stefano; Ivanov, Miroslav Penchev; Kitajima, Tomoya S; Peters, Jan-Michael; Ellenberg, Jan

2016-03-14

To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17's RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment. © 2016 Isokane et al.

Changes in Ect2 Localization Couple Actomyosin-Dependent Cell Shape Changes to Mitotic Progression

PubMed Central

Matthews, Helen K.; Delabre, Ulysse; Rohn, Jennifer L.; Guck, Jochen; Kunda, Patricia; Baum, Buzz

2012-01-01

Summary As they enter mitosis, animal cells undergo profound actin-dependent changes in shape to become round. Here we identify the Cdk1 substrate, Ect2, as a central regulator of mitotic rounding, thus uncovering a link between the cell-cycle machinery that drives mitotic entry and its accompanying actin remodeling. Ect2 is a RhoGEF that plays a well-established role in formation of the actomyosin contractile ring at mitotic exit, through the local activation of RhoA. We find that Ect2 first becomes active in prophase, when it is exported from the nucleus into the cytoplasm, activating RhoA to induce the formation of a mechanically stiff and rounded metaphase cortex. Then, at anaphase, binding to RacGAP1 at the spindle midzone repositions Ect2 to induce local actomyosin ring formation. Ect2 localization therefore defines the stage-specific changes in actin cortex organization critical for accurate cell division. PMID:22898780

PLK1 has tumor-suppressive potential in APC-truncated colon cancer cells.

PubMed

Raab, Monika; Sanhaji, Mourad; Matthess, Yves; Hörlin, Albrecht; Lorenz, Ioana; Dötsch, Christina; Habbe, Nils; Waidmann, Oliver; Kurunci-Csacsko, Elisabeth; Firestein, Ron; Becker, Sven; Strebhardt, Klaus

2018-03-16

The spindle assembly checkpoint (SAC) acts as a molecular safeguard in ensuring faithful chromosome transmission during mitosis, which is regulated by a complex interplay between phosphatases and kinases including PLK1. Adenomatous polyposis coli (APC) germline mutations cause aneuploidy and are responsible for familial adenomatous polyposis (FAP). Here we study the role of PLK1 in colon cancer cells with chromosomal instability promoted by APC truncation (APC-ΔC). The expression of APC-ΔC in colon cells reduces the accumulation of mitotic cells upon PLK1 inhibition, accelerates mitotic exit and increases the survival of cells with enhanced chromosomal abnormalities. The inhibition of PLK1 in mitotic, APC-∆C-expressing cells reduces the kinetochore levels of Aurora B and hampers the recruitment of SAC component suggesting a compromised mitotic checkpoint. Furthermore, Plk1 inhibition (RNAi, pharmacological compounds) promotes the development of adenomatous polyps in two independent Apc Min/+ mouse models. High PLK1 expression increases the survival of colon cancer patients expressing a truncated APC significantly.

Degradation of Hof1 by SCFGrr1 is important for actomyosin contraction during cytokinesis in yeast

PubMed Central

Blondel, Marc; Bach, Stéphane; Bamps, Sophie; Dobbelaere, Jeroen; Wiget, Philippe; Longaretti, Céline; Barral, Yves; Meijer, Laurent; Peter, Matthias

2005-01-01

SCF-type (SCF: Skp1–Cullin–F-box protein complex) E3 ligases regulate ubiquitin-dependent degradation of many cell cycle regulators, mainly at the G1/S transition. Here, we show that SCFGrr1 functions during cytokinesis by degrading the PCH protein Hof1. While Hof1 is required early in mitosis to assemble a functional actomyosin ring, it is specifically degraded late in mitosis and remains unstable during the entire G1 phase of the cell cycle. Degradation of Hof1 depends on its PEST motif and a functional 26S proteasome. Interestingly, degradation of Hof1 is independent of APCCdh1, but instead requires the SCFGrr1 E3 ligase. Grr1 is recruited to the mother–bud neck region after activation of the mitotic-exit network, and interacts with Hof1 in a PEST motif-dependent manner. Our results also show that downregulation of Hof1 at the end of mitosis is necessary to allow efficient contraction of the actomyosin ring and cell separation during cytokinesis. SCFGrr1-mediated degradation of Hof1 may thus represent a novel mechanism to couple exit from mitosis with initiation of cytokinesis. PMID:15775961

Functional Importance of the Anaphase-Promoting Complex-Cdh1-Mediated Degradation of TMAP/CKAP2 in Regulation of Spindle Function and Cytokinesis▿ †

PubMed Central

Hong, Kyung Uk; Park, Young Soo; Seong, Yeon-Sun; Kang, Dongmin; Bae, Chang-Dae; Park, Joobae

2007-01-01

Cytoskeleton-associated protein 2 (CKAP2), also known as tumor-associated microtubule-associated protein (TMAP), is a novel microtubule-associated protein that is frequently upregulated in various malignances. However, its cellular functions remain unknown. A previous study has shown that its protein level begins to increase during G1/S and peaks at G2/M, after which it decreases abruptly. Ectopic overexpression of TMAP/CKAP2 induced microtubule bundling related to increased microtubule stability. TMAP/CKAP2 overexpression also resulted in cell cycle arrest during mitosis due to a defect in centrosome separation and subsequent formation of a monopolar spindle. We also show that degradation of TMAP/CKAP2 during mitotic exit is mediated by the anaphase-promoting complex bound to Cdh1 and that the KEN box motif near the N terminus is necessary for its destruction. Compared to the wild type, expression of a nondegradable mutant of TMAP/CKAP2 significantly increased the occurrence of spindle defects and cytokinesis failure. These results suggest that TMAP/CKAP2 plays a role in the assembly and maintenance of mitotic spindles, presumably by regulating microtubule dynamics, and its destruction during mitotic exit serves an important role in the completion of cytokinesis and in the maintenance of spindle bipolarity in the next mitosis. PMID:17339342

Functional importance of the anaphase-promoting complex-Cdh1-mediated degradation of TMAP/CKAP2 in regulation of spindle function and cytokinesis.

PubMed

Hong, Kyung Uk; Park, Young Soo; Seong, Yeon-Sun; Kang, Dongmin; Bae, Chang-Dae; Park, Joobae

2007-05-01

Cytoskeleton-associated protein 2 (CKAP2), also known as tumor-associated microtubule-associated protein (TMAP), is a novel microtubule-associated protein that is frequently upregulated in various malignances. However, its cellular functions remain unknown. A previous study has shown that its protein level begins to increase during G(1)/S and peaks at G(2)/M, after which it decreases abruptly. Ectopic overexpression of TMAP/CKAP2 induced microtubule bundling related to increased microtubule stability. TMAP/CKAP2 overexpression also resulted in cell cycle arrest during mitosis due to a defect in centrosome separation and subsequent formation of a monopolar spindle. We also show that degradation of TMAP/CKAP2 during mitotic exit is mediated by the anaphase-promoting complex bound to Cdh1 and that the KEN box motif near the N terminus is necessary for its destruction. Compared to the wild type, expression of a nondegradable mutant of TMAP/CKAP2 significantly increased the occurrence of spindle defects and cytokinesis failure. These results suggest that TMAP/CKAP2 plays a role in the assembly and maintenance of mitotic spindles, presumably by regulating microtubule dynamics, and its destruction during mitotic exit serves an important role in the completion of cytokinesis and in the maintenance of spindle bipolarity in the next mitosis.

The PP2AB56 phosphatase promotes the association of Cdc20 with APC/C in mitosis.

PubMed

Lee, Sun Joo; Rodriguez-Bravo, Veronica; Kim, Hyunjung; Datta, Sutirtha; Foley, Emily A

2017-05-15

PP2A comprising B56 regulatory subunit isoforms (PP2A B56 ) is a serine/threonine phosphatase essential for mitosis. At the kinetochore, PP2A B56 both stabilizes microtubule binding and promotes silencing of the spindle assembly checkpoint (SAC) through its association with the SAC protein BubR1. Cells depleted of the B56 regulatory subunits of PP2A are delayed in activation of Cdc20-containing APC/C (APC/C Cdc20 ), which is an essential step for mitotic exit. It has been hypothesized that this delay arises from increased production of the mitotic checkpoint complex (MCC), an APC/C Cdc20 inhibitor formed at unattached kinetochores through SAC signaling. In contrast to this prediction, we show that depletion of B56 subunits does not increase the amount or stability of the MCC. Rather, delays in APC/C Cdc20 activation in B56-depleted cells correlate with impaired Cdc20 binding to APC/C. Stimulation of APC/C Cdc20 assembly does not require binding between PP2A B56 and BubR1, and thus this contribution of PP2A B56 towards mitotic exit is distinct from its functions at kinetochores. PP2A B56 associates with APC/C constitutively in a BubR1-independent manner. A mitotic phosphorylation site on Cdc20, known to be a substrate of PP2A B56 , modulates APC/C Cdc20 assembly. These results elucidate the contributions of PP2A B56 towards completion of mitosis. © 2017. Published by The Company of Biologists Ltd.

Interkinetic nuclear migration and basal tethering facilitates post-mitotic daughter separation in intestinal organoids

PubMed Central

Carroll, Thomas D.; Langlands, Alistair J.; Osborne, James M.; Newton, Ian P.; Appleton, Paul L.

2017-01-01

ABSTRACT Homeostasis of renewing tissues requires balanced proliferation, differentiation and movement. This is particularly important in the intestinal epithelium where lineage tracing suggests that stochastic differentiation choices are intricately coupled to the position of a cell relative to a niche. To determine how position is achieved, we followed proliferating cells in intestinal organoids and discovered that the behaviour of mitotic sisters predicted long-term positioning. We found that, normally, 70% of sisters remain neighbours, while 30% lose contact and separate after cytokinesis. These post-mitotic placements predict longer term differences in positions assumed by sisters: adjacent sisters reach similar positions over time; in a pair of separating sisters, one remains close to its birthplace while the other is displaced upward. Computationally modelling crypt dynamics confirmed that post-mitotic separation leads to sisters reaching different compartments. We show that interkinetic nuclear migration, cell size and asymmetric tethering by a process extending from the basal side of cells contribute to separations. These processes are altered in adenomatous polyposis coli (Apc) mutant epithelia where separation is lost. We conclude that post-mitotic placement contributes to stochastic niche exit and, when defective, supports the clonal expansion of Apc mutant cells. PMID:28982714

Activation of the yeast Hippo pathway by phosphorylation-dependent assembly of signaling complexes.

PubMed

Rock, Jeremy M; Lim, Daniel; Stach, Lasse; Ogrodowicz, Roksana W; Keck, Jamie M; Jones, Michele H; Wong, Catherine C L; Yates, John R; Winey, Mark; Smerdon, Stephen J; Yaffe, Michael B; Amon, Angelika

2013-05-17

Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.

Retention of Chs2p in the ER requires N-terminal CDK1-phosphorylation sites.

PubMed

Teh, Ee Mei; Chai, Chuan Chung; Yeong, Foong May

2009-09-15

In budding yeast, the secretory pathway is constitutively transporting cargoes such as invertase and alpha-factor throughout the cell division cycle. However, chitin synthase 2 (Chs2p), another cargo of the secretory pathway, is retained at the endoplasmic reticulum (ER) during mitosis when the mitotic kinase activity is high. Chs2p is exported from the ER to the mother-daughter neck only upon mitotic kinase destruction, indicating that the mitotic kinase activity is critical for the ER retention of Chs2p. However, a key question is whether the mitotic kinase acts directly upon Chs2p to prevent its ER export. We report here that mutation of Ser residues to Glu at 4 perfect CDK1-phosphorylation sites at the N-terminus of Chs2p leads to its retention in the ER when the mitotic kinase activity is absent. Conversely, Ser-to-Ala mutations result in the loss of Chs2p ER retention even when mitotic kinase activity is high. The mere overexpression of the non-destructible form of the mitotic cyclin in G(1) cells can confine the wild-type Chs2p but not the Ser-to-Ala mutant in the ER. Furthermore, overexpression of the Ser-to-Ala mutant kills cells. Time-lapsed imaging revealed that Chs2p is exported from the ER rapidly and synchronously to the Golgi upon metaphase release. Our data indicate that direct phosphorylation of Chs2p by the mitotic CDK1 helps restrain it in the ER during mitosis to prevent its rapid export in an untimely manner until after sister chromatid occurs and mitotic exit executed.

Proliferating cell nuclear antigen (PCNA)-associated KIAA0101/PAF15 protein is a cell cycle-regulated anaphase-promoting complex/cyclosome substrate.

PubMed

Emanuele, Michael J; Ciccia, Alberto; Elia, Andrew E H; Elledge, Stephen J

2011-06-14

The anaphase-promoting complex/cyclosome (APC/C) is a cell cycle-regulated E3 ubiquitin ligase that controls the degradation of substrate proteins at mitotic exit and throughout the G1 phase. We have identified an APC/C substrate and cell cycle-regulated protein, KIAA0101/PAF15. PAF15 protein levels peak in the G2/M phase of the cell cycle and drop rapidly at mitotic exit in an APC/C- and KEN-box-dependent fashion. PAF15 associates with proliferating cell nuclear antigen (PCNA), and depletion of PAF15 decreases the number of cells in S phase, suggesting a role for it in cell cycle regulation. Following irradiation, PAF15 colocalized with γH2AX foci at sites of DNA damage through its interaction with PCNA. Finally, PAF15 depletion led to an increase in homologous recombination-mediated DNA repair, and overexpression caused sensitivity to UV-induced DNA damage. We conclude that PAF15 is an APC/C-regulated protein involved in both cell cycle progression and the DNA damage response.

Bub2 regulation of cytokinesis and septation in budding yeast

PubMed Central

Park, Su Young; Cable, Addie E; Blair, Jessica; Stockstill, Katherine E; Shannnon, Katie B

2009-01-01

Background The mitotic exit network (MEN) is required for events at the end of mitosis such as degradation of mitotic cyclins and cytokinesis. Bub2 and its binding partner Bfa1 act as a GTPase activating protein (GAP) to negatively regulate the MEN GTPase Tem1. The Bub2/Bfa1 checkpoint pathway is required to delay the cell cycle in response to mispositioned spindles. In addition to its role in mitotic exit, Tem1 is required for actomyosin ring contraction. Results To test the hypothesis that the Bub2 pathway prevents premature actin ring assembly, we compared the timing of actin ring formation in wild type, bub2Δ, mad2Δ, and bub2Δmad2Δ cells both with and without microtubules. There was no difference in the timing of actin ring formation between wild type and mutant cells in a synchronized cell cycle. In the presence of nocodazole, both bub2Δ and mad2Δ cells formed rings after a delay of the same duration. Double mutant bub2Δmad2Δ and bfa1Δmad2Δ cells formed rings at the same time with and without nocodazole. To determine if Bub2 has an effect on actomyosin ring contraction through its regulation of Tem1, we used live cell imaging of Myo1-GFP in a bub2Δ strain. We found a significant decrease in the total time of contraction and an increase in rate of contraction compared to wild type cells. We also examined myosin contraction using Myo1-GFP in cells overexpressing an epitope tagged Bub2. Surprisingly, overexpression of Bub2 also led to a significant increase in the rate of contraction, as well as morphological defects. The chained cell phenotype caused by Bub2 overexpression could be rescued by co-overexpression of Tem1, and was not rescued by deletion of BFA1. Conclusion Our data indicate that the Bub2 checkpoint pathway does not have a specific role in delaying actin ring formation. The observed increase in the rate of myosin contraction in the bub2Δ strain provides evidence that the MEN regulates actomyosin ring contraction. Our data suggest that the overexpression of the Bub2 fusion protein acts as a dominant negative, leading to septation defects by a mechanism that is Tem1-dependent. PMID:19490645

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Bo; Huang Bo; School of Public Health, University of South China, Hengyang, Hunan 421001

Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe,more » such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3{sigma} and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe. - Graphical abstract: Display Omitted Research highlights: > 6-bromoisovanillin induced spindle disruption and sustained mitotic arrest, consequently resulted in mitotic catastrophe. > Proteomic profiling identified 137 differentially expressed proteins associated mitotic catastrophe. > The 14-3-3-mediated signaling network was the most significantly enriched for the altered proteins. > The macromolecule complex assembly, cell cycle, chromatin remodeling and DNA repair, tubulin organization were also shown involved in mitotic catastrophe.« less

The master Greatwall kinase, a critical regulator of mitosis and meiosis.

PubMed

Vigneron, Suzanne; Robert, Perle; Hached, Khaled; Sundermann, Lena; Charrasse, Sophie; Labbé, Jean-Claude; Castro, Anna; Lorca, Thierry

2016-01-01

Entry into mitosis requires the coordinated activation of various protein kinases and phosphatases that together activate sequential signaling pathways allowing entry, progression and exit of mitosis. The limiting step is thought to be the activation of the mitotic Cdk1-cyclin B kinase. However, this model has recently evolved with new data showing that in addition to the Cdk1-cyclin B complex, Greatwall (Gwl) kinase is also required to enter into and maintain mitosis. This new concept proposes that entry into mitosis is now based on the combined activation of both kinases Cdk1-cyclin B and Gwl, the former promoting massive phosphorylation of mitotic substrates and the latter inhibiting PP2A-B55 phosphatase responsible for dephosphorylation of these substrates. Activated Gwl phosphorylates both Arpp19 and ENSA, which associate and inhibit PP2A-B55. This pathway seems relatively well conserved from yeast to humans, although some differences appear based on models or techniques used. While Gwl is activated by phosphorylation, its inactivation requires dephosphorylation of critical residues. Several phosphatases such as PP1, PP2A-B55 and FCP1 are required to control the dephosphorylation and inactivation of Gwl and a properly regulated mitotic exit. Gwl has also been reported to be involved in cancer processes and DNA damage recovery. These new findings support the idea that the Gwl-Arpp19/ENSA-PP2A-B55 pathway is essential to achieve an efficient division of cells and to maintain genomic stability.

Controlling the response to DNA damage by the APC/C-Cdh1.

PubMed

de Boer, H Rudolf; Guerrero Llobet, S; van Vugt, Marcel A T M

2016-03-01

Proper cell cycle progression is safeguarded by the oscillating activities of cyclin/cyclin-dependent kinase complexes. An important player in the regulation of mitotic cyclins is the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase. Prior to entry into mitosis, the APC/C remains inactive, which allows the accumulation of mitotic regulators. APC/C activation requires binding to either the Cdc20 or Cdh1 adaptor protein, which sequentially bind the APC/C and facilitate targeting of multiple mitotic regulators for proteasomal destruction, including Securin and Cyclin B, to ensure proper chromosome segregation and mitotic exit. Emerging data have indicated that the APC/C, particularly in association with Cdh1, also functions prior to mitotic entry. Specifically, the APC/C-Cdh1 is activated in response to DNA damage in G2 phase cells. These observations are in line with in vitro and in vivo genetic studies, in which cells lacking Cdh1 expression display various defects, including impaired DNA repair and aberrant cell cycle checkpoints. In this review, we summarize the current literature on APC/C regulation in response to DNA damage, the functions of APC/C-Cdh1 activation upon DNA damage, and speculate how APC/C-Cdh1 can control cell fate in the context of persistent DNA damage.

Radmis, a Novel Mitotic Spindle Protein that Functions in Cell Division of Neural Progenitors

PubMed Central

Yumoto, Takahito; Nakadate, Kazuhiko; Nakamura, Yuki; Sugitani, Yoshinobu; Sugitani-Yoshida, Reiko; Ueda, Shuichi; Sakakibara, Shin-ichi

2013-01-01

Developmental dynamics of neural stem/progenitor cells (NSPCs) are crucial for embryonic and adult neurogenesis, but its regulatory factors are not fully understood. By differential subtractive screening with NSPCs versus their differentiated progenies, we identified the radmis (radial fiber and mitotic spindle)/ckap2l gene, a novel microtubule-associated protein (MAP) enriched in NSPCs. Radmis is a putative substrate for the E3-ubiquitin ligase, anaphase promoting complex/cyclosome (APC/C), and is degraded via the KEN box. Radmis was highly expressed in regions of active neurogenesis throughout life, and its distribution was dynamically regulated during NSPC division. In embryonic and perinatal brains, radmis localized to bipolar mitotic spindles and radial fibers (basal processes) of dividing NSPCs. As central nervous system development proceeded, radmis expression was lost in most brain regions, except for several neurogenic regions. In adult brain, radmis expression persisted in the mitotic spindles of both slowly-dividing stem cells and rapid amplifying progenitors. Overexpression of radmis in vitro induced hyper-stabilization of microtubules, severe defects in mitotic spindle formation, and mitotic arrest. In vivo gain-of-function using in utero electroporation revealed that radmis directed a reduction in NSPC proliferation and a concomitant increase in cell cycle exit, causing a reduction in the Tbr2-positive basal progenitor population and shrinkage of the embryonic subventricular zone. Besides, radmis loss-of-function by shRNAs induced the multipolar mitotic spindle structure, accompanied with the catastrophe of chromosome segregation including the long chromosome bridge between two separating daughter nuclei. These findings uncover the indispensable role of radmis in mitotic spindle formation and cell-cycle progression of NSPCs. PMID:24260314

The histone acetyltransferase component TRRAP is targeted for destruction during the cell cycle.

PubMed

Ichim, G; Mola, M; Finkbeiner, M G; Cros, M-P; Herceg, Z; Hernandez-Vargas, H

2014-01-09

Chromosomes are dynamic structures that must be reversibly condensed and unfolded to accommodate mitotic division and chromosome segregation. Histone modifications are involved in the striking chromatin reconfiguration taking place during mitosis. However, the mechanisms that regulate activity and function of histone-modifying factors as cells enter and exit mitosis are poorly understood. Here, we show that the anaphase-promoting complex or cyclosome (APC/C) is involved in the mitotic turnover of TRRAP (TRansformation/tRanscription domain-Associated Protein), a common component of histone acetyltransferase (HAT) complexes, and that the pre-mitotic degradation of TRRAP is mediated by the APC/C ubiquitin ligase activators Cdc20 and Cdh1. Ectopic expression of both Cdh1 and Cdc20 reduced the levels of coexpressed TRRAP protein and induced its ubiquitination. TRRAP overexpression or stabilization induces multiple mitotic defects, including lagging chromosomes, chromosome bridges and multipolar spindles. In addition, lack of sister chromatid cohesion and impaired chromosome condensation were found after TRRAP overexpression or stabilization. By using a truncated form of TRRAP, we show that mitotic delay is associated with a global histone H4 hyperacetylation induced by TRRAP overexpression. These results demonstrate that the chromatin modifier TRRAP is targeted for destruction in a cell cycle-dependent fashion. They also suggest that degradation of TRRAP by the APC/C is necessary for a proper condensation of chromatin and proper chromosome segregation. Chromatin compaction mediated by histone modifiers may represent a fundamental arm for APC/C orchestration of the mitotic machinery.

Influence of acute promyelocytic leukemia therapeutic drugs on nuclear pore complex density and integrity.

PubMed

Lång, Anna; Øye, Alexander; Eriksson, Jens; Rowe, Alexander D; Lång, Emma; Bøe, Stig Ove

2018-05-15

During cell division, a large number of nuclear proteins are released into the cytoplasm due to nuclear envelope breakdown. Timely nuclear import of these proteins following exit from mitosis is critical for establishment of the G1 nuclear environment. Dysregulation of post-mitotic nuclear import may affect the fate of newly divided stem or progenitor cells and may lead to cancer. Acute promyelocytic leukemia (APL) is a malignant disorder that involves a defect in blood cell differentiation at the promyelocytic stage. Recent studies suggest that pharmacological concentrations of the APL therapeutic drugs, all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), affect post-mitotic nuclear import of the APL-associated oncoprotein PML/RARA. In the present study, we have investigated the possibility that ATRA and ATO affect post-mitotic nuclear import through interference with components of the nuclear import machinery. We observe reduced density and impaired integrity of nuclear pore complexes after ATRA and/or ATO exposure. Using a post-mitotic nuclear import assay, we demonstrate distinct import kinetics among different nuclear import pathways while nuclear import rates were similar in the presence or absence of APL therapeutic drugs. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Dnmt1-dependent Chk1 pathway suppression is protective against neuron division.

PubMed

Oshikawa, Mio; Okada, Kei; Tabata, Hidenori; Nagata, Koh-Ichi; Ajioka, Itsuki

2017-09-15

Neuronal differentiation and cell-cycle exit are tightly coordinated, even in pathological situations. When pathological neurons re-enter the cell cycle and progress through the S phase, they undergo cell death instead of division. However, the mechanisms underlying mitotic resistance are mostly unknown. Here, we have found that acute inactivation of retinoblastoma (Rb) family proteins (Rb, p107 and p130) in mouse postmitotic neurons leads to cell death after S-phase progression. Checkpoint kinase 1 (Chk1) pathway activation during the S phase prevented the cell death, and allowed the division of cortical neurons that had undergone acute Rb family inactivation, oxygen-glucose deprivation (OGD) or in vivo hypoxia-ischemia. During neurogenesis, cortical neurons became protected from S-phase Chk1 pathway activation by the DNA methyltransferase Dnmt1, and underwent cell death after S-phase progression. Our results indicate that Chk1 pathway activation overrides mitotic safeguards and uncouples neuronal differentiation from mitotic resistance. © 2017. Published by The Company of Biologists Ltd.

Predicting network modules of cell cycle regulators using relative protein abundance statistics.

PubMed

Oguz, Cihan; Watson, Layne T; Baumann, William T; Tyson, John J

2017-02-28

Parameter estimation in systems biology is typically done by enforcing experimental observations through an objective function as the parameter space of a model is explored by numerical simulations. Past studies have shown that one usually finds a set of "feasible" parameter vectors that fit the available experimental data equally well, and that these alternative vectors can make different predictions under novel experimental conditions. In this study, we characterize the feasible region of a complex model of the budding yeast cell cycle under a large set of discrete experimental constraints in order to test whether the statistical features of relative protein abundance predictions are influenced by the topology of the cell cycle regulatory network. Using differential evolution, we generate an ensemble of feasible parameter vectors that reproduce the phenotypes (viable or inviable) of wild-type yeast cells and 110 mutant strains. We use this ensemble to predict the phenotypes of 129 mutant strains for which experimental data is not available. We identify 86 novel mutants that are predicted to be viable and then rank the cell cycle proteins in terms of their contributions to cumulative variability of relative protein abundance predictions. Proteins involved in "regulation of cell size" and "regulation of G1/S transition" contribute most to predictive variability, whereas proteins involved in "positive regulation of transcription involved in exit from mitosis," "mitotic spindle assembly checkpoint" and "negative regulation of cyclin-dependent protein kinase by cyclin degradation" contribute the least. These results suggest that the statistics of these predictions may be generating patterns specific to individual network modules (START, S/G2/M, and EXIT). To test this hypothesis, we develop random forest models for predicting the network modules of cell cycle regulators using relative abundance statistics as model inputs. Predictive performance is assessed by the areas under receiver operating characteristics curves (AUC). Our models generate an AUC range of 0.83-0.87 as opposed to randomized models with AUC values around 0.50. By using differential evolution and random forest modeling, we show that the model prediction statistics generate distinct network module-specific patterns within the cell cycle network.

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

PubMed

Colin, Didier J; Hain, Karolina O; Allan, Lindsey A; Clarke, Paul R

2015-03-01

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins

PubMed Central

Colin, Didier J.; Hain, Karolina O.; Allan, Lindsey A.; Clarke, Paul R.

2015-01-01

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies. PMID:25761368

Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila.

PubMed

Sousa-Nunes, Rita; Yee, Lih Ling; Gould, Alex P

2011-03-24

Many stem, progenitor and cancer cells undergo periods of mitotic quiescence from which they can be reactivated. The signals triggering entry into and exit from this reversible dormant state are not well understood. In the developing Drosophila central nervous system, multipotent self-renewing progenitors called neuroblasts undergo quiescence in a stereotypical spatiotemporal pattern. Entry into quiescence is regulated by Hox proteins and an internal neuroblast timer. Exit from quiescence (reactivation) is subject to a nutritional checkpoint requiring dietary amino acids. Organ co-cultures also implicate an unidentified signal from an adipose/hepatic-like tissue called the fat body. Here we provide in vivo evidence that Slimfast amino-acid sensing and Target of rapamycin (TOR) signalling activate a fat-body-derived signal (FDS) required for neuroblast reactivation. Downstream of this signal, Insulin-like receptor signalling and the Phosphatidylinositol 3-kinase (PI3K)/TOR network are required in neuroblasts for exit from quiescence. We demonstrate that nutritionally regulated glial cells provide the source of Insulin-like peptides (ILPs) relevant for timely neuroblast reactivation but not for overall larval growth. Conversely, ILPs secreted into the haemolymph by median neurosecretory cells systemically control organismal size but do not reactivate neuroblasts. Drosophila thus contains two segregated ILP pools, one regulating proliferation within the central nervous system and the other controlling tissue growth systemically. Our findings support a model in which amino acids trigger the cell cycle re-entry of neural progenitors via a fat-body-glia-neuroblasts relay. This mechanism indicates that dietary nutrients and remote organs, as well as local niches, are key regulators of transitions in stem-cell behaviour.

The flavonoid eupatorin inactivates the mitotic checkpoint leading to polyploidy and apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salmela, Anna-Leena; Turku Graduate School of Biomedical Sciences, Turku; Turku Centre for Biotechnology, P.O. Box 123, University of Turku

The spindle assembly checkpoint (SAC) is a conserved mechanism that ensures the fidelity of chromosome distribution in mitosis by preventing anaphase onset until the correct bipolar microtubule-kinetochore attachments are formed. Errors in SAC function may contribute to tumorigenesis by inducing numerical chromosome anomalies (aneuploidy). On the other hand, total disruption of SAC can lead to massive genomic imbalance followed by cell death, a phenomena that has therapeutic potency. We performed a cell-based high-throughput screen with a compound library of 2000 bioactives for novel SAC inhibitors and discovered a plant-derived phenolic compound eupatorin (3 Prime ,5-dihydroxy-4 Prime ,6,7-trimethoxyflavone) as an anti-mitoticmore » flavonoid. The premature override of the microtubule drug-imposed mitotic arrest by eupatorin is dependent on microtubule-kinetochore attachments but not interkinetochore tension. Aurora B kinase activity, which is essential for maintenance of normal SAC signaling, is diminished by eupatorin in cells and in vitro providing a mechanistic explanation for the observed forced mitotic exit. Eupatorin likely has additional targets since eupatorin treatment of pre-mitotic cells causes spindle anomalies triggering a transient M phase delay followed by impaired cytokinesis and polyploidy. Finally, eupatorin potently induces apoptosis in multiple cancer cell lines and suppresses cancer cell proliferation in organotypic 3D cell culture model.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick

Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugationmore » to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.« less

The Anaphase-Promoting Complex is a dual integrator that regulates both microRNA-mediated transcriptional regulation of Cyclin B1 and degradation of Cyclin B1 during Arabidopsis male gametophyte development

USDA-ARS?s Scientific Manuscript database

The anaphase-promoting complex/cyclosome (APC/C), an essential ubiquitin protein ligase, regulates mitotic progression and exit by enhancing degradation of cell cycle regulatory proteins, such as CYCB1;1, whose transcripts are upregulated by DUO POLLEN1 (DUO1). DUO1 is required for cell division in ...

The Anaphase-Promoting Complex Is a Dual Integrator That Regulates Both MicroRNA-Mediated Transcriptional Regulation of Cyclin B1 and Degradation of Cyclin B1 during Arabidopsis Male Gametophyte Development

USDA-ARS?s Scientific Manuscript database

The anaphase-promoting complex/cyclosome (APC/C), an essential ubiquitin protein ligase, regulates mitotic progression and exit by enhancing degradation of cell cycle regulatory proteins, such as CYCB1;1, whose transcripts are upregulated by DUO POLLEN1 (DUO1). DUO1 is required for cell division in ...

Precommitment low-level Neurog3 expression defines a long-lived mitotic endocrine-biased progenitor pool that drives production of endocrine-committed cells

PubMed Central

Bechard, Matthew E.; Bankaitis, Eric D.; Hipkens, Susan B.; Ustione, Alessandro; Piston, David W.; Yang, Yu-Ping; Magnuson, Mark A.; Wright, Christopher V.E.

2016-01-01

The current model for endocrine cell specification in the pancreas invokes high-level production of the transcription factor Neurogenin 3 (Neurog3) in Sox9+ bipotent epithelial cells as the trigger for endocrine commitment, cell cycle exit, and rapid delamination toward proto-islet clusters. This model posits a transient Neurog3 expression state and short epithelial residence period. We show, however, that a Neurog3TA.LO cell population, defined as Neurog3 transcriptionally active and Sox9+ and often containing nonimmunodetectable Neurog3 protein, has a relatively high mitotic index and prolonged epithelial residency. We propose that this endocrine-biased mitotic progenitor state is functionally separated from a pro-ductal pool and endows them with long-term capacity to make endocrine fate-directed progeny. A novel BAC transgenic Neurog3 reporter detected two types of mitotic behavior in Sox9+ Neurog3TA.LO progenitors, associated with progenitor pool maintenance or derivation of endocrine-committed Neurog3HI cells, respectively. Moreover, limiting Neurog3 expression dramatically increased the proportional representation of Sox9+ Neurog3TA.LO progenitors, with a doubling of its mitotic index relative to normal Neurog3 expression, suggesting that low Neurog3 expression is a defining feature of this cycling endocrine-biased state. We propose that Sox9+ Neurog3TA.LO endocrine-biased progenitors feed production of Neurog3HI endocrine-committed cells during pancreas organogenesis. PMID:27585590

Two-phase deep convolutional neural network for reducing class skewness in histopathological images based breast cancer detection.

PubMed

Wahab, Noorul; Khan, Asifullah; Lee, Yeon Soo

2017-06-01

Different types of breast cancer are affecting lives of women across the world. Common types include Ductal carcinoma in situ (DCIS), Invasive ductal carcinoma (IDC), Tubular carcinoma, Medullary carcinoma, and Invasive lobular carcinoma (ILC). While detecting cancer, one important factor is mitotic count - showing how rapidly the cells are dividing. But the class imbalance problem, due to the small number of mitotic nuclei in comparison to the overwhelming number of non-mitotic nuclei, affects the performance of classification models. This work presents a two-phase model to mitigate the class biasness issue while classifying mitotic and non-mitotic nuclei in breast cancer histopathology images through a deep convolutional neural network (CNN). First, nuclei are segmented out using blue ratio and global binary thresholding. In Phase-1 a CNN is then trained on the segmented out 80×80 pixel patches based on a standard dataset. Hard non-mitotic examples are identified and augmented; mitotic examples are oversampled by rotation and flipping; whereas non-mitotic examples are undersampled by blue ratio histogram based k-means clustering. Based on this information from Phase-1, the dataset is modified for Phase-2 in order to reduce the effects of class imbalance. The proposed CNN architecture and data balancing technique yielded an F-measure of 0.79, and outperformed all the methods relying on specific handcrafted features, as well as those using a combination of handcrafted and CNN-generated features. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structure-Templated Predictions of Novel Protein Interactions from Sequence Information

PubMed Central

Betel, Doron; Breitkreuz, Kevin E; Isserlin, Ruth; Dewar-Darch, Danielle; Tyers, Mike; Hogue, Christopher W. V

2007-01-01

The multitude of functions performed in the cell are largely controlled by a set of carefully orchestrated protein interactions often facilitated by specific binding of conserved domains in the interacting proteins. Interacting domains commonly exhibit distinct binding specificity to short and conserved recognition peptides called binding profiles. Although many conserved domains are known in nature, only a few have well-characterized binding profiles. Here, we describe a novel predictive method known as domain–motif interactions from structural topology (D-MIST) for elucidating the binding profiles of interacting domains. A set of domains and their corresponding binding profiles were derived from extant protein structures and protein interaction data and then used to predict novel protein interactions in yeast. A number of the predicted interactions were verified experimentally, including new interactions of the mitotic exit network, RNA polymerases, nucleotide metabolism enzymes, and the chaperone complex. These results demonstrate that new protein interactions can be predicted exclusively from sequence information. PMID:17892321

Mathematical modeling and numerical simulation of the mitotic spindle orientation system.

PubMed

Ibrahim, Bashar

2018-05-21

The mitotic spindle orientation and position is crucial for the fidelity of chromosome segregation during asymmetric cell division to generate daughter cells with different sizes or fates. This mechanism is best understood in the budding yeast Saccharomyces cerevisiae, named the spindle position checkpoint (SPOC). The SPOC inhibits cells from exiting mitosis until the mitotic spindle is properly oriented along the mother-daughter polarity axis. Despite many experimental studies, the mechanisms underlying SPOC regulation remains elusive and unexplored theoretically. Here, a minimal mathematical is developed to describe SPOC activation and silencing having autocatalytic feedback-loop. Numerical simulations of the nonlinear ordinary differential equations (ODEs) model accurately reproduce the phenotype of SPOC mechanism. Bifurcation analysis of the nonlinear ODEs reveals the orientation dependency on spindle pole bodies, and how this dependence is altered by parameter values. These results provide for systems understanding on the molecular organization of spindle orientation system via mathematical modeling. The presented mathematical model is easy to understand and, within the above mentioned context, can be used as a base for further development of quantitative models in asymmetric cell-division. Copyright © 2018. Published by Elsevier Inc.

53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis.

PubMed

Fong, Chii Shyang; Mazo, Gregory; Das, Tuhin; Goodman, Joshua; Kim, Minhee; O'Rourke, Brian P; Izquierdo, Denisse; Tsou, Meng-Fu Bryan

2016-07-02

Mitosis occurs efficiently, but when it is disturbed or delayed, p53-dependent cell death or senescence is often triggered after mitotic exit. To characterize this process, we conducted CRISPR-mediated loss-of-function screens using a cell-based assay in which mitosis is consistently disturbed by centrosome loss. We identified 53BP1 and USP28 as essential components acting upstream of p53, evoking p21-dependent cell cycle arrest in response not only to centrosome loss, but also to other distinct defects causing prolonged mitosis. Intriguingly, 53BP1 mediates p53 activation independently of its DNA repair activity, but requiring its interacting protein USP28 that can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo. Moreover, 53BP1 can transduce prolonged mitosis to cell cycle arrest independently of the spindle assembly checkpoint (SAC), suggesting that while SAC protects mitotic accuracy by slowing down mitosis, 53BP1 and USP28 function in parallel to select against disturbed or delayed mitosis, promoting mitotic efficiency.

Mechanism of APC/CCDC20 activation by mitotic phosphorylation.

PubMed

Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A; Brunner, Michael R; Davidson, Iain F; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A; Peters, Jan-Michael

2016-05-10

Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/C(CDC20) activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/C(CDC20) activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/C(CDC20) activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis.

Mechanism of APC/CCDC20 activation by mitotic phosphorylation

PubMed Central

Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G.; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A.; Brunner, Michael R.; Davidson, Iain F.; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A.; Peters, Jan-Michael

2016-01-01

Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/CCDC20 activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/CCDC20 activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/CCDC20 activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis. PMID:27114510

Human Nek7-interactor RGS2 is required for mitotic spindle organization.

PubMed

de Souza, Edmarcia Elisa; Hehnly, Heidi; Perez, Arina Marina; Meirelles, Gabriela Vaz; Smetana, Juliana Helena Costa; Doxsey, Stephen; Kobarg, Jörg

2015-01-01

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.

Revised genetic requirements for the decatenation G2 checkpoint: the role of ATM

PubMed Central

Bower, Jacquelyn J.; Zhou, Yingchun; Zhou, Tong; Simpson, Dennis A.; Arlander, Sonnet J.; Paules, Richard S.; Cordeiro-Stone, Marila; Kaufmann, William K.

2010-01-01

The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in CHEK2, but no appreciable phosphorylation of Ser139 on H2AX. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage. PMID:20372057

Human Nek7-interactor RGS2 is required for mitotic spindle organization

PubMed Central

de Souza, Edmarcia Elisa; Hehnly, Heidi; Perez, Arina Marina; Meirelles, Gabriela Vaz; Smetana, Juliana Helena Costa; Doxsey, Stephen; Kobarg, Jörg

2015-01-01

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization. PMID:25664600

Blocking variant surface glycoprotein synthesis alters endoplasmic reticulum exit sites/Golgi homeostasis in Trypanosoma brucei.

PubMed

Ooi, Cher-Pheng; Smith, Terry K; Gluenz, Eva; Wand, Nadina Vasileva; Vaughan, Sue; Rudenko, Gloria

2018-06-01

The predominant secretory cargo of bloodstream form Trypanosoma brucei is variant surface glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre-cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post-mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of endoplasmic reticulum exit sites (ERES) in post-mitotic cells dropped from 3.9 ± 0.6 to 2.7 ± 0.1 eight hours after blocking VSG synthesis. The secretory pathway was still functional in these stalled cells, as monitored using Cathepsin L. Rates of phospholipid and glycosylphosphatidylinositol-anchor biosynthesis remained relatively unaffected, except for the level of sphingomyelin which increased. However, both endoplasmic reticulum and Golgi morphology became distorted, with the Golgi cisternae becoming significantly dilated, particularly at the trans-face. Membrane accumulation in these structures is possibly caused by reduced budding of nascent vesicles due to the drastic reduction in the total amount of secretory cargo, that is, VSG. These data argue that the total flux of secretory cargo impacts upon the biogenesis and maintenance of secretory structures and organelles in T. brucei, including the ERES and Golgi. © 2018 The Authors. Traffic published by John Wiley & Sons Ltd.

Inhibition of Bcl-xL sensitizes cells to mitotic blockers, but not mitotic drivers

PubMed Central

Bennett, Ailsa; Sloss, Olivia; Topham, Caroline; Nelson, Louisa; Tighe, Anthony

2016-01-01

Cell fate in response to an aberrant mitosis is governed by two competing networks: the spindle assembly checkpoint (SAC) and the intrinsic apoptosis pathway. The mechanistic interplay between these two networks is obscured by functional redundancy and the ability of cells to die either in mitosis or in the subsequent interphase. By coupling time-lapse microscopy with selective pharmacological agents, we systematically probe pro-survival Bcl-xL in response to various mitotic perturbations. Concentration matrices show that BH3-mimetic-mediated inhibition of Bcl-xL synergises with perturbations that induce an SAC-mediated mitotic block, including drugs that dampen microtubule dynamics, and inhibitors targeting kinesins and kinases required for spindle assembly. By contrast, Bcl-xL inhibition does not synergize with drugs which drive cells through an aberrant mitosis by overriding the SAC. This differential effect, which is explained by compensatory Mcl-1 function, provides opportunities for patient stratification and combination treatments in the context of cancer chemotherapy. PMID:27512141

Spatiotemporal Regulation of Nuclear Transport Machinery and Microtubule Organization

PubMed Central

Okada, Naoyuki; Sato, Masamitsu

2015-01-01

Spindle microtubules capture and segregate chromosomes and, therefore, their assembly is an essential event in mitosis. To carry out their mission, many key players for microtubule formation need to be strictly orchestrated. Particularly, proteins that assemble the spindle need to be translocated at appropriate sites during mitosis. A small GTPase (hydrolase enzyme of guanosine triphosphate), Ran, controls this translocation. Ran plays many roles in many cellular events: nucleocytoplasmic shuttling through the nuclear envelope, assembly of the mitotic spindle, and reorganization of the nuclear envelope at the mitotic exit. Although these events are seemingly distinct, recent studies demonstrate that the mechanisms underlying these phenomena are substantially the same as explained by molecular interplay of the master regulator Ran, the transport factor importin, and its cargo proteins. Our review focuses on how the transport machinery regulates mitotic progression of cells. We summarize translocation mechanisms governed by Ran and its regulatory proteins, and particularly focus on Ran-GTP targets in fission yeast that promote spindle formation. We also discuss the coordination of the spatial and temporal regulation of proteins from the viewpoint of transport machinery. We propose that the transport machinery is an essential key that couples the spatial and temporal events in cells. PMID:26308057

[Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

PubMed

Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

2012-01-01

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

A role for tubular networks and a COP I-independent pathway in the mitotic fragmentation of Golgi stacks in a cell-free system

PubMed Central

1995-01-01

Golgi stacks were previously shown to be converted into tubular networks when incubated in mitotic cytosol depleted of the coatomer subunit of COP I coats (Misteli and Warren, 1994). Similar, though smaller, networks are now shown to be an early intermediate on the Golgi fragmentation pathway both in vitro and in vivo. Their appearance mirrors the disappearance of Golgi cisternae and at their peak they constitute 35% of total Golgi membrane. They are consumed by two pathways, the first involving the budding of COP I-coated vesicles described previously (Misteli and Warren, 1994). The second involves a COP I-independent mechanism that leads eventually to a vesicle fraction that is larger in size and more heterogeneous than that produced by the COP I-mechanism. We suggest that both pathways operate concurrently at the onset of mitotic fragmentation. The COP I-independent pathway converts cisternae into tubular networks that then fragment. The COP I- dependent pathway partially consumes first the cisternae at the beginning of the incubation and then the tubular networks that form from them. PMID:7657690

Molecular networks linked by Moesin drive remodeling of the cell cortex during mitosis

PubMed Central

Roubinet, Chantal; Decelle, Barbara; Chicanne, Gaëtan; Dorn, Jonas F.; Payrastre, Bernard; Payre, François; Carreno, Sébastien

2011-01-01

The cortical mechanisms that drive the series of mitotic cell shape transformations remain elusive. In this paper, we identify two novel networks that collectively control the dynamic reorganization of the mitotic cortex. We demonstrate that Moesin, an actin/membrane linker, integrates these two networks to synergize the cortical forces that drive mitotic cell shape transformations. We find that the Pp1-87B phosphatase restricts high Moesin activity to early mitosis and down-regulates Moesin at the polar cortex, after anaphase onset. Overactivation of Moesin at the polar cortex impairs cell elongation and thus cytokinesis, whereas a transient recruitment of Moesin is required to retract polar blebs that allow cortical relaxation and dissipation of intracellular pressure. This fine balance of Moesin activity is further adjusted by Skittles and Pten, two enzymes that locally produce phosphoinositol 4,5-bisphosphate and thereby, regulate Moesin cortical association. These complementary pathways provide a spatiotemporal framework to explain how the cell cortex is remodeled throughout cell division. PMID:21969469

Interkinetic and migratory behavior of a cohort of neocortical neurons arising in the early embryonic murine cerebral wall

NASA Technical Reports Server (NTRS)

Takahashi, T.; Nowakowski, R. S.; Caviness, V. S. Jr

1996-01-01

Neocortical neuronogenesis occurs in the pseudostratified ventricular epithelium (PVE) where nuclei of proliferative cells undergo interkinetic nuclear movement. A fraction of daughter cells exits the cell cycle as neurons (the quiescent, or Q, fraction), whereas a complementary fraction remains in the cell cycle (the proliferative, or P, fraction). By means of sequential thymidine and bromodeoxyuridine injections in mouse on embryonic day 14, we have monitored the proliferative and post-mitotic migratory behaviors of 1 and 2 hr cohorts of PVE cells defined by the injection protocols. Soon after mitosis, the Q fraction partitions into a rapidly exiting (up to 50 microns/hr) subpopulation (Qr) and a more slowly exiting (6 microns/hr) subpopulation (Qs). Qr and Qs are separated as two distributions on exit from the ventricular zone with an interpeak distance of approximately 40 microns. Cells in Qr and Qs migrate through the intermediate zone with no significant change in the interpeak distance, suggesting that they migrate at approximately the same velocities. The rate of migration increases with ascent through the intermediate zone (average 2-6.4 microns/hr) slowing only transiently on entry into the developing cortex. Within the cortex, Qr and Qs merge to form a single distribution most concentrated over layer V.

Polo-like kinase 1 inhibits DNA damage response during mitosis

PubMed Central

Benada, Jan; Burdová, Kamila; Lidak, Tomáš; von Morgen, Patrick; Macurek, Libor

2015-01-01

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis. PMID:25607646

Polo-like kinase 1 inhibits DNA damage response during mitosis.

PubMed

Benada, Jan; Burdová, Kamila; Lidak, Tomáš; von Morgen, Patrick; Macurek, Libor

2015-01-01

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.

The Clathrin-dependent Spindle Proteome*

PubMed Central

Rao, Sushma R.; Flores-Rodriguez, Neftali; Page, Scott L.; Wong, Chin; Robinson, Phillip J.; Chircop, Megan

2016-01-01

The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a “moonlighting” role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation. PMID:27174698

The Clathrin-dependent Spindle Proteome.

PubMed

Rao, Sushma R; Flores-Rodriguez, Neftali; Page, Scott L; Wong, Chin; Robinson, Phillip J; Chircop, Megan

2016-08-01

The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a "moonlighting" role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Atomic structure of the APC/C and its mechanism of protein ubiquitination

PubMed Central

Yang, Jing; McLaughlin, Stephen H.; Barford, David

2015-01-01

The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex, and interphase inhibitor Emi1 ensures the correct order and timing of distinct cell cycle transitions. Here, we used cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module, and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of experiments to further investigate APC/C functions in vivo. PMID:26083744

Measuring APC/C-Dependent Ubiquitylation In Vitro.

PubMed

Jarvis, Marc A; Brown, Nicholas G; Watson, Edmond R; VanderLinden, Ryan; Schulman, Brenda A; Peters, Jan-Michael

2016-01-01

The anaphase-promoting complex/cyclosome (APC/C) is a 1.2 MDa ubiquitin ligase complex with important functions in both proliferating and post-mitotic differentiated cells. In proliferating cells, APC/C controls cell cycle progression by targeting inhibitors of chromosome segregation and mitotic exit for degradation by the 26S proteasome. To understand how APC/C recruits and ubiquitylates its substrate proteins and how these processes are controlled, it is essential to analyze APC/C activity in vitro. In the past, such experiments have been limited by the fact that large quantities of purified APC/C were difficult to obtain and that mutated versions of the APC/C could not be easily generated. In this chapter we review recent advances in generating and purifying recombinant forms of the human APC/C and its co-activators, using methods that are scalable and compatible with mutagenesis. We also describe a method that allows the quantitative analysis of APC/C activity using fluorescently labeled substrate proteins.

Micromechanics of human mitotic chromosomes

NASA Astrophysics Data System (ADS)

Sun, Mingxuan; Kawamura, Ryo; Marko, John F.

2011-02-01

Eukaryote cells dramatically reorganize their long chromosomal DNAs to facilitate their physical segregation during mitosis. The internal organization of folded mitotic chromosomes remains a basic mystery of cell biology; its understanding would likely shed light on how chromosomes are separated from one another as well as into chromosome structure between cell divisions. We report biophysical experiments on single mitotic chromosomes from human cells, where we combine micromanipulation, nano-Newton-scale force measurement and biochemical treatments to study chromosome connectivity and topology. Results are in accord with previous experiments on amphibian chromosomes and support the 'chromatin network' model of mitotic chromosome structure. Prospects for studies of chromosome-organizing proteins using siRNA expression knockdowns, as well as for differential studies of chromosomes with and without mutations associated with genetic diseases, are also discussed.

Micromechanical-biochemical studies of mitotic chromosome elasticity and structure

NASA Astrophysics Data System (ADS)

Poirier, Michael Guy

The structure of mitotic chromosomes was studied by combining micromechanical force measurements with microfluidic biochemical exposures. Our method is to use glass micropipettes attached to either end of a single chromosome to do mechanical experiments in the extracellular buffer. A third pipette can be used to locally 'spray' reactants so as to carry out dynamical mechanical-chemical experiments. The following elastic properties of mitotic chromosomes are found: Young's modulus, Y = 300 Pa; Poisson ratio, sigma = 0.1; Bending rigidity, B = 1 x 10 -22 J·m; Internal viscosity, eta' = 100 kg/m·sec; Volume fraction, ϕ = 0.7; Extensions of less than 3 times the relaxed length are linear and reversible; Extensions beyond 30 fold exhibit a force plateau at 15 nN and convert the chromosome to a disperse ghost-like state with little change in chromatin structure; Mitotic chromosomes are relatively isotropic; dsDNA cuts of at least every 3 kb cause the a mitotic chromosomes to fall apart; dsDNA cuts less frequently than every 50 kb do not affect mitotic chromosome structure. These results lead to the conclusion that mitotic chromosomes are a network crosslinked every 50 kb between which chromatin is fold by chromatin folding proteins, which are likely to be condensins.

Downsides and benefits of unicellularity in budding yeast

NASA Astrophysics Data System (ADS)

Balazsi, Gabor; Chen, Lin; Kuzdzal-Fick, Jennie

Yeast cells that do not separate after cell division form clumps. Clumping was shown to aid utilization of certain sugars, but its effects in stressful conditions are unknown. Generally speaking, what are the costs and benefits of unicellularity versus clumping multicellularity in normal and stressful conditions? To address this question, we evolved clumping yeast towards unicellularity by continuously propagating only those cells that remain suspended in liquid culture after settling. Whole-genome sequencing indicated that mutations in the AMN1 (antagonist of mitotic exit network) gene underlie the changes from clumping to unicellular phenotypes in these evolved yeast cells. Simple models predict that clumping should hinder growth in normal conditions while being protective in stress. Accordingly, we find experimentally that yeast clumps are more resistant to freeze/thaw, hydrogen peroxide, and ethanol stressors than their unicellular counterparts. On the other hand, unicellularity seems to be advantageous in normal conditions. Overall, these results reveal the downsides and benefits of unicellularity in different environmental conditions and uncover its genetic bases in yeast. This research was supported by the NIH Director's New Innovator Award Program (1DP2 OD006481-01), by NSF/IOS 1021675 and the Laufer Center for Physical & Quantitative Biology.

Amphiastral Mitotic Spindle Assembly in Vertebrate Cells Lacking Centrosomes

PubMed Central

Hornick, Jessica E.; Mader, Christopher C.; Tribble, Emily K.; Bagne, Cydney C.; Vaughan, Kevin T.; Shaw, Sidney L.; Hinchcliffe, Edward H.

2011-01-01

Summary The role of centrosomes/centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles [1, 2, 3]. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays – termed an amphiaster (“a star on both sides” [4]) – that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB) [5]. Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC re-assembled, and prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split/separate before NEB, and these entered mitosis with persistent monastral spindles. The chromatin-mediated RAN-GTP pathway could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but in its absence, the fidelity of bipolar spindle assembly is highly compromised. PMID:21439826

3D analysis of mitosis distribution highlights the longitudinal zonation and diarch symmetry in proliferation activity of the Arabidopsis thaliana root meristem.

PubMed

Lavrekha, Viktoriya V; Pasternak, Taras; Ivanov, Victor B; Palme, Klaus; Mironova, Victoria V

2017-12-01

To date CYCB1;1 marker and cortex cell lengths have been conventionally used to determine the proliferation activity of the Arabidopsis root meristem. By creating a 3D map of mitosis distribution we showed that these markers overlooked that stele and endodermis save their potency to divide longer than the cortex and epidermis. Cessation of cell divisions is not a random process, so that mitotic activity within the endodermis and stele shows a diarch pattern. Mitotic activity of all root tissues peaked at the same distance from the quiescent center (QC); however, different tissues stopped dividing at different distances, with cells of the protophloem exiting the cell cycle first and the procambial cells being the last. The robust profile of mitotic activity in the root tip defines the longitudinal zonation in the meristem with the proliferation domain, where all cells are able to divide; and the transition domain, where the cell files cease to divide. 3D analysis of cytokinin deficient and cytokinin signaling mutants showed that their proliferation domain is similar to that of the wild type, but the transition domain is much longer. Our data suggest a strong inhibitory effect of cytokinin on anticlinal cell divisions in the stele. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Electro-Acoustic Behavior of the Mitotic Spindle: A Semi-Classical Coarse-Grained Model

PubMed Central

Havelka, Daniel; Kučera, Ondřej; Deriu, Marco A.; Cifra, Michal

2014-01-01

The regulation of chromosome separation during mitosis is not fully understood yet. Microtubules forming mitotic spindles are targets of treatment strategies which are aimed at (i) the triggering of the apoptosis or (ii) the interruption of uncontrolled cell division. Despite these facts, only few physical models relating to the dynamics of mitotic spindles exist up to now. In this paper, we present the first electromechanical model which enables calculation of the electromagnetic field coupled to acoustic vibrations of the mitotic spindle. This electromagnetic field originates from the electrical polarity of microtubules which form the mitotic spindle. The model is based on the approximation of resonantly vibrating microtubules by a network of oscillating electric dipoles. Our computational results predict the existence of a rapidly changing electric field which is generated by either driven or endogenous vibrations of the mitotic spindle. For certain values of parameters, the intensity of the electric field and its gradient reach values which may exert a not-inconsiderable force on chromosomes which are aligned in the spindle midzone. Our model may describe possible mechanisms of the effects of ultra-short electrical and mechanical pulses on dividing cells—a strategy used in novel methods for cancer treatment. PMID:24497952

Phospho-Bcl-xL(Ser62) influences spindle assembly and chromosome segregation during mitosis.

PubMed

Wang, Jianfang; Beauchemin, Myriam; Bertrand, Richard

2014-01-01

Functional analysis of a series of phosphorylation mutants reveals that Bcl-xL(Ser62Ala) influences cell entry into anaphase and mitotic exit in taxol-exposed cells compared with cells expressing wild-type Bcl-xL or a series of other phosphorylation mutants, an effect that appears to be independent of its anti-apoptotic activity. During normal mitosis progression, Bcl-xL(Ser62) is strongly phosphorylated by PLK1 and MAPK14/SAPKp38α at the prometaphase, metaphase, and the anaphase boundaries, while it is de-phosphorylated at telophase and cytokinesis. Phospho-Bcl-xL(Ser62) localizes in centrosomes with γ-tubulin and in the mitotic cytosol with some spindle-assembly checkpoint signaling components, including PLK1, BubR1, and Mad2. In taxol- and nocodazole-exposed cells, phospho-Bcl-xL(Ser62) also binds to Cdc20- Mad2-, BubR1-, and Bub3-bound complexes, while Bcl-xL(Ser62Ala) does not. Silencing Bcl-xL expression and expressing the phosphorylation mutant Bcl-xL(Ser62Ala) lead to an increased number of cells harboring mitotic spindle defects including multipolar spindle, chromosome lagging and bridging, aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h. Together, the data indicate that during mitosis, Bcl-xL(Ser62) phosphorylation impacts on spindle assembly and chromosome segregation, influencing chromosome stability. Observations of mitotic cells harboring aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h were also made with cells expressing the phosphorylation mutant Bcl-xL(Ser49Ala) and dual mutant Bcl-xL(Ser49/62Ala).

Dropped out or pushed out? Insurance market exit and provider market power in Medicare Advantage.

PubMed

Pelech, Daria

2017-01-01

This paper explores how provider and insurer market power affect which markets an insurer chooses to operate in. A 2011 policy change required that certain private insurance plans in Medicare form provider networks de novo; in response, insurers cancelled two-thirds of the affected plans. Using detailed data on pre-policy provider and insurer market structure, I compare markets where insurers built networks to those they exited. Overall, insurers in the most concentrated hospital and physician markets were 9 and 13 percentage points more likely to exit, respectively, than those in the least concentrated markets. Conversely, insurers with more market power were less likely to exit than those with less, and an insurer's market power had the largest effect on exit in concentrated hospital markets. These findings suggest that concentrated provider markets contribute to insurer exit and that insurers with less market power have more difficulty surviving in concentrated provider markets. Published by Elsevier B.V.

A nonlinear q-voter model with deadlocks on the Watts-Strogatz graph

NASA Astrophysics Data System (ADS)

Sznajd-Weron, Katarzyna; Michal Suszczynski, Karol

2014-07-01

We study the nonlinear $q$-voter model with deadlocks on a Watts-Strogats graph. Using Monte Carlo simulations, we obtain so called exit probability and exit time. We determine how network properties, such as randomness or density of links influence exit properties of a model.

Drosophila Polo regulates the spindle assembly checkpoint through Mps1-dependent BubR1 phosphorylation.

PubMed

Conde, Carlos; Osswald, Mariana; Barbosa, João; Moutinho-Santos, Tatiana; Pinheiro, Diana; Guimarães, Sofia; Matos, Irina; Maiato, Helder; Sunkel, Claudio E

2013-06-12

Maintenance of genomic stability during eukaryotic cell division relies on the spindle assembly checkpoint (SAC) that prevents mitotic exit until all chromosomes are properly attached to the spindle. Polo is a mitotic kinase proposed to be involved in SAC function, but its role has remained elusive. We demonstrate that Polo and Aurora B functional interdependency comprises a positive feedback loop that promotes Mps1 kinetochore localization and activity. Expression of constitutively active Polo restores normal Mps1 kinetochore levels even after Aurora B inhibition, highlighting a role for Polo in Mps1 recruitment to unattached kinetochores downstream of Aurora B. We also show that Mps1 kinetochore localization is required for BubR1 hyperphosphorylation and formation of the 3F3/2 phosphoepitope. This is essential to allow recruitment of Cdc20 to unattached kinetochores and the assembly of anaphase-promoting complex/cyclosome-inhibitory complexes to levels that ensure long-term SAC activity. We propose a model in which Polo controls Mps1-dependent BubR1 phosphorylation to promote Cdc20 kinetochore recruitment and sustained SAC function.

Drosophila Polo regulates the spindle assembly checkpoint through Mps1-dependent BubR1 phosphorylation

PubMed Central

Conde, Carlos; Osswald, Mariana; Barbosa, João; Moutinho-Santos, Tatiana; Pinheiro, Diana; Guimarães, Sofia; Matos, Irina; Maiato, Helder; Sunkel, Claudio E

2013-01-01

Maintenance of genomic stability during eukaryotic cell division relies on the spindle assembly checkpoint (SAC) that prevents mitotic exit until all chromosomes are properly attached to the spindle. Polo is a mitotic kinase proposed to be involved in SAC function, but its role has remained elusive. We demonstrate that Polo and Aurora B functional interdependency comprises a positive feedback loop that promotes Mps1 kinetochore localization and activity. Expression of constitutively active Polo restores normal Mps1 kinetochore levels even after Aurora B inhibition, highlighting a role for Polo in Mps1 recruitment to unattached kinetochores downstream of Aurora B. We also show that Mps1 kinetochore localization is required for BubR1 hyperphosphorylation and formation of the 3F3/2 phosphoepitope. This is essential to allow recruitment of Cdc20 to unattached kinetochores and the assembly of anaphase-promoting complex/cyclosome-inhibitory complexes to levels that ensure long-term SAC activity. We propose a model in which Polo controls Mps1-dependent BubR1 phosphorylation to promote Cdc20 kinetochore recruitment and sustained SAC function. PMID:23685359

INPP5E Preserves Genomic Stability through Regulation of Mitosis.

PubMed

Sierra Potchanant, Elizabeth A; Cerabona, Donna; Sater, Zahi Abdul; He, Ying; Sun, Zejin; Gehlhausen, Jeff; Nalepa, Grzegorz

2017-03-15

The partially understood phosphoinositide signaling cascade regulates multiple aspects of cellular metabolism. Previous studies revealed that INPP5E, the inositol polyphosphate-5-phosphatase that is mutated in the developmental disorders Joubert and MORM syndromes, is essential for the function of the primary cilium and maintenance of phosphoinositide balance in nondividing cells. Here, we report that INPP5E further contributes to cellular homeostasis by regulating cell division. We found that silencing or genetic knockout of INPP5E in human and murine cells impairs the spindle assembly checkpoint, centrosome and spindle function, and maintenance of chromosomal integrity. Consistent with a cell cycle regulatory role, we found that INPP5E expression is cell cycle dependent, peaking at mitotic entry. INPP5E localizes to centrosomes, chromosomes, and kinetochores in early mitosis and shuttles to the midzone spindle at mitotic exit. Our findings identify the previously unknown, essential role of INPP5E in mitosis and prevention of aneuploidy, providing a new perspective on the function of this phosphoinositide phosphatase in health and development. Copyright © 2017 Sierra Potchanant et al.

Assays for the spindle assembly checkpoint in cell culture.

PubMed

Marcozzi, Chiara; Pines, Jonathon

2018-01-01

The spindle assembly checkpoint (SAC) is crucial to maintain genomic stability since it prevents premature separation of sister chromatids in mitosis and ensures the fidelity of chromosome segregation. The SAC arrests cells in mitosis and is not satisfied until all kinetochores are stably attached to the mitotic spindle. Improperly attached kinetochores activate the SAC and catalyze the formation of the mitotic checkpoint complex (MCC), containing Mad2, Cdc20, BubR1, and Bub3 proteins. The MCC binds and thereby inhibits the APC/C E3 ubiquitin ligase until the last kinetochore has attached to microtubules. Once the SAC is satisfied, the APC/C promptly activates and targets cyclin B1 and securin for degradation, thus allowing sister chromatids to separate and the cell to exit mitosis. Our understanding of SAC signaling has increased thanks to the development of new genetic, biochemical, molecular, and structural biology techniques. Here, we describe how live-cell imaging microscopy in combination with gene-targeting strategies and biochemical assays can be exploited to investigate the intrinsic properties of the SAC in mammalian cultured cells. © 2018 Elsevier Inc. All rights reserved.

Discovery of benzo[e]pyridoindolones as kinase inhibitors that disrupt mitosis exit while erasing AMPK-Thr172 phosphorylation on the spindle.

PubMed

Le, Ly-Thuy-Tram; Couvet, Morgane; Favier, Bertrand; Coll, Jean-Luc; Nguyen, Chi-Hung; Molla, Annie

2015-09-08

Aurora kinases play an essential role in mitotic progression and are attractive targets in cancer therapy. The first generation of benzo[e]pyridoindole exhibited powerful aurora kinase inhibition but their low solubility limited further development. Grafting a pyperidine-ethoxy group gives rise to a hydrosoluble inhibitor: compound C5M.C5M could efficiently inhibit the proliferation of cells from different origins. C5M prevented cell cycling, induced a strong mitotic arrest then, cells became polyploid and finally died. C5M did not impair the spindle checkpoint, the separation of the sister chromatids and the transfer of aurora B on the mid-zone. C5M prevented histone H3 phosphorylation at mitotic entry and erased AMPK-Thr172 phosphorylation in late mitosis. With this unique profile of inhibition, C5M could be useful for understanding the role of phospho-Thr172-AMPK in abscission and the relationship between the chromosomal complex and the energy sensing machinery.C5M is a multikinase inhibitor with interesting preclinical characteristics: high hydro-solubility and a good stability in plasma. A single dose prevents the expansion of multicellular spheroids. C5M can safely be injected to mice and reduces significantly the development of xenograft. The next step will be to define the protocol of treatment and the cancer therapeutic field of this new anti-proliferative drug.

Novel Mad2-targeting miR-493-3p controls mitotic fidelity and cancer cells' sensitivity to paclitaxel.

PubMed

Tambe, Mahesh; Pruikkonen, Sofia; Mäki-Jouppila, Jenni; Chen, Ping; Elgaaen, Bente Vilming; Straume, Anne Hege; Huhtinen, Kaisa; Cárpen, Olli; Lønning, Per Eystein; Davidson, Ben; Hautaniemi, Sampsa; Kallio, Marko J

2016-03-15

The molecular pathways that contribute to the proliferation and drug response of cancer cells are highly complex and currently insufficiently characterized. We have identified a previously unknown microRNA-based mechanism that provides cancer cells means to stimulate tumorigenesis via increased genomic instability and, at the same time, evade the action of clinically utilized microtubule drugs. We demonstrate miR-493-3p to be a novel negative regulator of mitotic arrest deficient-2 (MAD2), an essential component of the spindle assembly checkpoint that monitors the fidelity of chromosome segregation. The microRNA targets the 3' UTR of Mad2 mRNA thereby preventing translation of the Mad2 protein. In cancer cells, overexpression of miR-493-3p induced a premature mitotic exit that led to increased frequency of aneuploidy and cellular senescence in the progeny cells. Importantly, excess of the miR-493-3p conferred resistance of cancer cells to microtubule drugs. In human neoplasms, miR-493-3p and Mad2 expression alterations correlated with advanced ovarian cancer forms and high miR-493-3p levels were associated with reduced survival of ovarian and breast cancer patients with aggressive tumors, especially in the paclitaxel therapy arm. Our results suggest that intratumoral profiling of miR-493-3p and Mad2 levels can have diagnostic value in predicting the efficacy of taxane chemotherapy.

Automatic, time-interval traffic counts for recreation area management planning

Treesearch

D. L. Erickson; C. J. Liu; H. K. Cordell

1980-01-01

Automatic, time-interval recorders were used to count directional vehicular traffic on a multiple entry/exit road network in the Red River Gorge Geological Area, Daniel Boone National Forest. Hourly counts of entering and exiting traffic differed according to recorder location, but an aggregated distribution showed a delayed peak in exiting traffic thought to be...

Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

PubMed

Subramaniam, Sindhu; Sreenivas, Prethish; Cheedipudi, Sirisha; Reddy, Vatrapu Rami; Shashidhara, Lingadahalli Subrahmanya; Chilukoti, Ravi Kumar; Mylavarapu, Madhavi; Dhawan, Jyotsna

2014-01-01

Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological states such as cancer and degenerative disease.

Stability of kinetochore-microtubule attachment and the role of different KMN network components in Drosophila.

PubMed

Feijão, Tália; Afonso, Olga; Maia, André F; Sunkel, Claudio E

2013-10-01

Kinetochores bind spindle microtubules and also act as signaling centers that monitor this interaction. Defects in kinetochore assembly lead to chromosome missegregation and aneuploidy. The interaction between microtubules and chromosomes involves a conserved super-complex of proteins, known as the KNL1Mis12Ndc80 (KMN) network, composed by the KNL1 (Spc105), Mis12, and Ndc80 complexes. Previous studies indicate that all components of the network are required for kinetochore-microtubule attachment and all play relevant functions in chromosome congression, biorientation, and segregation. Here, we report a comparative study addressing the role of the different KMN components using dsRNA and in vivo fluorescence microscopy in Drosophila S2 cells allowing us to suggest that different KMN network components might perform different roles in chromosome segregation and the mitotic checkpoint signaling. Depletion of different components results in mostly lateral kinetochore-microtubule attachments that are relatively stable on depletion of Mis12 or Ndc80 but very unstable after Spc105 depletion. In vivo analysis on depletion of Mis12, Ndc80, and to some extent Spc105, shows that lateral kinetochore-microtubule interactions are still functional allowing poleward kinetochore movement. We also find that different KMN network components affect differently the localization of spindle assembly checkpoint (SAC) proteins at kinetochores. Depletion of Ndc80 and Spc105 abolishes the mitotic checkpoint, whereas depletion of Mis12 causes a delay in mitotic progression. Taken together, our results suggest that Mis12 and Ndc80 complexes help to properly orient microtubule attachment, whereas Spc105 plays a predominant role in the kinetochore-microtubule attachment as well as in the poleward movement of chromosomes, SAC response, and cell viability. Copyright © 2013 Wiley Periodicals, Inc.

Cell cycle-coupled expansion of AR activity promotes cancer progression.

PubMed

McNair, C; Urbanucci, A; Comstock, C E S; Augello, M A; Goodwin, J F; Launchbury, R; Zhao, S G; Schiewer, M J; Ertel, A; Karnes, J; Davicioni, E; Wang, L; Wang, Q; Mills, I G; Feng, F Y; Li, W; Carroll, J S; Knudsen, K E

2017-03-23

The androgen receptor (AR) is required for prostate cancer (PCa) survival and progression, and ablation of AR activity is the first line of therapeutic intervention for disseminated disease. While initially effective, recurrent tumors ultimately arise for which there is no durable cure. Despite the dependence of PCa on AR activity throughout the course of disease, delineation of the AR-dependent transcriptional network that governs disease progression remains elusive, and the function of AR in mitotically active cells is not well understood. Analyzing AR activity as a function of cell cycle revealed an unexpected and highly expanded repertoire of AR-regulated gene networks in actively cycling cells. New AR functions segregated into two major clusters: those that are specific to cycling cells and retained throughout the mitotic cell cycle ('Cell Cycle Common'), versus those that were specifically enriched in a subset of cell cycle phases ('Phase Restricted'). Further analyses identified previously unrecognized AR functions in major pathways associated with clinical PCa progression. Illustrating the impact of these unmasked AR-driven pathways, dihydroceramide desaturase 1 was identified as an AR-regulated gene in mitotically active cells that promoted pro-metastatic phenotypes, and in advanced PCa proved to be highly associated with development of metastases, recurrence after therapeutic intervention and reduced overall survival. Taken together, these findings delineate AR function in mitotically active tumor cells, thus providing critical insight into the molecular basis by which AR promotes development of lethal PCa and nominate new avenues for therapeutic intervention.

Degradation of the human mitotic checkpoint kinase Mps1 is cell cycle-regulated by APC-cCdc20 and APC-cCdh1 ubiquitin ligases.

PubMed

Cui, Yongping; Cheng, Xiaolong; Zhang, Ce; Zhang, Yanyan; Li, Shujing; Wang, Chuangui; Guadagno, Thomas M

2010-10-22

Mps1 is a dual specificity protein kinase with key roles in regulating the spindle assembly checkpoint and chromosome-microtubule attachments. Consistent with these mitotic functions, Mps1 protein levels fluctuate during the cell cycle, peaking at early mitosis and abruptly declining during mitotic exit and progression into the G(1) phase. Although evidence in budding yeast indicates that Mps1 is targeted for degradation at anaphase by the anaphase-promoting complex (APC)-c(Cdc20) complex, little is known about the regulatory mechanisms that govern Mps1 protein levels in human cells. Here, we provide evidence for the ubiquitin ligase/proteosome pathway in regulating human Mps1 levels during late mitosis through G(1) phase. First, we showed that treatment of HEK 293T cells with the proteosome inhibitor MG132 resulted in an increase in both the polyubiquitination and the accumulation of Mps1 protein levels. Next, Mps1 was shown to co-precipitate with APC and its activators Cdc20 and Cdh1 in a cell cycle-dependent manner. Consistent with this, overexpression of Cdc20 or Cdh1 led to a marked reduction of endogenous Mps1 levels during anaphase or G(1) phase, respectively. In contrast, depletion of Cdc20 or Cdh1 by RNAi treatment both led to the stabilization of Mps1 protein during mitosis or G(1) phase, respectively. Finally, we identified a single D-box motif in human Mps1 that is required for its ubiquitination and degradation. Failure to appropriately degrade Mps1 is sufficient to trigger centrosome amplification and mitotic abnormalities in human cells. Thus, our results suggest that the sequential actions of the APC-c(Cdc20) and APC-c(Cdh1) ubiquitin ligases regulate the clearance of Mps1 levels and are critical for Mps1 functions during the cell cycle in human cells.

THE MECHANISM OF 5-AMINOURACIL-INDUCED SYNCHRONY OF CELL DIVISION IN VI CIA FABA ROOT MERISTEMS

PubMed Central

Prensky, Wolf; Smith, Harold H.

1965-01-01

Cessation of mitosis was brought about in Vicia faba roots incubated for 24 hours in the thymine analogue, 5-aminouracil. Recovery of mitotic activity began 8 hours after removal from 5-aminouracil and reached a peak at 15 hours. If colchicine was added 4 hours before the peak of mitoses, up to 80 per cent of all cells accumulated in mitotic division stages. By use of single and double labeling techniques, it was shown that synchrony of cell divisions resulted from depression in the rate of DNA synthesis by 5-aminouracil, which brought about an accumulation of cells in the S phase of the cell cycle. Treatment with 5-aminouracil may have also caused a delay in the rate of exit of cells from the G2 period. It appeared to have no effect on the duration of the G1 period. When roots were removed from 5-aminouracil, DNA synthesis resumed in all cells in the S phase. Although thymidine antagonized the effects of 5-aminouracil, an exogenous supply of it was not necessary for the resumption of DNA synthesis, as shown by incorporation studies with tritiated deoxycytidine. PMID:19866644

Metaphase to Anaphase (mat) Transition–Defective Mutants inCaenorhabditis elegans

PubMed Central

Golden, Andy; Sadler, Penny L.; Wallenfang, Matthew R.; Schumacher, Jill M.; Hamill, Danielle R.; Bates, Gayle; Bowerman, Bruce; Seydoux, Geraldine; Shakes, Diane C.

2000-01-01

The metaphase to anaphase transition is a critical stage of the eukaryotic cell cycle, and, thus, it is highly regulated. Errors during this transition can lead to chromosome segregation defects and death of the organism. In genetic screens for temperature-sensitive maternal effect embryonic lethal (Mel) mutants, we have identified 32 mutants in the nematode Caenorhabditis elegans in which fertilized embryos arrest as one-cell embryos. In these mutant embryos, the oocyte chromosomes arrest in metaphase of meiosis I without transitioning to anaphase or producing polar bodies. An additional block in M phase exit is evidenced by the failure to form pronuclei and the persistence of phosphohistone H3 and MPM-2 antibody staining. Spermatocyte meiosis is also perturbed; primary spermatocytes arrest in metaphase of meiosis I and fail to produce secondary spermatocytes. Analogous mitotic defects cause M phase delays in mitotic germline proliferation. We have named this class of mutants “mat” for metaphase to anaphase transition defective. These mutants, representing six different complementation groups, all map near genes that encode subunits of the anaphase promoting complex or cyclosome, and, here, we show that one of the genes, emb-27, encodes the C. elegans CDC16 ortholog. PMID:11134076

Temporal Regulation of Lipin Activity Diverged to Account for Differences in Mitotic Programs

PubMed Central

Makarova, Maria; Gu, Ying; Chen, Jun-Song; Beckley, Janel Renée; Gould, Kathleen Louise; Oliferenko, Snezhana

2016-01-01

Summary Eukaryotes remodel the nucleus during mitosis using a variety of mechanisms that differ in the timing and the extent of nuclear envelope (NE) breakdown. Here, we probe the principles enabling this functional diversity by exploiting the natural divergence in NE management strategies between the related fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus [1, 2, 3]. We show that inactivation of Ned1, the phosphatidic acid phosphatase of the lipin family, by CDK phosphorylation is both necessary and sufficient to promote NE expansion required for “closed” mitosis in S. pombe. In contrast, Ned1 is not regulated during division in S. japonicus, thus limiting membrane availability and necessitating NE breakage. Interspecies gene swaps result in phenotypically normal divisions with the S. japonicus lipin acquiring an S. pombe-like mitotic phosphorylation pattern. Our results provide experimental evidence for the mitotic regulation of phosphatidic acid flux and suggest that the regulatory networks governing lipin activity diverged in evolution to give rise to strikingly dissimilar mitotic programs. PMID:26774782

Atypical and Malignant Meningioma: Outcome and Prognostic Factors in 119 Irradiated Patients. A Multicenter, Retrospective Study of the Rare Cancer Network

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pasquier, David; Bijmolt, Stefan; Veninga, Theo

2008-08-01

Purpose: To retrospectively analyze and assess the outcomes and prognostic factors in a large number of patients with atypical and malignant meningiomas. Methods and Materials: Ten academic medical centers participating in this Rare Cancer Network contributed 119 cases of patients with atypical or malignant meningiomas treated with external beam radiotherapy (EBRT) after surgery or for recurrence. Eligibility criteria were histologically proven atypical or anaplastic (malignant) meningioma (World Health Organization Grade 2 and 3) treated with fractionated EBRT after initial resection or for recurrence, and age >18 years. Sex ratio (male/female) was 1.3, and mean ({+-}SD) age was 57.6 {+-} 12more » years. Surgery was macroscopically complete (Simpson Grades 1-3) in 71% of patients; histology was atypical and malignant in 69% and 31%, respectively. Mean dose of EBRT was 54.6 {+-} 5.1 Gy (range, 40-66 Gy). Median follow-up was 4.1 years. Results: The 5- and 10-year actuarial overall survival rates were 65% and 51%, respectively, and were significantly influenced by age >60 years (p = 0.005), Karnofsky performance status (KPS) (p = 0.01), and high mitotic rate (p = 0.047) on univariate analysis. On multivariate analysis age >60 years (p = 0.001) and high mitotic rate (p = 0.02) remained significant adverse prognostic factors. The 5- and 10-year disease-free survival rates were 58% and 48%, respectively, and were significantly influenced by KPS (p 0.04) and high mitotic rate (p = 0.003) on univariate analysis. On multivariate analysis only high mitotic rate (p = 0.003) remained a significant prognostic factor. Conclusions: In this multicenter retrospective study, age, KPS, and mitotic rate influenced outcome. Multicenter prospective studies are necessary to clarify the management and prognostic factors of such a rare disease.« less

Exit probability of the one-dimensional q-voter model: Analytical results and simulations for large networks

NASA Astrophysics Data System (ADS)

Timpanaro, André M.; Prado, Carmen P. C.

2014-05-01

We discuss the exit probability of the one-dimensional q-voter model and present tools to obtain estimates about this probability, both through simulations in large networks (around 107 sites) and analytically in the limit where the network is infinitely large. We argue that the result E(ρ )=ρq/ρq+(1-ρ)q, that was found in three previous works [F. Slanina, K. Sznajd-Weron, and P. Przybyła, Europhys. Lett. 82, 18006 (2008), 10.1209/0295-5075/82/18006; R. Lambiotte and S. Redner, Europhys. Lett. 82, 18007 (2008), 10.1209/0295-5075/82/18007, for the case q =2; and P. Przybyła, K. Sznajd-Weron, and M. Tabiszewski, Phys. Rev. E 84, 031117 (2011), 10.1103/PhysRevE.84.031117, for q >2] using small networks (around 103 sites), is a good approximation, but there are noticeable deviations that appear even for small systems and that do not disappear when the system size is increased (with the notable exception of the case q =2). We also show that, under some simple and intuitive hypotheses, the exit probability must obey the inequality ρq/ρq+(1-ρ)≤E(ρ)≤ρ/ρ +(1-ρ)q in the infinite size limit. We believe this settles in the negative the suggestion made [S. Galam and A. C. R. Martins, Europhys. Lett. 95, 48005 (2001), 10.1209/0295-5075/95/48005] that this result would be a finite size effect, with the exit probability actually being a step function. We also show how the result that the exit probability cannot be a step function can be reconciled with the Galam unified frame, which was also a source of controversy.

Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

PubMed Central

Ferrero, Macarena; Ferragud, Juan; Orlando, Leonardo; Valero, Luz; Sánchez del Pino, Manuel; Farràs, Rosa; Font de Mora, Jaime

2011-01-01

Background Although the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis. Methodology/Principal Findings Mitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell. Conclusions Collectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the transcriptional activity of AIB1. PMID:22163316

Kif11 dependent cell cycle progression in radial glial cells is required for proper neurogenesis in the zebrafish neural tube.

PubMed

Johnson, Kimberly; Moriarty, Chelsea; Tania, Nessy; Ortman, Alissa; DiPietrantonio, Kristina; Edens, Brittany; Eisenman, Jean; Ok, Deborah; Krikorian, Sarah; Barragan, Jessica; Golé, Christophe; Barresi, Michael J F

2014-03-01

Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-L-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226× delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of compensatory regulation may exist to maintain overall proportions in the neural tube. We propose a model in which Kif11 normally functions during mitotic spindle formation to facilitate the progression of radial glia through mitosis, which leads to the maturation of progeny into specific secondary neuronal and glial lineages in the developing neural tube. Copyright © 2014. Published by Elsevier Inc.

Kif11 dependent cell cycle progression in radial glial cells is required for proper neurogenesis in the zebrafish neural tube

PubMed Central

Johnson, Kimberly; Moriarty, Chelsea; Tania, Nessy; Ortman, Alissa; DiPietrantonio, Kristina; Edens, Brittany; Eisenman, Jean; Ok, Deborah; Krikorian, Sarah; Barragan, Jessica; Gole, Christophe; Barresi, Michael J.F.

2014-01-01

Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-L-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226x delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of compensatory regulation may exist to maintain overall proportions in the neural tube. We propose a model in which Kif11 normally functions during mitotic spindle formation to facilitate the progression of radial glia through mitosis, which leads to the maturation of progeny into specific secondary neuronal and glial lineages in the developing neural tube. PMID:24370453

Kinetochore localized Mad2 and Cdc20 is itself insufficient for triggering the mitotic checkpoint when Mps1 is low in Drosophila melanogaster neuroblasts.

PubMed

Herriott, Ashleigh; Sweeney, Michele; Whitaker, Michael; Taggart, Michael; Huang, Jun-Yong

2012-12-15

The relationships between the kinetochore and checkpoint control remain unresolved. Here, we report the characterization of the in vivo behavior of Cdc20 and Mad2 and the relevant spindle assembly checkpoint (SAC) functions in the neuroblasts of a Drosophila Mps1 weak allele (ald (B4-2) ). ald (B4-2) third instar larvae brain samples contain only around 16% endogenous Mps1 protein, and the SAC function is abolished. However, this does not lead to rapid anaphase onset and mitotic exit, in contrast to the loss of Mad2 alone in a mad2 (EY) mutant. The level of GFP-Cdc20 recruitment to the kinetochore is unaffected in ald (B4-2) neuroblasts, while the level of GFP-Mad2 is reduced to just about 20%. Cdc20 and Mad2 display only monophasic exponential kinetics at the kinetochores. The ald (B4-2) heterozygotes expressed approximately 65% of normal Mps1 protein levels, and this is enough to restore the SAC function. The kinetochore recruitment of GFP-Mad2 in response to SAC activation increases by around 80% in heterozygotes, compared with just about 20% in ald (B4-2) mutant. This suggests a correlation between Mps1 levels and Mad2 kinetochore localization and perhaps the existence of a threshold level at which Mps1 is fully functional. The failure to arrest the mitotic progression in ald (B4-2) neuroblasts in response to colchicine treatment suggests that when Mps1 levels are low, approximately 20% of normal GFP-Mad2, alongside normal levels of GFP-Cdc20 kinetochore recruitments, is insufficient for triggering SAC signal propagation.

Influence of the bud neck on nuclear envelope fission in Saccharomyces cerevisiae.

PubMed

Melloy, Patricia G; Rose, Mark D

2017-09-15

Studies have shown that nuclear envelope fission (karyokinesis) in budding yeast depends on cytokinesis, but not distinguished whether this was a direct requirement, indirect, because of cell cycle arrest, or due to bud neck-localized proteins impacting both processes. To determine the requirements for karyokinesis, we examined mutants conditionally defective for bud emergence and/or nuclear migration. The common mutant phenotype was completion of the nuclear division cycle within the mother cell, but karyokinesis did not occur. In the cdc24 swe1 mutant, at the non-permissive temperature, multiple nuclei accumulated within the unbudded cell, with connected nuclear envelopes. Upon return to the permissive temperature, the cdc24 swe1 mutant initiated bud emergence, but only the nucleus spanning the neck underwent fission suggesting that the bud neck region is important for fission initiation. The neck may be critical for either mechanical reasons, as the contractile ring might facilitate fission, or for regulatory reasons, as the site of a protein network regulating nuclear envelope fission, mitotic exit, and cytokinesis. We also found that 77-85% of pairs of septin mutant nuclei completed nuclear envelope fission. In addition, 27% of myo1Δ mutant nuclei completed karyokinesis. These data suggested that fission is not dependent on mechanical contraction at the bud neck, but was instead controlled by regulatory proteins there. Copyright © 2017 Elsevier Inc. All rights reserved.

The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

PubMed Central

Magron, Audrey; Elowe, Sabine; Carreau, Madeleine

2015-01-01

The Fanconi anemia (FA) proteins are involved in a signaling network that assures the safeguard of chromosomes. To understand the function of FA proteins in cellular division events, we investigated the interaction between Stathmin-1 (STMN1) and the FA group C (FANCC) protein. STMN1 is a ubiquitous cytosolic protein that regulates microtubule dynamics. STMN1 activities are regulated through phosphorylation-dephosphorylation mechanisms that control assembly of the mitotic spindle, and dysregulation of STMN1 phosphorylation is associated with mitotic aberrancies leading to chromosome instability and cancer progression. Using different biochemical approaches, we showed that FANCC interacts and co-localizes with STMN1 at centrosomes during mitosis. We also showed that FANCC is required for STMN1 phosphorylation, as mutations in FANCC reduced serine 16- and 38-phosphorylated forms of STMN1. Phosphorylation of STMN1 at serine 16 is likely an event dependent on a functional FA pathway, as it is reduced in FANCA- and FANCD2-mutant cells. Furthermore, FA-mutant cells exhibited mitotic spindle anomalies such as supernumerary centrosomes and shorter mitotic spindles. These results suggest that FA proteins participate in the regulation of cellular division via the microtubule-associated protein STMN1. PMID:26466335

Withaferin A modulates the Spindle assembly checkpoint by degradation of Mad2-Cdc20 complex in colorectal cancer cell lines.

PubMed

Das, Tania; Roy, Kumar Singha; Chakrabarti, Tulika; Mukhopadhyay, Sibabrata; Roychoudhury, Susanta

2014-09-01

Withania somnifera L. Dunal (Ashwagandha) is used over centuries in the ayurvedic medicines in India. Withaferin A, a withanolide, is the major compound present in leaf extract of the plant which shows anticancer activity against leukemia, breast cancer and colorectal cancer. It arrests the ovarian cancer cells in the G2/M phase in dose dependent manner. In the current study we show the effect of Withaferin A on cell cycle regulation of colorectal cancer cell lines HCT116 and SW480 and its effect on cell fate. Treatment of these cells with this compound leads to apoptosis in a dose dependent manner. It causes the G2/M arrest in both the cell lines. We show that Withaferin A (WA) causes mitotic delay by blocking Spindle assembly checkpoint (SAC) function. Apoptosis induced by Withaferin A is associated with proteasomal degradation of Mad2 and Cdc20, an important constituent of the Spindle Checkpoint Complex. Further overexpression of Mad2 partially rescues the deleterious effect of WA by restoring proper anaphase initiation and keeping more number of cells viable. We hypothesize that Withaferin A kills cancer cells by delaying the mitotic exit followed by inducing chromosome instability. Copyright © 2014 Elsevier Inc. All rights reserved.

Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sigala, Barbara; Edwards, Mina; Puri, Teena

2005-11-01

TIP48 is a highly conserved eukaryotic AAA{sup +} protein which is an essential cofactor for several complexes involved in chromatin acetylation and remodeling, transcriptional and developmental regulation and nucleolar organization and trafficking. We show that TIP48 abundance in HeLa cells did not change during the cell cycle, nor did its distribution in various biochemical fractions. However, we observed distinct changes in the subcellular localization of TIP48 during M phase using immunofluorescence microscopy. Our studies demonstrate that in interphase cells TIP48 was found mainly in the nucleus and exhibited a distinct localization in the nuclear periphery. As the cells entered mitosis,more » TIP48 was excluded from the condensing chromosomes but showed association with the mitotic apparatus. During anaphase, some TIP48 was detected in the centrosome colocalizing with tubulin but the strongest staining appeared in the mitotic equator associated with the midzone central spindle. Accumulation of TIP48 in the midzone and the midbody was observed in late telophase and cytokinesis. This redeployment of TIP48 during anaphase and cytokinesis was independent of microtubule assembly. The relocation of endogenous TIP48 to the midzone/midbody under physiological conditions suggests a novel and distinct function for TIP48 in mitosis and possible involvement in the exit of mitosis.« less

[Characterization of access to normal childbirth care in Bahia State, Brazil, based on graph theory].

PubMed

Sousa, Ludmilla Monfort Oliveira; Araújo, Edna Maria de; Miranda, José Garcia Vivas

2017-12-18

Origin-destination flow is a phenomenon that can be modeled as a network. Graph theory is a mathematical tool to characterize a network and thus allows studying the topological properties and temporal and spatial development of a set of related elements. The article aims to estimate the topological evolution of an inter-municipal network of normal deliveries. We selected the admissions for normal deliveries in the Hospital Information System of the Brazilian Unified National Health System, from 2008 to 2014, for women residing in Bahia State, Brazil. The following indices were applied: entry degree (from how many municipalities the women came for childbirth), exit degree (to how many municipalities they left), entry flow (how many women came), exit flow (how many women left), and the mean size of the exit edge (distance traveled). Analyses between macro-regions used the following indicators: proportion of normal deliveries performed outside the municipality of residence and mean size of the exit edge. The results indicate an increase in deliveries performed outside the municipality of residence, in addition to the persistence of concentration of deliveries in the hub municipalities in the Health Regions, and an increase in the distance between the municipality of residence and the municipality where the delivery took place. The organization of networks for normal childbirth poses an on-going challenge. It is important to analyze the flow of women for childbirth care in order to support the establishment of inter-municipal references to guarantee safe labor and childbirth. In conclusion, it is necessary to develop a regionalized network to meet the demand by pregnant women in the territory with universal and equitable coverage.

Experimental models for Murray’s law

NASA Astrophysics Data System (ADS)

Akita, Dai; Kunita, Itsuki; Fricker, Mark D.; Kuroda, Shigeru; Sato, Katsuhiko; Nakagaki, Toshiyuki

2017-01-01

Transport networks are ubiquitous in multicellular organisms and include leaf veins, fungal mycelia and blood vessels. While transport of materials and signals through the network plays a crucial role in maintaining the living system, the transport capacity of the network can best be understood in terms of hydrodynamics. We report here that plasmodium from the large, single-celled amoeboid Physarum was able to construct a hydrodynamically optimized vein-network when evacuating biomass from confined arenas of various shapes through a narrow exit. Increasingly thick veins developed towards the exit, and the network spanned the arena via repetitive bifurcations to give a branching tree. The Hausdorff distance from all parts of the plasmodium to the vein network was kept low, whilst the hydrodynamic conductivity from distal parts of the network to the exit was equivalent, irrespective of the arena shape. This combination of spatial patterning and differential vein thickening served to evacuate biomass at an equivalent rate across the entire arena. The scaling relationship at the vein branches was determined experimentally to be 2.53-3.29, consistent with predictions from Murray’s law. Furthermore, we show that mathematical models for self-organised, adaptive transport in Physarum simulate the experimental network organisation well if the scaling coefficient of the current-reinforcement rule is set to 3. In simulations, this resulted in rapid development of an optimal network that minimised the combined volume and frictional energy in comparison with other scaling coefficients. This would predict that the boundary shear forces within each vein are constant throughout the network, and would be consistent with a feedback mechanism based on a sensing a threshold shear at the vein wall.

DeepMitosis: Mitosis detection via deep detection, verification and segmentation networks.

PubMed

Li, Chao; Wang, Xinggang; Liu, Wenyu; Latecki, Longin Jan

2018-04-01

Mitotic count is a critical predictor of tumor aggressiveness in the breast cancer diagnosis. Nowadays mitosis counting is mainly performed by pathologists manually, which is extremely arduous and time-consuming. In this paper, we propose an accurate method for detecting the mitotic cells from histopathological slides using a novel multi-stage deep learning framework. Our method consists of a deep segmentation network for generating mitosis region when only a weak label is given (i.e., only the centroid pixel of mitosis is annotated), an elaborately designed deep detection network for localizing mitosis by using contextual region information, and a deep verification network for improving detection accuracy by removing false positives. We validate the proposed deep learning method on two widely used Mitosis Detection in Breast Cancer Histological Images (MITOSIS) datasets. Experimental results show that we can achieve the highest F-score on the MITOSIS dataset from ICPR 2012 grand challenge merely using the deep detection network. For the ICPR 2014 MITOSIS dataset that only provides the centroid location of mitosis, we employ the segmentation model to estimate the bounding box annotation for training the deep detection network. We also apply the verification model to eliminate some false positives produced from the detection model. By fusing scores of the detection and verification models, we achieve the state-of-the-art results. Moreover, our method is very fast with GPU computing, which makes it feasible for clinical practice. Copyright © 2018 Elsevier B.V. All rights reserved.

Fifteen years of APC/cyclosome: a short and impressive biography.

PubMed

Simpson-Lavy, Kobi J; Oren, Yifat S; Feine, Oren; Sajman, Julia; Listovsky, Tammy; Brandeis, Michael

2010-02-01

The APC/C (anaphase-promoting complex/cyclosome) discovered exactly 15 years ago by Avram Heshko and Marc Kirschner is by far the most complex ubiquitin ligase discovered so far. The APC/C is composed of roughly a dozen subunits and measures a massive 1.5 MDa. This huge complex, as well as its multiple modes of regulation, boasts impressive evolutionary conservation. One of its most puzzling features is its split personality: regulation of mitotic exit events on the one hand, and its ongoing activity during G(1)-phase, G(0)-phase and in terminally differentiated cells. The present short review is intended to provide a basic description of our current understanding of the APC/C, focusing on recent findings concerning its role in G(1)-phase and in differentiated cells.

Intelligent Exit-Selection Behaviors during a Room Evacuation

NASA Astrophysics Data System (ADS)

Zarita, Zainuddin; Lim Eng, Aik

2012-01-01

A modified version of the existing cellular automata (CA) model is proposed to simulate an evacuation procedure in a classroom with and without obstacles. Based on the numerous literature on the implementation of CA in modeling evacuation motions, it is notable that most of the published studies do not take into account the pedestrian's ability to select the exit route in their models. To resolve these issues, we develop a CA model incorporating a probabilistic neural network for determining the decision-making ability of the pedestrians, and simulate an exit-selection phenomenon in the simulation. Intelligent exit-selection behavior is observed in our model. From the simulation results, it is observed that occupants tend to select the exit closest to them when the density is low, but if the density is high they will go to an alternative exit so as to avoid a long wait. This reflects the fact that occupants may not fully utilize multiple exits during evacuation. The improvement in our proposed model is valuable for further study and for upgrading the safety aspects of building designs.

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression

PubMed Central

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-01-01

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division. PMID:24714560

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression.

PubMed

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-05-02

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.

Spatial Rule-Based Modeling: A Method and Its Application to the Human Mitotic Kinetochore

PubMed Central

Ibrahim, Bashar; Henze, Richard; Gruenert, Gerd; Egbert, Matthew; Huwald, Jan; Dittrich, Peter

2013-01-01

A common problem in the analysis of biological systems is the combinatorial explosion that emerges from the complexity of multi-protein assemblies. Conventional formalisms, like differential equations, Boolean networks and Bayesian networks, are unsuitable for dealing with the combinatorial explosion, because they are designed for a restricted state space with fixed dimensionality. To overcome this problem, the rule-based modeling language, BioNetGen, and the spatial extension, SRSim, have been developed. Here, we describe how to apply rule-based modeling to integrate experimental data from different sources into a single spatial simulation model and how to analyze the output of that model. The starting point for this approach can be a combination of molecular interaction data, reaction network data, proximities, binding and diffusion kinetics and molecular geometries at different levels of detail. We describe the technique and then use it to construct a model of the human mitotic inner and outer kinetochore, including the spindle assembly checkpoint signaling pathway. This allows us to demonstrate the utility of the procedure, show how a novel perspective for understanding such complex systems becomes accessible and elaborate on challenges that arise in the formulation, simulation and analysis of spatial rule-based models. PMID:24709796

A Clb/Cdk1-mediated regulation of Fkh2 synchronizes CLB expression in the budding yeast cell cycle.

PubMed

Linke, Christian; Chasapi, Anastasia; González-Novo, Alberto; Al Sawad, Istabrak; Tognetti, Silvia; Klipp, Edda; Loog, Mart; Krobitsch, Sylvia; Posas, Francesc; Xenarios, Ioannis; Barberis, Matteo

2017-01-01

Precise timing of cell division is achieved by coupling waves of cyclin-dependent kinase (Cdk) activity with a transcriptional oscillator throughout cell cycle progression. Although details of transcription of cyclin genes are known, it is unclear which is the transcriptional cascade that modulates their expression in a timely fashion. Here, we demonstrate that a Clb/Cdk1-mediated regulation of the Fkh2 transcription factor synchronizes the temporal mitotic CLB expression in budding yeast. A simplified kinetic model of the cyclin/Cdk network predicts a linear cascade where a Clb/Cdk1-mediated regulation of an activator molecule drives CLB3 and CLB2 expression. Experimental validation highlights Fkh2 as modulator of CLB3 transcript levels, besides its role in regulating CLB2 expression. A Boolean model based on the minimal number of interactions needed to capture the information flow of the Clb/Cdk1 network supports the role of an activator molecule in the sequential activation, and oscillatory behavior, of mitotic Clb cyclins. This work illustrates how transcription and phosphorylation networks can be coupled by a Clb/Cdk1-mediated regulation that synchronizes them.

Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis

PubMed Central

Hua, Hui; Namdar, Mandana; Ganier, Olivier; Gregan, Juraj; Méchali, Marcel; Kearsey, Stephen E.

2013-01-01

Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I–II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry. PMID:23303250

CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis.

PubMed

Zitouni, Sihem; Francia, Maria E; Leal, Filipe; Montenegro Gouveia, Susana; Nabais, Catarina; Duarte, Paulo; Gilberto, Samuel; Brito, Daniela; Moyer, Tyler; Kandels-Lewis, Steffi; Ohta, Midori; Kitagawa, Daiju; Holland, Andrew J; Karsenti, Eric; Lorca, Thierry; Lince-Faria, Mariana; Bettencourt-Dias, Mónica

2016-05-09

Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nuclear Migration During Retinal Development

PubMed Central

Baye, Lisa M.; Link, Brian A.

2009-01-01

In this review we focus on the mechanisms, regulation, and cellular consequences of nuclear migration in the developing retina. In the nervous system, nuclear migration is prominent during both proliferative and post-mitotic phases of development. Interkinetic nuclear migration is the process where the nucleus oscillates from the apical to basal surfaces in proliferative neuroepithelia. Proliferative nuclear movement occurs in step with the cell cycle, with M-phase being confined to the apical surface and G1-, S-, and G2-phases occurring at more basal locations. Later, following cell cycle exit, some neuron precursors migrate by nuclear translocation. In this mode of cellular migration, nuclear movement is the driving force for motility. Following discussion of the key components and important regulators for each of these processes, we present an emerging model where interkinetic nuclear migration functions to distinguish cell fates among retinal neuroepithelia. PMID:17560964

Mitotic Arrest in Teratoma Susceptible Fetal Male Germ Cells

PubMed Central

Western, Patrick S.; Ralli, Rachael A.; Wakeling, Stephanie I.; Lo, Camden; van den Bergen, Jocelyn A.; Miles, Denise C.; Sinclair, Andrew H.

2011-01-01

Formation of germ cell derived teratomas occurs in mice of the 129/SvJ strain, but not in C57Bl/6 inbred or CD1 outbred mice. Despite this, there have been few comparative studies aimed at determining the similarities and differences between teratoma susceptible and non-susceptible mouse strains. This study examines the entry of fetal germ cells into the male pathway and mitotic arrest in 129T2/SvJ mice. We find that although the entry of fetal germ cells into mitotic arrest is similar between 129T2/SvJ, C57Bl/6 and CD1 mice, there were significant differences in the size and germ cell content of the testis cords in these strains. In 129T2/SvJ mice germ cell mitotic arrest involves upregulation of p27KIP1, p15INK4B, activation of RB, the expression of male germ cell differentiation markers NANOS2, DNMT3L and MILI and repression of the pluripotency network. The germ-line markers DPPA2 and DPPA4 show reciprocal repression and upregulation, respectively, while FGFR3 is substantially enriched in the nucleus of differentiating male germ cells. Further understanding of fetal male germ cell differentiation promises to provide insight into disorders of the testis and germ cell lineage, such as testis tumour formation and infertility. PMID:21674058

A Minimal Anaphase Promoting Complex/Cyclosome (APC/C) in Trypanosoma brucei

PubMed Central

Bessat, Mohamed; Knudsen, Giselle; Burlingame, Alma L.; Wang, Ching C.

2013-01-01

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C. PMID:23533609

Camera Network Topology Discovery Literature Review

DTIC Science & Technology

2011-11-01

essential for 21st century military, enviromental and surveillance applications [Devarajan, Cheng & Radke 2008]. Computer networks pose several research...starting and ending points of object trajectories into entry/exit regions [Makris & Ellis 2003]. 3LDA is a new standard for document analysis. The model

Acid Rain

MedlinePlus

... Clean Air Status and Trends Network (CASTNET) Surface Water Monitoring National Atmospheric Deposition Program (NADP) Exit Interstate Air Pollution Transport Contact Us to ask a question, provide ...

NAT10, a nucleolar protein, localizes to the midbody and regulates cytokinesis and acetylation of microtubules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Qi; Zheng, Xingzheng; McNutt, Michael A.

2009-06-10

The midbody is a structural organelle formed in late phase mitosis which is responsible for completion of cytokinesis. Although various kinds of proteins have been found to distribute or immigrate to this organelle, their functions have still not been completely worked out. In this study, we demonstrated that NAT10 (N-acetyltransferase 10, NAT10) is not only predominantly distributed in the nucleolus in interphase, but is also concentrated in the mitotic midbody during telophase. The domain in N-terminal residues 549-834 of NAT10 specifically mediated its subcellular localization. Treatment with genotoxic agents or irradiation increased concentration of NAT10 in both the nucleolus andmore » midbody. Moreover, DNA damage induced increase of NAT10 in the midbody apparently accompanied by in situ elevation of the level of acetylated {alpha}-tubulin, suggesting that it plays a role in maintaining or enhancing stability of {alpha}-tubulin. The depletion of NAT10 induced defects in nucleolar assembly, cytokinesis and decreased acetylated {alpha}-tubulin, leading to G2/M cell cycle arrest or delay of mitotic exit. In addition, over-expression of NAT10 was found in a variety of soft tissue sarcomas, and correlated with tumor histological grading. These results indicate that NAT10 may play an important role in cell division through facilitating reformation of the nucleolus and midbody in the late phase of cell mitosis, and stabilization of microtubules.« less

Collective synchronization of divisions in Drosophila development

NASA Astrophysics Data System (ADS)

Vergassola, Massimo

Mitoses in the early development of most metazoans are rapid and synchronized across the entire embryo. While diffusion is too slow, in vitro experiments have shown that waves of the cell-cycle regulator Cdk1 can transfer information rapidly across hundreds of microns. However, the signaling dynamics and the physical properties of chemical waves during embryonic development remain unclear. We develop FRET biosensors for the activity of Cdk1 and the checkpoint kinase Chk1 in Drosophila embryos and exploit them to measure waves in vivo. We demonstrate that Cdk1 chemical waves control mitotic waves and that their speed is regulated by the activity of Cdk1 during the S-phase (and not mitosis). We quantify the progressive slowdown of the waves with developmental cycles and identify its underlying control mechanism by the DNA replication checkpoint through the Chk1/Wee1 pathway. The global dynamics of the mitotic signaling network illustrates a novel control principle: the S-phase activity of Cdk1 regulates the speed of the mitotic wave, while the Cdk1 positive feedback ensures an invariantly rapid onset of mitosis. Mathematical modeling captures the speed of the waves and predicts a fundamental distinction between the S-phase Cdk1 trigger waves and the mitotic phase waves, which is illustrated by embryonic ablation experiments. In collaboration with Victoria Deneke1, Anna Melbinger2, and Stefano Di Talia1 1 Department of Cell Biology, Duke University Medical Center 2 Department of Physics, University of California San Diego.

CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging.

PubMed

Held, Michael; Schmitz, Michael H A; Fischer, Bernd; Walter, Thomas; Neumann, Beate; Olma, Michael H; Peter, Matthias; Ellenberg, Jan; Gerlich, Daniel W

2010-09-01

Fluorescence time-lapse imaging has become a powerful tool to investigate complex dynamic processes such as cell division or intracellular trafficking. Automated microscopes generate time-resolved imaging data at high throughput, yet tools for quantification of large-scale movie data are largely missing. Here we present CellCognition, a computational framework to annotate complex cellular dynamics. We developed a machine-learning method that combines state-of-the-art classification with hidden Markov modeling for annotation of the progression through morphologically distinct biological states. Incorporation of time information into the annotation scheme was essential to suppress classification noise at state transitions and confusion between different functional states with similar morphology. We demonstrate generic applicability in different assays and perturbation conditions, including a candidate-based RNA interference screen for regulators of mitotic exit in human cells. CellCognition is published as open source software, enabling live-cell imaging-based screening with assays that directly score cellular dynamics.

Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function.

PubMed

Kwiatkowski, Nicholas; Jelluma, Nannette; Filippakopoulos, Panagis; Soundararajan, Meera; Manak, Michael S; Kwon, Mijung; Choi, Hwan Geun; Sim, Taebo; Deveraux, Quinn L; Rottmann, Sabine; Pellman, David; Shah, Jagesh V; Kops, Geert J P L; Knapp, Stefan; Gray, Nathanael S

2010-05-01

Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and for the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, the development of a potent, selective small-molecule inhibitor of Mps1 should facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 cocrystal structures of new, selective small-molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells having extra centrosomes (an abnormality found in some cancers), Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.

APLP2 regulates neuronal stem cell differentiation during cortical development.

PubMed

Shariati, S Ali M; Lau, Pierre; Hassan, Bassem A; Müller, Ulrike; Dotti, Carlos G; De Strooper, Bart; Gärtner, Annette

2013-03-01

Expression of amyloid precursor protein (APP) and its two paralogues, APLP1 and APLP2 during brain development coincides with key cellular events such as neuronal differentiation and migration. However, genetic knockout and shRNA studies have led to contradictory conclusions about their role during embryonic brain development. To address this issue, we analysed in depth the role of APLP2 during neurogenesis by silencing APLP2 in vivo in an APP/APLP1 double knockout mouse background. We find that under these conditions cortical progenitors remain in their undifferentiated state much longer, displaying a higher number of mitotic cells. In addition, we show that neuron-specific APLP2 downregulation does not impact the speed or position of migrating excitatory cortical neurons. In summary, our data reveal that APLP2 is specifically required for proper cell cycle exit of neuronal progenitors, and thus has a distinct role in priming cortical progenitors for neuronal differentiation.

How does the Xenopus laevis embryonic cell cycle avoid spatial chaos?

PubMed Central

Gelens, Lendert; Huang, Kerwyn Casey; Ferrell, James E.

2015-01-01

Summary Theoretical studies have shown that a deterministic biochemical oscillator can become chaotic when operating over a sufficiently large volume, and have suggested that the Xenopus laevis cell cycle oscillator operates close to such a chaotic regime. To experimentally test this hypothesis, we decreased the speed of the post-fertilization calcium wave, which had been predicted to generate chaos. However, cell divisions were found to develop normally and eggs developed into normal tadpoles. Motivated by these experiments, we carried out modeling studies to understand the prerequisites for the predicted spatial chaos. We showed that this type of spatial chaos requires oscillatory reaction dynamics with short pulse duration, and postulated that the mitotic exit in Xenopus laevis is likely slow enough to avoid chaos. In systems with shorter pulses, chaos may be an important hazard, as in cardiac arrhythmias, or a useful feature, as in the pigmentation of certain mollusk shells. PMID:26212326

Developmental regulation of CYCA2s contributes to tissue-specific proliferation in Arabidopsis

PubMed Central

Vanneste, Steffen; Coppens, Frederik; Lee, EunKyoung; Donner, Tyler J; Xie, Zidian; Van Isterdael, Gert; Dhondt, Stijn; De Winter, Freya; De Rybel, Bert; Vuylsteke, Marnik; De Veylder, Lieven; Friml, Jiří; Inzé, Dirk; Grotewold, Erich; Scarpella, Enrico; Sack, Fred; Beemster, Gerrit T S; Beeckman, Tom

2011-01-01

In multicellular organisms, morphogenesis relies on a strict coordination in time and space of cell proliferation and differentiation. In contrast to animals, plant development displays continuous organ formation and adaptive growth responses during their lifespan relying on a tight coordination of cell proliferation. How developmental signals interact with the plant cell-cycle machinery is largely unknown. Here, we characterize plant A2-type cyclins, a small gene family of mitotic cyclins, and show how they contribute to the fine-tuning of local proliferation during plant development. Moreover, the timely repression of CYCA2;3 expression in newly formed guard cells is shown to require the stomatal transcription factors FOUR LIPS/MYB124 and MYB88, providing a direct link between developmental programming and cell-cycle exit in plants. Thus, transcriptional downregulation of CYCA2s represents a critical mechanism to coordinate proliferation during plant development. PMID:21772250

The Drosophila Pericentrin-like-protein (PLP) cooperates with Cnn to maintain the integrity of the outer PCM

PubMed Central

Richens, Jennifer H.; Barros, Teresa P.; Lucas, Eliana P.; Peel, Nina; Pinto, David Miguel Susano; Wainman, Alan; Raff, Jordan W.

2015-01-01

ABSTRACT Centrosomes comprise a pair of centrioles surrounded by a matrix of pericentriolar material (PCM). In vertebrate cells, Pericentrin plays an important part in mitotic PCM assembly, but the Drosophila Pericentrin-like protein (PLP) appears to have a more minor role in mitotic fly cells. Here we investigate the function of PLP during the rapid mitotic cycles of the early Drosophila embryo. Unexpectedly, we find that PLP is specifically enriched in the outer-most regions of the PCM, where it largely co-localizes with the PCM scaffold protein Cnn. In the absence of PLP the outer PCM appears to be structurally weakened, and it rapidly disperses along the centrosomal microtubules (MTs). As a result, centrosomal MTs are subtly disorganized in embryos lacking PLP, although mitosis is largely unperturbed and these embryos develop and hatch at near-normal rates. Y2H analysis reveals that PLP can potentially form multiple interactions with itself and with the PCM recruiting proteins Asl, Spd-2 and Cnn. A deletion analysis suggests that PLP participates in a complex network of interactions that ultimately help to strengthen the PCM. PMID:26157019

The Drosophila Pericentrin-like-protein (PLP) cooperates with Cnn to maintain the integrity of the outer PCM.

PubMed

Richens, Jennifer H; Barros, Teresa P; Lucas, Eliana P; Peel, Nina; Pinto, David Miguel Susano; Wainman, Alan; Raff, Jordan W

2015-07-08

Centrosomes comprise a pair of centrioles surrounded by a matrix of pericentriolar material (PCM). In vertebrate cells, Pericentrin plays an important part in mitotic PCM assembly, but the Drosophila Pericentrin-like protein (PLP) appears to have a more minor role in mitotic fly cells. Here we investigate the function of PLP during the rapid mitotic cycles of the early Drosophila embryo. Unexpectedly, we find that PLP is specifically enriched in the outer-most regions of the PCM, where it largely co-localizes with the PCM scaffold protein Cnn. In the absence of PLP the outer PCM appears to be structurally weakened, and it rapidly disperses along the centrosomal microtubules (MTs). As a result, centrosomal MTs are subtly disorganized in embryos lacking PLP, although mitosis is largely unperturbed and these embryos develop and hatch at near-normal rates. Y2H analysis reveals that PLP can potentially form multiple interactions with itself and with the PCM recruiting proteins Asl, Spd-2 and Cnn. A deletion analysis suggests that PLP participates in a complex network of interactions that ultimately help to strengthen the PCM. © 2015. Published by The Company of Biologists Ltd.

Leiodermatolide, a novel marine natural product, has potent cytotoxic and anti-mitotic activity against cancer cells, appears to affect microtubule dynamics, and exhibits anti-tumor activity

PubMed Central

Guzmán, Esther A.; Xu, Qunli; Pitts, Tara P.; Mitsuhashi, Kaoru Ogawa; Baker, Cheryl; Linley, Patricia A.; Oestreicher, Judy; Tendyke, Karen; Winder, Priscilla L.; Suh, Edward M.; Wright, Amy E.

2016-01-01

Pancreatic cancer, the fourth leading cause of cancer death in the United States, has a negative prognosis because metastasis occurs before symptoms manifest. Leiodermatolide, a polyketide macrolide with antimitotic activity isolated from a deep water sponge of the genus Leiodermatium, exhibits potent and selective cytotoxicity towards the pancreatic cancer cell lines AsPC-1, PANC-1, BxPC-3, and MIA PaCa-2, and potent cytotoxicity against skin, breast and colon cancer cell lines. Induction of apoptosis by leiodermatolide was confirmed in the AsPC-1, BxPC-3 and MIA PaCa-2 cells. Leiodermatolide induces cell cycle arrest but has no effects on in vitro polymerization or depolymerization of tubulin alone, while it enhances polymerization of tubulin containing microtubule associated proteins (MAPs). Observations through confocal microscopy show that leiodermatolide, at low concentrations, causes minimal effects on polymerization or depolymerization of the microtubule network in interphase cells, but disruption of spindle formation in mitotic cells. At higher concentrations, depolymerization of the microtubule network is observed. Visualization of the growing microtubule in HeLa cells expressing GFP-tagged plus end binding protein EB-1 showed that leiodermatolide stopped the polymerization of tubulin. These results suggest that leiodermatolide may affect tubulin dynamics without directly interacting with tubulin and hint at a unique mechanism of action. In a mouse model of metastatic pancreatic cancer, leiodermatolide exhibited significant tumor reduction when compared to gemcitabine and controls. The anti-tumor activities of leiodermatolide, as well as the proven utility of anti-mitotic compounds against cancer, make leiodermatolide an interesting compound with potential chemotherapeutic effects that may merit further research. PMID:27376928

Inference of genetic network of Xenopus frog egg: improved genetic algorithm.

PubMed

Wu, Shinq-Jen; Chou, Chia-Hsien; Wu, Cheng-Tao; Lee, Tsu-Tian

2006-01-01

An improved genetic algorithm (IGA) is proposed to achieve S-system gene network modeling of Xenopus frog egg. Via the time-courses training datasets from Michaelis-Menten model, the optimal parameters are learned. The S-system can clearly describe activative and inhibitory interaction between genes as generating and consuming process. We concern the mitotic control in cell-cycle of Xenopus frog egg to realize cyclin-Cdc2 and Cdc25 for MPF activity. The proposed IGA can achieve global search with migration and keep the best chromosome with elitism operation. The generated gene regulatory networks can provide biological researchers for further experiments in Xenopus frog egg cell cycle control.

A stochastic and dynamical view of pluripotency in mouse embryonic stem cells

PubMed Central

Lee, Esther J.

2018-01-01

Pluripotent embryonic stem cells are of paramount importance for biomedical sciences because of their innate ability for self-renewal and differentiation into all major cell lines. The fateful decision to exit or remain in the pluripotent state is regulated by complex genetic regulatory networks. The rapid growth of single-cell sequencing data has greatly stimulated applications of statistical and machine learning methods for inferring topologies of pluripotency regulating genetic networks. The inferred network topologies, however, often only encode Boolean information while remaining silent about the roles of dynamics and molecular stochasticity inherent in gene expression. Herein we develop a framework for systematically extending Boolean-level network topologies into higher resolution models of networks which explicitly account for the promoter architectures and gene state switching dynamics. We show the framework to be useful for disentangling the various contributions that gene switching, external signaling, and network topology make to the global heterogeneity and dynamics of transcription factor populations. We find the pluripotent state of the network to be a steady state which is robust to global variations of gene switching rates which we argue are a good proxy for epigenetic states of individual promoters. The temporal dynamics of exiting the pluripotent state, on the other hand, is significantly influenced by the rates of genetic switching which makes cells more responsive to changes in extracellular signals. PMID:29451874

U.S. Army Research Laboratory 2010 Annual Review

DTIC Science & Technology

2010-12-01

Translation Between Scales battlefield neuroscience Neuro-Cognitive Measurement Cognitive/Information – Decision Making Neurally Inspired Systems...the areas of Bioscience, Neuroscience , Network Science of Decision Making, Nanoscience, GaN High Power Electronics, Power for Microsystems, Graphene...source project POF, and NSA Trickler have also demonstrated that networks can be understood through passive observation of traffic as it exits a

Ectopic Expression of Nolz-1 in Neural Progenitors Promotes Cell Cycle Exit/Premature Neuronal Differentiation Accompanying with Abnormal Apoptosis in the Developing Mouse Telencephalon

PubMed Central

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU−, Ki67− and phospho-histone 3-positive cells in E11.5–12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon. PMID:24073229

Ectopic expression of nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation accompanying with abnormal apoptosis in the developing mouse telencephalon.

PubMed

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU-, Ki67- and phospho-histone 3-positive cells in E11.5-12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon.

Prognostic value of mitotic counts in breast cancer of Saudi Arabian patients.

PubMed

Buhmeida, Abdelbaset; Al-Maghrabi, Jaudah; Merdad, Adnan; Al-Thubaity, Fatima; Chaudhary, Adeel; Gari, Mamdooh; Abuzenadah, Adel; Collan, Yrjö; Syrjänen, Kari; Al-Qahtani, Mohammed

2011-01-01

Quantitative methods in combination with other objective prognostic criteria can improve the evaluation of a cancer patient's prognosis, and possibly predict response to therapy. One of the important prognostic and predictive markers is the mitotic count, which has proven valuable in many aspects. In this study, the prognostic value of the mitotic count was assessed in breast cancer (BC) patients in Saudi Arabia. The study comprised a series of 87 patients diagnosed and treated for breast cancer at the Departments of Surgery and Oncology, King Abdul-Aziz University Hospital, between 2000 and 2008. Mitotic counts were carried out using a standard laboratory microscope (objective, × 40; field diameter, 420 μm). The number of mitotic figures in 10 consecutive high-power fields (hpf) from the most cellular area of the sample gave the mitotic activity index (MAI, mitotic figures/10 hpf). The standardized mitotic index (SMI) recorded the mitotic count as the number of mitotic figures by area of the neoplastic tissue in the microscopic field, thus the number of mitoses in 10 consecutive fields was corrected for the volume fraction and field size (mitotic figures/mm²). The means of MAI and SMI of the tumors in the entire series of 87 patients were 15 mitotic figures/10 hpf (range 4-45) and 4 mitotic figures/mm² (range 1-9), respectively. The mitotic counts were higher in advanced stages than in early cancer (p < 0.04). The mitotic counts were significantly larger in patients with high-grade tumor (p < 0.004) and in cases with tumor metastasis (p < 0.004). The mitotic counts were also significantly larger in the recurrent cases than in non-recurrent ones (p < 0.02). The quantitatively measurable mitotic counts of cancer cell nuclei are of significant prognostic value in invasive ductal carcinoma of the breast in Saudi Arabia and the mean cut-off values of MAI and SMI can be applied as objective (quantitative) criteria to distinguish breast cancer patients into groups with favorable and less favorable prognosis.

Role of Structural Asymmetry in Controlling Drop Spacing in Microfluidic Ladder Networks

NASA Astrophysics Data System (ADS)

Wang, William; Maddala, Jeevan; Vanapalli, Siva; Rengasamy, Raghunathan

2012-02-01

Manipulation of drop spacing is crucial to many processes in microfluidic devices including drop coalescence, detection and storage. Microfluidic ladder networks ---where two droplet-carrying parallel channels are connected by narrow bypass channels through which the motion of drops is forbidden---have been proposed as a means to control relative separation between pairs of drops. Prior studies in microfluidic ladder networks with vertical bypasses, which possess fore-aft structural symmetry, have revealed that pairs of drops can only undergo reduction in drop spacing at the ladder exit. We investigate the dynamics of drops in microfluidic ladder networks with both vertical and slanted bypasses. Our analytical results indicate that unlike symmetric ladder networks, structural asymmetry introduced by a single slanted bypass can be used to modulate the relative spacing between drops, enabling them to contract, synchronize, expand or even flip at the ladder exit. Our experiments confirm all the behaviors predicted by theory. Numerical analysis further shows that ladders containing several identical bypasses can only linearly transform the input drop spacing. Finally, we find that ladders with specific combinations of vertical and slanted bypasses can generate non-linear transformation of input drop spacing, despite the absence of drop decision-making events at the bypass junctions.

Arsenite inhibits mitotic division and perturbs spindle dynamics in HeLa S3 cells.

PubMed

Huang, S C; Lee, T C

1998-05-01

Arsenical compounds, known to be human carcinogens, were shown to disturb cell cycle progression and induce cytogenetic alterations in a variety of cell systems. We report here that a 24 h treatment of arsenite induced mitotic accumulation in human cell lines. HeLa S3 and KB cells were most susceptible: 35% of the total cell population was arrested at the mitotic stage after treatment with 5 microM sodium arsenite in HeLa S3 cells and after 10 microM in KB cells. Under a microscope, we observed abnormal mitotic figures in arsenite-arrested mitotic cells, including deranged chromosome congression, elongated polar distance of mitotic spindle, and enhanced microtubule immunofluorescence. The spindle microtubules of arsenite-arrested mitotic cells were more resistant to nocodazole-induced dissolution than those of control mitotic cells. According to turbidity assay, arsenite at concentrations below 100 microM significantly enhanced polymerization of tubulins. Since spindle dynamics play a crucial role in mitotic progression, our results suggest that arsenite-induced mitotic arrest may be due to arsenite's effects on attenuation of spindle dynamics.

Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko

Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, butmore » F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.« less

Specifying peripheral heterochromatin during nuclear lamina reassembly

PubMed Central

Poleshko, Andrey; Katz, Richard A

2014-01-01

A conserved organizational feature of eukaryotic nuclei is the peripheral heterochromatin compartment, which provides a protected area for epigenetically silent genes and gene-poor DNA. In metazoan cells this compartment is associated with the nuclear lamina, the protein meshwork at the inner edge of the nucleus. Heterochromatin-nuclear lamina interactions promote epigenetic gene silencing, which may drive many normal and diseased biological processes. We recently obtained evidence that a previously unstudied human protein, PRR14, participates in the tethering of heterochromatin to the inner nuclear periphery. PRR14 associates with the nuclear lamina and attaches to heterochromatin through its binding partner, heterochromatin protein 1 (HP1). After disassembly early in mitosis, PRR14 reassembles in two steps, first binding to anaphase chromosomes through HP1, followed by association with the nuclear lamina in telophase. PRR14 may thereby play a role in specifying HP1-bound heterochromatin for reattachment to the nuclear lamina at mitotic exit. Here we review the relevant literature, summarize our initial work, and provide additional comments and findings. PMID:24637393

Specifying peripheral heterochromatin during nuclear lamina reassembly.

PubMed

Poleshko, Andrey; Katz, Richard A

2014-01-01

A conserved organizational feature of eukaryotic nuclei is the peripheral heterochromatin compartment, which provides a protected area for epigenetically silent genes and gene-poor DNA. In metazoan cells this compartment is associated with the nuclear lamina, the protein meshwork at the inner edge of the nucleus. Heterochromatin-nuclear lamina interactions promote epigenetic gene silencing, which may drive many normal and diseased biological processes. We recently obtained evidence that a previously unstudied human protein, PRR14, participates in the tethering of heterochromatin to the inner nuclear periphery. PRR14 associates with the nuclear lamina and attaches to heterochromatin through its binding partner, heterochromatin protein 1 (HP1). After disassembly early in mitosis, PRR14 reassembles in two steps, first binding to anaphase chromosomes through HP1, followed by association with the nuclear lamina in telophase. PRR14 may thereby play a role in specifying HP1-bound heterochromatin for reattachment to the nuclear lamina at mitotic exit. Here we review the relevant literature, summarize our initial work, and provide additional comments and findings.

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing.

PubMed

Moura, Margarida; Osswald, Mariana; Leça, Nelson; Barbosa, João; Pereira, António J; Maiato, Helder; Sunkel, Claudio E; Conde, Carlos

2017-05-02

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

Representing perturbed dynamics in biological network models

NASA Astrophysics Data System (ADS)

Stoll, Gautier; Rougemont, Jacques; Naef, Felix

2007-07-01

We study the dynamics of gene activities in relatively small size biological networks (up to a few tens of nodes), e.g., the activities of cell-cycle proteins during the mitotic cell-cycle progression. Using the framework of deterministic discrete dynamical models, we characterize the dynamical modifications in response to structural perturbations in the network connectivities. In particular, we focus on how perturbations affect the set of fixed points and sizes of the basins of attraction. Our approach uses two analytical measures: the basin entropy H and the perturbation size Δ , a quantity that reflects the distance between the set of fixed points of the perturbed network and that of the unperturbed network. Applying our approach to the yeast-cell-cycle network introduced by Li [Proc. Natl. Acad. Sci. U.S.A. 101, 4781 (2004)] provides a low-dimensional and informative fingerprint of network behavior under large classes of perturbations. We identify interactions that are crucial for proper network function, and also pinpoint functionally redundant network connections. Selected perturbations exemplify the breadth of dynamical responses in this cell-cycle model.

Multi-scale computational study of the mechanical regulation of cell mitotic rounding in epithelia

PubMed Central

Xu, Zhiliang; Zartman, Jeremiah J.; Alber, Mark

2017-01-01

Mitotic rounding during cell division is critical for preventing daughter cells from inheriting an abnormal number of chromosomes, a condition that occurs frequently in cancer cells. Cells must significantly expand their apical area and transition from a polygonal to circular apical shape to achieve robust mitotic rounding in epithelial tissues, which is where most cancers initiate. However, how cells mechanically regulate robust mitotic rounding within packed tissues is unknown. Here, we analyze mitotic rounding using a newly developed multi-scale subcellular element computational model that is calibrated using experimental data. Novel biologically relevant features of the model include separate representations of the sub-cellular components including the apical membrane and cytoplasm of the cell at the tissue scale level as well as detailed description of cell properties during mitotic rounding. Regression analysis of predictive model simulation results reveals the relative contributions of osmotic pressure, cell-cell adhesion and cortical stiffness to mitotic rounding. Mitotic area expansion is largely driven by regulation of cytoplasmic pressure. Surprisingly, mitotic shape roundness within physiological ranges is most sensitive to variation in cell-cell adhesivity and stiffness. An understanding of how perturbed mechanical properties impact mitotic rounding has important potential implications on, amongst others, how tumors progressively become more genetically unstable due to increased chromosomal aneuploidy and more aggressive. PMID:28531187

Genome-wide Expression Profiling, In Vivo DNA Binding Analysis, and Probabilistic Motif Prediction Reveal Novel Abf1 Target Genes during Fermentation, Respiration, and Sporulation in Yeast

PubMed Central

Schlecht, Ulrich; Erb, Ionas; Demougin, Philippe; Robine, Nicolas; Borde, Valérie; van Nimwegen, Erik; Nicolas, Alain

2008-01-01

The autonomously replicating sequence binding factor 1 (Abf1) was initially identified as an essential DNA replication factor and later shown to be a component of the regulatory network controlling mitotic and meiotic cell cycle progression in budding yeast. The protein is thought to exert its functions via specific interaction with its target site as part of distinct protein complexes, but its roles during mitotic growth and meiotic development are only partially understood. Here, we report a comprehensive approach aiming at the identification of direct Abf1-target genes expressed during fermentation, respiration, and sporulation. Computational prediction of the protein's target sites was integrated with a genome-wide DNA binding assay in growing and sporulating cells. The resulting data were combined with the output of expression profiling studies using wild-type versus temperature-sensitive alleles. This work identified 434 protein-coding loci as being transcriptionally dependent on Abf1. More than 60% of their putative promoter regions contained a computationally predicted Abf1 binding site and/or were bound by Abf1 in vivo, identifying them as direct targets. The present study revealed numerous loci previously unknown to be under Abf1 control, and it yielded evidence for the protein's variable DNA binding pattern during mitotic growth and meiotic development. PMID:18305101

Novel Mps1 Kinase Inhibitors with Potent Antitumor Activity.

PubMed

Wengner, Antje M; Siemeister, Gerhard; Koppitz, Marcus; Schulze, Volker; Kosemund, Dirk; Klar, Ulrich; Stoeckigt, Detlef; Neuhaus, Roland; Lienau, Philip; Bader, Benjamin; Prechtl, Stefan; Raschke, Marian; Frisk, Anna-Lena; von Ahsen, Oliver; Michels, Martin; Kreft, Bertolt; von Nussbaum, Franz; Brands, Michael; Mumberg, Dominik; Ziegelbauer, Karl

2016-04-01

Monopolar spindle 1 (Mps1) has been shown to function as the key kinase that activates the spindle assembly checkpoint (SAC) to secure proper distribution of chromosomes to daughter cells. Here, we report the structure and functional characterization of two novel selective Mps1 inhibitors, BAY 1161909 and BAY 1217389, derived from structurally distinct chemical classes. BAY 1161909 and BAY 1217389 inhibited Mps1 kinase activity with IC50 values below 10 nmol/L while showing an excellent selectivity profile. In cellular mechanistic assays, both Mps1 inhibitors abrogated nocodazole-induced SAC activity and induced premature exit from mitosis ("mitotic breakthrough"), resulting in multinuclearity and tumor cell death. Both compounds efficiently inhibited tumor cell proliferation in vitro (IC50 nmol/L range). In vivo, BAY 1161909 and BAY 1217389 achieved moderate efficacy in monotherapy in tumor xenograft studies. However, in line with its unique mode of action, when combined with paclitaxel, low doses of Mps1 inhibitor reduced paclitaxel-induced mitotic arrest by the weakening of SAC activity. As a result, combination therapy strongly improved efficacy over paclitaxel or Mps1 inhibitor monotreatment at the respective MTDs in a broad range of xenograft models, including those showing acquired or intrinsic paclitaxel resistance. Both Mps1 inhibitors showed good tolerability without adding toxicity to paclitaxel monotherapy. These preclinical findings validate the innovative concept of SAC abrogation for cancer therapy and justify clinical proof-of-concept studies evaluating the Mps1 inhibitors BAY 1161909 and BAY 1217389 in combination with antimitotic cancer drugs to enhance their efficacy and potentially overcome resistance. Mol Cancer Ther; 15(4); 583-92. ©2016 AACR. ©2016 American Association for Cancer Research.

Xanthium strumarium extract inhibits mammalian cell proliferation through mitotic spindle disruption mediated by xanthatin.

PubMed

Sánchez-Lamar, Angel; Piloto-Ferrer, Janet; Fiore, Mario; Stano, Pasquale; Cozzi, Renata; Tofani, Daniela; Cundari, Enrico; Francisco, Marbelis; Romero, Aylema; González, Maria L; Degrassi, Francesca

2016-12-24

Xanthium strumarium L. is a member of the Asteraceae family popularly used with multiple therapeutic purposes. Whole extracts of this plant have shown anti-mitotic activity in vitro suggesting that some components could induce mitotic arrest in proliferating cells. Aim of the present work was to characterize the anti-mitotic properties of the X. strumarium whole extract and to isolate and purify active molecule(s). The capacity of the whole extract to inhibit mitotic progression in mammalian cultured cells was investigated to identify its anti-mitotic activity. Isolation of active component(s) was performed using a bioassay-guided multistep separation procedure in which whole extract was submitted to a progressive process of fractionation and fractions were challenged for their anti-mitotic activity. Our results show for the first time that X. strumarium whole extract inhibits assembly of the mitotic spindle and spindle-pole separation, thereby heavily affecting mitosis, impairing the metaphase to anaphase transition and inducing apoptosis. The purification procedure led to a fraction with an anti-mitotic activity comparable to that of the whole extract. Chemical analysis of this fraction showed that its major component was xanthatin. The present work shows a new activity of X. strumarium extract, i.e. the alteration of the mitotic apparatus in cultured cells that may be responsible for the anti-proliferative activity of the extract. Anti-mitotic activity is shown to be mainly exerted by xanthatin. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte exit from quiescence

PubMed Central

Garcia, Thomas Xavier; DeFalco, Tony; Capel, Blanche; Hofmann, Marie-Claude

2013-01-01

Notch signaling components have long been detected in Sertoli and germ cells in the developing and mature testis. However, the role of this pathway in testis development and spermatogenesis remains unknown. Using reporter mice expressing green fluorescent protein following Notch receptor activation, we found that Notch signaling was active in Sertoli cells at various fetal, neonatal, and adult stages. Since Notch signaling specifies stem cell fate in many developing and mature organ systems, we hypothesized that maintenance and differentiation of gonocytes and/or spermatogonial stem cells would be modulated through this pathway in Sertoli cells. To this end, we generated mutant mice constitutively expressing the active, intracellular domain of NOTCH1 (NICD1) in Sertoli cells. We found that mutant Sertoli cells were morphologically normal before and after birth, but presented a number of functional changes that drastically affected gonocyte numbers and physiology. We observed aberrant exit of gonocytes from mitotic arrest, migration toward cord periphery, and premature differentiation before birth. These events, presumably unsupported by the cellular microenvironment, were followed by gonocyte apoptosis and near complete disappearance of the gonocytes by day 2 after birth. Molecular analysis demonstrated that these effects are correlated with a dysregulation of Sertoli-expressed genes that are required for germ cell maintenance, such as Cyp26b1 and Gdnf. Taken together, our results demonstrate that Notch signaling is active in Sertoli cells throughout development and that proper regulation of Notch signaling in Sertoli cells is required for the maintenance of gonocytes in an undifferentiated state during fetal development. PMID:23391689

Quasi-2D Unsteady Flow Procedure for Real Fluids (PREPRINT)

DTIC Science & Technology

2006-05-17

water /steam/ oil piping networks, refinery systems, gas-turbine secondary flow -path and cooling networks...friction factor, f, which is a function of the local Reynolds number and the wall surface roughness . For the viscous flow examples presented below, the...3.5 4 4.5 Time ( s ) V el oc ity (m / s ) Line 2 Inlet 25% 50% 75% Exit Velocity Figure 4. Water transient viscous pipe flow using

Analytical expression for the exit probability of the q -voter model in one dimension

NASA Astrophysics Data System (ADS)

Timpanaro, André M.; Galam, Serge

2015-07-01

We present in this paper an approximation that is able to give an analytical expression for the exit probability of the q -voter model in one dimension. This expression gives a better fit for the more recent data about simulations in large networks [A. M. Timpanaro and C. P. C. do Prado, Phys. Rev. E 89, 052808 (2014), 10.1103/PhysRevE.89.052808] and as such departs from the expression ρ/qρq+(1-ρ ) q found in papers that investigated small networks only [R. Lambiotte and S. Redner, Europhys. Lett. 82, 18007 (2008), 10.1209/0295-5075/82/18007; P. Przybyła et al., Phys. Rev. E 84, 031117 (2011), 10.1103/PhysRevE.84.031117; F. Slanina et al., Europhys. Lett. 82, 18006 (2008), 10.1209/0295-5075/82/18006]. The approximation consists in assuming a large separation on the time scales at which active groups of agents convince inactive ones and the time taken in the competition between active groups. Some interesting findings are that for q =2 we still have ρ/2ρ2+(1-ρ ) 2 as the exit probability and for q >2 we can obtain a lower-order approximation of the form ρ/sρs+(1-ρ ) s with s varying from q for low values of q to q -1/2 for large values of q . As such, this work can also be seen as a deduction for why the exit probability ρ/qρq+(1-ρ ) q gives a good fit, without relying on mean-field arguments or on the assumption that only the first step is nondeterministic, as q and q -1/2 will give very similar results when q →∞ .

Inhibition of the Ras-ERK pathway in mitotic COS7 cells is due to the inability of EGFR/Raf to transduce EGF signaling to downstream proteins.

PubMed

Shi, Huaiping; Zhang, Tianying; Yi, Yongqing; Ma, Yue

2016-06-01

Although previous studies have shown that Ras-ERK signaling in mitosis is closed due to the inhibition of signal transduction, the events involved in the molecular mechanisms are still unclear. In the present study, we investigated the Ras-ERK signaling pathway in mitotic COS7 cells. The results demonstrated that treatment with epidermal growth factor (EGF) failed to increase the endocytosis of EGF-EGFR (EGF receptor) complexes in mitotic COS7 cells, although a large amount of endosomes were found in asynchronous COS7 cells. Clathrin expression levels in mitotic COS7 cells were inhibited whereas caveolin expression levels in mitotic COS7 cells were almost unaffected. Y1068 and Y1086 residues of EGFR in the mitotic COS7 cells were activated. However, Grb2 and Shc in the mitotic COS7 cells did not bind to activated EGFR. Ras activity was inhibited in the mitotic COS7 cells whereas its downstream protein, Raf, was obviously phosphorylated by EGF in mitosis. Treatment with phorbol 12-myristate 13-acetate (PMA) also increased the phosphorylation levels of Raf in the mitotic COS7 cells. Nevertheless, Raf phosphorylation in mitosis was significantly inhibited by AG1478. Lastly, activation of EGF-mediated MEK and ERK in the mitotic COS7 cells was obviously inhibited. In summary, our results suggest that the Ras-ERK pathway is inhibited in mitotic COS7 cells which may be the dual result of the difficulty in the transduction of EGF signaling by EGFR or Raf to downstream proteins.

A robust and tunable mitotic oscillator in artificial cells

PubMed Central

Wang, Shiyuan; Barnes, Patrick M; Liu, Xuwen; Xu, Haotian; Jin, Minjun; Liu, Allen P

2018-01-01

Single-cell analysis is pivotal to deciphering complex phenomena like heterogeneity, bistability, and asynchronous oscillations, where a population ensemble cannot represent individual behaviors. Bulk cell-free systems, despite having unique advantages of manipulation and characterization of biochemical networks, lack the essential single-cell information to understand a class of out-of-steady-state dynamics including cell cycles. Here, by encapsulating Xenopus egg extracts in water-in-oil microemulsions, we developed artificial cells that are adjustable in sizes and periods, sustain mitotic oscillations for over 30 cycles, and function in forms from the simplest cytoplasmic-only to the more complicated ones involving nuclear dynamics, mimicking real cells. Such innate flexibility and robustness make it key to studying clock properties like tunability and stochasticity. Our results also highlight energy as an important regulator of cell cycles. We demonstrate a simple, powerful, and likely generalizable strategy of integrating strengths of single-cell approaches into conventional in vitro systems to study complex clock functions. PMID:29620527

Senescence, apoptosis or autophagy? When a damaged cell must decide its path--a mini-review.

PubMed

Vicencio, José Miguel; Galluzzi, Lorenzo; Tajeddine, Nicolas; Ortiz, Carla; Criollo, Alfredo; Tasdemir, Ezgi; Morselli, Eugenia; Ben Younes, Amena; Maiuri, Maria Chiara; Lavandero, Sergio; Kroemer, Guido

2008-01-01

Many features of aging result from the incapacity of cells to adapt to stress conditions. When damage accumulates irreversibly, mitotic cells from renewable tissues rely on either of two mechanisms to avoid replication. They can permanently arrest the cell cycle (cellular senescence) or trigger cell death programs. Apoptosis (self-killing) is the best-described form of programmed cell death, but autophagy (self-eating), which is a lysosomal degradation pathway essential for homeostasis, reportedly contributes to cell death as well. Unlike mitotic cells, postmitotic cells like neurons or cardiomyocytes cannot become senescent since they are already terminally differentiated. The fate of these cells entirely depends on their ability to cope with stress. Autophagy then operates as a major homeostatic mechanism to eliminate damaged organelles, long-lived or aberrant proteins and superfluous portions of the cytoplasm. In this mini-review, we briefly summarize the molecular networks that allow damaged cells either to adapt to stress or to engage in programmed-cell-death pathways. (c) 2008 S. Karger AG, Basel.

Cell Death During Crisis Is Mediated by Mitotic Telomere Deprotection

PubMed Central

Hayashi, Makoto T.; Cesare, Anthony J.; Rivera, Teresa; Karlseder, Jan

2015-01-01

Tumour formation is blocked by two barriers, replicative senescence and crisis1. Senescence is triggered by short telomeres and is bypassed by disruption of tumour suppressive pathways. After senescence bypass, cells undergo crisis, during which almost all of the cells in the population die. Cells that escape crisis harbor unstable genomes and other parameters of transformation. The mechanism of cell death during crisis remained elusive. We show that cells in crisis undergo spontaneous mitotic arrest, resulting in death during mitosis or in the following cell cycle. The phenotype was induced by loss of p53 function, and suppressed by telomerase overexpression. Telomere fusions triggered mitotic arrest in p53-compromised non-crisis cells, indicating such fusions as the underlying cause. Exacerbation of mitotic telomere deprotection by partial TRF2 knockdown2 increased the ratio of cells that died during mitotic arrest and sensitized cancer cells to mitotic poisons. We propose a crisis pathway wherein chromosome fusions induce mitotic arrest, resulting in mitotic telomere deprotection and cell death, thereby eliminating precancerous cells from the population. PMID:26108857

ATP depletion during mitotic arrest induces mitotic slippage and APC/CCdh1-dependent cyclin B1 degradation.

PubMed

Park, Yun Yeon; Ahn, Ju-Hyun; Cho, Min-Guk; Lee, Jae-Ho

2018-04-27

ATP depletion inhibits cell cycle progression, especially during the G1 phase and the G2 to M transition. However, the effect of ATP depletion on mitotic progression remains unclear. We observed that the reduction of ATP after prometaphase by simultaneous treatment with 2-deoxyglucose and NaN 3 did not arrest mitotic progression. Interestingly, ATP depletion during nocodazole-induced prometaphase arrest resulted in mitotic slippage, as indicated by a reduction in mitotic cells, APC/C-dependent degradation of cyclin B1, increased cell attachment, and increased nuclear membrane reassembly. Additionally, cells successfully progressed through the cell cycle after mitotic slippage, as indicated by EdU incorporation and time-lapse imaging. Although degradation of cyclin B during normal mitotic progression is primarily regulated by APC/C Cdc20 , we observed an unexpected decrease in Cdc20 prior to degradation of cyclin B during mitotic slippage. This decrease in Cdc20 was followed by a change in the binding partner preference of APC/C from Cdc20 to Cdh1; consequently, APC/C Cdh1 , but not APC/C Cdc20 , facilitated cyclin B degradation following ATP depletion. Pulse-chase analysis revealed that ATP depletion significantly abrogated global translation, including the translation of Cdc20 and Cdh1. Additionally, the half-life of Cdh1 was much longer than that of Cdc20. These data suggest that ATP depletion during mitotic arrest induces mitotic slippage facilitated by APC/C Cdh1 -dependent cyclin B degradation, which follows a decrease in Cdc20 resulting from reduced global translation and the differences in the half-lives of the Cdc20 and Cdh1 proteins.

Cytogenetic Analysis of the South American Fruit Fly Anastrepha fraterculus (Diptera:Tephritidae) Species Complex: Construction of Detailed Photographic Polytene Chromosome Maps of the Argentinian Af. sp.1 Member

PubMed Central

Augustinos, Antonios A.; Drosopoulou, Elena; Lanzavecchia, Silvia B.; Cladera, Jorge L.; Caceres, Carlos; Bourtzis, Kostas; Mavragani-Tsipidou, Penelope; Zacharopoulou, Antigone

2016-01-01

Genetic and cytogenetic studies constitute a significant basis for understanding the biology of insect pests and the design and the construction of genetic tools for biological control strategies. Anastrepha fraterculus is an important pest of the Tephritidae family. It is distributed from southern Texas through eastern Mexico, Central America and South America causing significant crop damage and economic losses. Currently it is considered as a species complex; until now seven members have been described based on multidisciplinary approaches. Here we report the cytogenetic analysis of an Argentinian population characterized as Af. sp.1 member of the Anastrepha fraterculus species complex. The mitotic karyotype and the first detailed photographic maps of the salivary gland polytene chromosomes are presented. The mitotic metaphase complement consists of six (6) pairs of chromosomes, including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes that correspond to the five autosomes of the mitotic karyotype and a heterochromatic network corresponding to the sex chromosomes. Comparison of the polytene chromosome maps between this species and Anastrepha ludens shows significant similarity. The polytene maps presented here are suitable for cytogenetic studies that could shed light on the species limits within this species complex and support the development of genetic tools for sterile insect technique (SIT) applications. PMID:27362546

Strategies for Transporting Data Between Classified and Unclassified Networks

DTIC Science & Technology

2016-03-01

datagram protocol (UDP) must be used. The UDP is typically used when speed is a higher priority than data integrity, such as in music or video streaming ...and the exit point of data are separate and can be tightly controlled. This does effectively prevent the comingling of data and is used in industry to...perform functions such as streaming video and audio from secure to insecure networks (ref. 1). A second disadvantage lies in the fact that the

Decreased Expression of the Early Mitotic Gene, CHFR, Contributes to the Acquisition of Breast Cancer Phenotypes

DTIC Science & Technology

2008-03-01

immortalization with the human papilloma virus (HPV) E6 and E7 proteins, which had decreased CHFR expression by RNAi did not have an altered apoptotic response...septation initiation network (SIN). Dev Cell 2002; 3:779–90. 40. Band V, Zajchowski D, Kulesa V, Sager R. Human papilloma virus DNAs immortalize normal... papilloma virus (HPV)–immortalized series of nontumorigenic mammary cell lines were developed and provided by S.P. Ethier, Karmanos Cancer Institute

The radiation hypersensitivity of cells at mitosis.

PubMed

Stobbe, C C; Park, S J; Chapman, J D

2002-12-01

Mitotic cells are hypersensitive to ionizing radiation, exhibiting single-hit inactivation coefficients near to those of repair deficient cell lines and lymphocytes. To elucidate possible mechanisms for this hypersensitivity, the kinetics of oxygen radiosensitization, the proportion of indirect effect by OH radicals and the kinetics of radiation-induced DNA strand breakage in the chromatin of mitotic cells were investigated. Synchronized populations of >90% mitotic HT-29 cells were obtained by the mitotic shake-off method. Cells were irradiated at < or =4 degrees C with (137)Cs gamma-rays. Cellular oxygen concentration was varied by gassing cell suspensions prior to and during irradiation with mixtures of pure N(2) that contained 5% CO(2) and measured quantities of O(2). The indirect effect of OH radicals was investigated with the radical scavenger, DMSO. DNA strand breakage was measured by the comet assay. Mitotic HT-29 cell inactivation is well described by a single-hit inactivation coefficient (alpha) of 1.14 +/- 0.06 Gy(-1). The oxygen enhancement ratio of mitotic cells (at 10% survival) was found to be approximately 2.0, significantly lower than the value of 2.8 measured for interphase (asynchronous) cells. More than 60% of mitotic cell killing was eliminated when the media contained 2 M DMSO, indicating that indirect effect is as important in the killing of mitotic cells as it is for interphase cells. The chromatin in mitotic cells was found to be ~2.8 times more sensitive to radiation-induced DNA single-strand breakage than the chromatin of interphase cells. The alpha-inactivation coefficient of mitotic HT-29 cells was ~30 times larger than that of interphase cells. Mitotic cell chromatin appears to contain intrinsic DNA breaks that are not lethal. In addition, chromatin in mitotic cells was found to be more susceptible to radiation-induced DNA strand-breakage than the dispersed chromatin of interphase cells. How the enhanced production of these simple DNA lesions (that are usually reparable) translates into the lethal (non-reparable) events associated with alpha-inactivation is not known. The compaction/dispersion status of DNA throughout the cell cycle appears to be an important factor for determining intrinsic cell radiosensitivity and might be manipulated for radiotherapeutic advantage.

The p90 ribosomal S6 kinase 2 specifically affects mitotic progression by regulating the basal level, distribution and stability of mitotic spindles

PubMed Central

Park, Yun Yeon; Nam, Hyun-Ja; Do, Mihyang; Lee, Jae-Ho

2016-01-01

RSK2, also known as RPS6KA3 (ribosomal protein S6 kinase, 90 kDa, polypeptide 3), is a downstream kinase of the mitogen-activated protein kinase (MAPK) pathway, which is important in regulating survival, transcription, growth and proliferation. However, its biological role in mitotic progression is not well understood. In this study, we examined the potential involvement of RSK2 in the regulation of mitotic progression. Interestingly, depletion of RSK2, but not RSK1, caused the accumulation of mitotic cells. Time-lapse analysis revealed that mitotic duration, particularly the duration for metaphase-to-anaphase transition was prolonged in RSK2-depleted cells, suggesting activation of spindle assembly checkpoint (SAC). Indeed, more BubR1 (Bub1-related kinase) was present on metaphase plate kinetochores in RSK2-depleted cells, and depletion of BubR1 abolished the mitotic accumulation caused by RSK2 depletion, confirming BubR1-dependent SAC activation. Along with the shortening of inter-kinetochore distance, these data suggested that weakening of the tension across sister kinetochores by RSK2 depletion led to the activation of SAC. To test this, we analyzed the RSK2 effects on the stability of kinetochore–microtubule interactions, and found that RSK2-depleted cells formed less kinetochore–microtubule fibers. Moreover, RSK2 depletion resulted in the decrease of basal level of microtubule as well as an irregular distribution of mitotic spindles, which might lead to observed several mitotic progression defects such as increase in unaligned chromosomes, defects in chromosome congression and a decrease in pole-to-pole distance in these cells. Taken together, our data reveal that RSK2 affects mitotic progression by regulating the distribution, basal level and the stability of mitotic spindles. PMID:27491410

DOE Office of Scientific and Technical Information (OSTI.GOV)

Galán-Malo, Patricia; Vela, Laura; Gonzalo, Oscar

Microtubule poisons and other anti-mitotic drugs induce tumor death but the molecular events linking mitotic arrest to cell death are still not fully understood. We have analyzed cell fate after mitotic arrest produced by the microtubule-destabilizing drug vincristine in a panel of human tumor cell lines showing different response to vincristine. In Jurkat, RPMI 8226 and HeLa cells, apoptosis was triggered shortly after vincristine-induced mitotic arrest. However, A549 cells, which express a great amount of Bcl-x{sub L} and undetectable amounts of Bak, underwent mitotic slippage prior to cell death. However, when Bcl-x{sub L} gene was silenced in A549 cells, vincristinemore » induced apoptosis during mitotic arrest. Another different behavior was found in MiaPaca2 cells, where vincristine caused death by mitotic catastrophe that switched to apoptosis when cyclin B1 degradation was prevented by proteasome inhibition. Overexpression of Bcl-x{sub L} or silencing Bax and Bak expression delayed the onset of apoptosis in Jurkat and RPMI 8226 cells, enabling mitotic slippage and endoreduplication. In HeLa cells, overexpression of Bcl-x{sub L} switched cell death from apoptosis to mitotic catastrophe. Mcl-1 offered limited protection to vincristine-induced cell death and Mcl-1 degradation was not essential for vincristine-induced death. All these results, taken together, indicate that the Bcl-x{sub L}/Bak ratio and the ability to degrade cyclin B1 determine cell fate after mitotic arrest in the different tumor cell types. Highlights: ► Vincristine induces cell death by apoptosis or mitotic catastrophe. ► Apoptosis-proficient cells die by apoptosis during mitosis upon vincristine treatment. ► p53wt apoptosis-deficient cells undergo apoptosis from a G1-like tetraploid state. ► p53mt apoptosis-deficient cells can survive and divide giving rise to 8N cells.« less

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

PubMed Central

Moura, Margarida; Osswald, Mariana; Leça, Nelson; Barbosa, João; Pereira, António J; Maiato, Helder; Sunkel, Claudio E; Conde, Carlos

2017-01-01

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit. DOI: http://dx.doi.org/10.7554/eLife.25366.001 PMID:28463114

Prophase pathway-dependent removal of cohesin from human chromosomes requires opening of the Smc3–Scc1 gate

PubMed Central

Buheitel, Johannes; Stemmann, Olaf

2013-01-01

Faithful transmission of chromosomes during eukaryotic cell division requires sister chromatids to be paired from their generation in S phase until their separation in M phase. Cohesion is mediated by the cohesin complex, whose Smc1, Smc3 and Scc1 subunits form a tripartite ring that entraps both DNA double strands. Whereas centromeric cohesin is removed in late metaphase by Scc1 cleavage, metazoan cohesin at chromosome arms is displaced already in prophase by proteolysis-independent signalling. Which of the three gates is triggered by the prophase pathway to open has remained enigmatic. Here, we show that displacement of human cohesin from early mitotic chromosomes requires dissociation of Smc3 from Scc1 but no opening of the other two gates. In contrast, loading of human cohesin onto chromatin in telophase occurs through the Smc1–Smc3 hinge. We propose that the use of differently regulated gates for loading and release facilitates unidirectionality of DNA's entry into and exit from the cohesin ring. PMID:23361318

The mammalian Doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells

PubMed Central

Matson, Clinton K.; Murphy, Mark W.; Griswold, Michael D.; Yoshida, Shosei; Bardwell, Vivian J.; Zarkower, David

2010-01-01

Summary The switch from mitosis to meiosis is a unique feature of germ cell development. In mammals, meiotic initiation requires retinoic acid (RA), which activates meiotic inducers including Stra8, but how the switch to meiosis is controlled in male germ cells (spermatogonia) remains poorly understood. Here we examine the role of the Doublesex-related transcription factor DMRT1 in adult spermatogenesis using conditional gene targeting in the mouse. Loss of Dmrt1 causes spermatogonia to precociously exit the spermatogonial program and enter meiosis. Dmrt1 therefore determines whether male germ cells undergo mitosis and spermatogonial differentiation or meiosis. Loss of Dmrt1 in spermatogonia also disrupts cyclical gene expression in Sertoli cells. DMRT1 acts in spermatogonia to restrict RA responsiveness, directly repress Stra8 transcription, and activate transcription of the spermatogonial differentiation factor Sohlh1, thereby preventing meiosis and promoting spermatogonial development. By coordinating spermatogonial development and mitotic amplification with meiosis, DMRT1 allows abundant, continuous production of sperm. PMID:20951351

The initiation of lateral roots in the primary roots of maize (Zea mays L.) implies a reactivation of cell proliferation in a group of founder pericycle cells.

PubMed

Alarcón, M Victoria; Lloret, Pedro G; Martín-Partido, Gervasio; Salguero, Julio

2016-03-15

The initiation of lateral roots (LRs) has generally been viewed as a reactivation of proliferative activity in pericycle cells that are committed to initiate primordia. However, it is also possible that pericycle founder cells that initiate LRs never cease proliferative activity but rather are displaced to the most distal root zones while undertaking successive stages of LR initiation. In this study, we tested these two alternative hypotheses by examining the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of meristematic root cells of Zea mays. According to the values for the length of the cell cycle and values for cell displacement along the maize root, our results strongly suggest that pericycle cells that initiate LR primordia ceased proliferative activity upon exiting the meristematic zone. This finding is supported by the existence of a root zone between 4 and 20mm from the root cap junction, in which neither mitotic cells nor labelled nuclei were observed in phloem pericycle cells. Copyright © 2016 Elsevier GmbH. All rights reserved.

The Drosophila Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

PubMed Central

Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

2013-01-01

Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs. PMID:23593258

Mitotic rate is associated with positive lymph nodes in patients with thin melanomas.

PubMed

Wheless, Lee; Isom, Chelsea A; Hooks, Mary A; Kauffmann, Rondi M

2018-05-01

The American Joint Commission on Cancer will remove mitotic rate from its staging guidelines in 2018. Using a large nationally representative cohort, we examined the association between mitotic rate and lymph node positivity among thin melanomas. A total of 149,273 thin melanomas in the National Cancer Database were examined for their association of high-risk features of mitotic rate, ulceration, and Breslow depth with lymph node status. Among 17,204 patients with thin melanomas with data on Breslow depth, ulceration, and mitotic rate who underwent a lymph node biopsy, there was a strong linear relationship between odds of having a positive lymph node and mitotic rate (R 2  = 0.96, P < .0001, β = 3.31). The odds of having a positive node increased by 19% with each 1-point increase in mitotic rate (odds ratio, 1.19; 95% confidence interval, 1.17-1.21). Cases with negative nodes had a mean mitotic rate of 1.54 plus or minus 2.07 mitoses/mm 2 compared with 3.30 plus or minus 3.54 mitoses/mm 2 for those with positive nodes (P < .0001). The data collected do not allow for survival analyses. Mitotic rate was strongly associated with the odds of having a positive lymph node and should continue to be reported on pathology reports. Copyright © 2017 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

Spatial Reorganization of the Endoplasmic Reticulum during Mitosis Relies on Mitotic Kinase Cyclin A in the Early Drosophila Embryo

PubMed Central

Bergman, Zane J.; Mclaurin, Justin D.; Eritano, Anthony S.; Johnson, Brittany M.; Sims, Amanda Q.; Riggs, Blake

2015-01-01

Mitotic cyclin-dependent kinase with their cyclin partners (cyclin:Cdks) are the master regulators of cell cycle progression responsible for regulating a host of activities during mitosis. Nuclear mitotic events, including chromosome condensation and segregation have been directly linked to Cdk activity. However, the regulation and timing of cytoplasmic mitotic events by cyclin:Cdks is poorly understood. In order to examine these mitotic cytoplasmic events, we looked at the dramatic changes in the endoplasmic reticulum (ER) during mitosis in the early Drosophila embryo. The dynamic changes of the ER can be arrested in an interphase state by inhibition of either DNA or protein synthesis. Here we show that this block can be alleviated by micro-injection of Cyclin A (CycA) in which defined mitotic ER clusters gathered at the spindle poles. Conversely, micro-injection of Cyclin B (CycB) did not affect spatial reorganization of the ER, suggesting CycA possesses the ability to initiate mitotic ER events in the cytoplasm. Additionally, RNAi-mediated simultaneous inhibition of all 3 mitotic cyclins (A, B and B3) blocked spatial reorganization of the ER. Our results suggest that mitotic ER reorganization events rely on CycA and that control and timing of nuclear and cytoplasmic events during mitosis may be defined by release of CycA from the nucleus as a consequence of breakdown of the nuclear envelope. PMID:25689737

DOE Office of Scientific and Technical Information (OSTI.GOV)

Komura, Jun-ichiro, E-mail: junkom@med.tohoku.ac.jp; Ikehata, Hironobu; Mori, Toshio

During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40 years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cellsmore » was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression. -- Highlights: Black-Right-Pointing-Pointer Global genome repair of (6-4) photoproducts is fully active in mitotic cells. Black-Right-Pointing-Pointer DNA in condensed mitotic chromatin does not seem inaccessible or inert. Black-Right-Pointing-Pointer Mitotic transcriptional repression may impair transcription-coupled repair.« less

Phosphorylation of histone H3 on Ser-10 by Aurora B is essential for chromosome condensation in porcine embryos during the first mitotic division.

PubMed

Chen, Changchao; Zhang, Zixiao; Cui, Panpan; Liao, Yaya; Zhang, Yue; Yao, Lingyun; Rui, Rong; Ju, Shiqiang

2017-07-01

Phosphorylation of histone H3 on Ser-10 (H3S10ph) is involved in regulating mitotic chromosome condensation and decondensation, which plays an important regulatory role during mitotic cell cycle progression in mammalian cells. However, whether H3S10ph plays a similar role in early porcine embryos during the first mitotic division remains uncertain. In this study, the subcellular localization and possible roles of H3S10ph were evaluated in the first mitotic cell cycle progression of porcine embryos using western blot, indirect immunofluorescence and barasertib (H3S10ph upstream regulator Aurora-B inhibitor) treatments. H3S10ph exhibited a dynamic localization pattern and was localized to chromosomes from prometaphase to anaphase stages. Treatment of porcine embryos with barasertib inhibited mitotic division at the prophase stage and was associated with a defect in chromosome condensation accompanied by the reduction of H3S10ph. These results indicated that H3S10ph is involved in the first mitotic division in porcine embryos through its regulatory function in chromosome condensation, which further affects porcine embryo cell cycle progression during mitotic division.

The Notch pathway regulates the Second Mitotic Wave cell cycle independently of bHLH proteins.

PubMed

Bhattacharya, Abhishek; Li, Ke; Quiquand, Manon; Rimesso, Gerard; Baker, Nicholas E

2017-11-15

Notch regulates both neurogenesis and cell cycle activity to coordinate precursor cell generation in the differentiating Drosophila eye. Mosaic analysis with mitotic clones mutant for Notch components was used to identify the pathway of Notch signaling that regulates the cell cycle in the Second Mitotic Wave. Although S phase entry depends on Notch signaling and on the transcription factor Su(H), the transcriptional co-activator Mam and the bHLH repressor genes of the E(spl)-Complex were not essential, although these are Su(H) coactivators and targets during the regulation of neurogenesis. The Second Mitotic Wave showed little dependence on ubiquitin ligases neuralized or mindbomb, and although the ligand Delta is required non-autonomously, partial cell cycle activity occurred in the absence of known Notch ligands. We found that myc was not essential for the Second Mitotic Wave. The Second Mitotic Wave did not require the HLH protein Extra macrochaetae, and the bHLH protein Daughterless was required only cell-nonautonomously. Similar cell cycle phenotypes for Daughterless and Atonal were consistent with requirement for neuronal differentiation to stimulate Delta expression, affecting Notch activity in the Second Mitotic Wave indirectly. Therefore Notch signaling acts to regulate the Second Mitotic Wave without activating bHLH gene targets. Copyright © 2017 Elsevier Inc. All rights reserved.

An automated fluorescence videomicroscopy assay for the detection of mitotic catastrophe

PubMed Central

Rello-Varona, S; Kepp, O; Vitale, I; Michaud, M; Senovilla, L; Jemaà, M; Joza, N; Galluzzi, L; Castedo, M; Kroemer, G

2010-01-01

Mitotic catastrophe can be defined as a cell death mode that occurs during or shortly after a prolonged/aberrant mitosis, and can show apoptotic or necrotic features. However, conventional procedures for the detection of apoptosis or necrosis, including biochemical bulk assays and cytofluorometric techniques, cannot discriminate among pre-mitotic, mitotic and post-mitotic death, and hence are inappropriate to monitor mitotic catastrophe. To address this issue, we generated isogenic human colon carcinoma cell lines that differ in ploidy and p53 status, yet express similar amounts of fluorescent biosensors that allow for the visualization of chromatin (histone H2B coupled to green fluorescent protein (GFP)) and centrosomes (centrin coupled to the Discosoma striata red fluorescent protein (DsRed)). By combining high-resolution fluorescence videomicroscopy and automated image analysis, we established protocols and settings for the simultaneous assessment of ploidy, mitosis, centrosome number and cell death (which in our model system occurs mainly by apoptosis). Time-lapse videomicroscopy showed that this approach can be used for the high-throughput detection of mitotic catastrophe induced by three mechanistically distinct anti-mitotic agents (dimethylenastron (DIMEN), nocodazole (NDZ) and paclitaxel (PTX)), and – in this context – revealed an important role of p53 in the control of centrosome number. PMID:21364633

JMJD5 (Jumonji Domain-containing 5) Associates with Spindle Microtubules and Is Required for Proper Mitosis.

PubMed

He, Zhimin; Wu, Junyu; Su, Xiaonan; Zhang, Ye; Pan, Lixia; Wei, Huimin; Fang, Qiang; Li, Haitao; Wang, Da-Liang; Sun, Fang-Lin

2016-02-26

Precise mitotic spindle assembly is a guarantee of proper chromosome segregation during mitosis. Chromosome instability caused by disturbed mitosis is one of the major features of various types of cancer. JMJD5 has been reported to be involved in epigenetic regulation of gene expression in the nucleus, but little is known about its function in mitotic process. Here we report the unexpected localization and function of JMJD5 in mitotic progression. JMJD5 partially accumulates on mitotic spindles during mitosis, and depletion of JMJD5 results in significant mitotic arrest, spindle assembly defects, and sustained activation of the spindle assembly checkpoint (SAC). Inactivating SAC can efficiently reverse the mitotic arrest caused by JMJD5 depletion. Moreover, JMJD5 is found to interact with tubulin proteins and associate with microtubules during mitosis. JMJD5-depleted cells show a significant reduction of α-tubulin acetylation level on mitotic spindles and fail to generate enough interkinetochore tension to satisfy the SAC. Further, JMJD5 depletion also increases the susceptibility of HeLa cells to the antimicrotubule agent. Taken together, these results suggest that JMJD5 plays an important role in regulating mitotic progression, probably by modulating the stability of spindle microtubules. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Fast Recall for Complex-Valued Hopfield Neural Networks with Projection Rules.

PubMed

Kobayashi, Masaki

2017-01-01

Many models of neural networks have been extended to complex-valued neural networks. A complex-valued Hopfield neural network (CHNN) is a complex-valued version of a Hopfield neural network. Complex-valued neurons can represent multistates, and CHNNs are available for the storage of multilevel data, such as gray-scale images. The CHNNs are often trapped into the local minima, and their noise tolerance is low. Lee improved the noise tolerance of the CHNNs by detecting and exiting the local minima. In the present work, we propose a new recall algorithm that eliminates the local minima. We show that our proposed recall algorithm not only accelerated the recall but also improved the noise tolerance through computer simulations.

The Ribosome Shape Directs mRNA Translocation through Entrance and Exit Dynamics

USDA-ARS?s Scientific Manuscript database

The protein-synthesizing ribosome undergoes large motions to effect the translocation of tRNAs (transfer ribonucleic acids) and mRNA (messenger ribonucleic acid); here the domain motions of this system are explored with a coarse-grained elastic network model using normal mode analysis. Crystal struc...

Effects of hypergravity on rat liver regeneration

NASA Technical Reports Server (NTRS)

Feller, D. D.

1982-01-01

The effects of centrifugation on liver regrowth were examined by measuring mitotic activity. The results indicate that the increased gravity caused a delay in the onset of mitotic activity and a significant decrease in overall mitotic activity.

Helping Survivors of Human Trafficking: A Systematic Review of Exit and Postexit Interventions.

PubMed

Dell, Nathaniel A; Maynard, Brandy R; Born, Kara R; Wagner, Elizabeth; Atkins, Bonnie; House, Whitney

2017-01-01

Human trafficking is a global problem and results in deleterious psychological, social, and physical effects on the lives of those who are trafficked; however, it is not clear how to best intervene with survivors. The purpose of this review was to synthesize the evidence of exit and postexit intervention programs for survivors of human trafficking to inform practice and research. Systematic review methods were used to search, select, and extract data from published and unpublished experimental, quasi-experimental, and preexperimental studies that assessed the effects of any exit or postexit interventions for victims of human trafficking. The authors searched eight databases, reviewed bibliographies, and conducted forward citation searches from relevant reports and prior reviews to find studies authored between 2005 and 2015. The search yielded six eligible studies that included 155 female and 6 male survivors from four countries. Interventions were diverse, with three using a trauma-informed approach. Authors measured a myriad of outcomes, including mental health, social network, community reintegration, and employment; however, the quality of most studies was poor. Evidence of effects of exit and postexit interventions is sparse, and much of the research is poorly designed and executed; however, the needs of trafficking survivors are complex and effective interventions are desperately needed. Implications for practice and research are discussed.

Analysis of mitosis and antimitotic drug responses in tumors by in vivo microscopy and single-cell pharmacodynamics.

PubMed

Orth, James D; Kohler, Rainer H; Foijer, Floris; Sorger, Peter K; Weissleder, Ralph; Mitchison, Timothy J

2011-07-01

Cancer relies upon frequent or abnormal cell division, but how the tumor microenvironment affects mitotic processes in vivo remains unclear, largely due to the technical challenges of optical access, spatial resolution, and motion. We developed high-resolution in vivo microscopy methods to visualize mitosis in a murine xenograft model of human cancer. Using these methods, we determined whether the single-cell response to the antimitotic drug paclitaxel (Ptx) was the same in tumors as in cell culture, observed the impact of Ptx on the tumor response as a whole, and evaluated the single-cell pharmacodynamics (PD) of Ptx (by in vivo PD microscopy). Mitotic initiation was generally less frequent in tumors than in cell culture, but subsequently it proceeded normally. Ptx treatment caused spindle assembly defects and mitotic arrest, followed by slippage from mitotic arrest, multinucleation, and apoptosis. Compared with cell culture, the peak mitotic index in tumors exposed to Ptx was lower and the tumor cells survived longer after mitotic arrest, becoming multinucleated rather than dying directly from mitotic arrest. Thus, the tumor microenvironment was much less proapoptotic than cell culture. The morphologies associated with mitotic arrest were dose and time dependent, thereby providing a semiquantitative, single-cell measure of PD. Although many tumor cells did not progress through Ptx-induced mitotic arrest, tumor significantly regressed in the model. Our findings show that in vivo microscopy offers a useful tool to visualize mitosis during tumor progression, drug responses, and cell fate at the single-cell level. ©2011 AACR.

[Immunohistochemical observation on keratin filaments of cultured tumor cells by ABC staining].

PubMed

Wang, J; Yang, F

1991-06-01

Avidin-Biotin Peroxidase complex technique, ABV staining, was employed by using monoclonal anti-keratin antibody HK2 in this study. The organization and dynamics of keratins in both interphase and mitotic T56 and HeLa cells were analysed. We also observed the effects of microtubule (MT) and microfilament (MF) inhibitors, colchicine and cytochalasin B, on the organization of keratin filaments in T56 and HeLa cells. The results showed that a significant alteration in the structural organization and distribution of keratin filaments occurred during mitosis, and an extensive rearrangement of keratin networks of the two cell lines was induced in interphase after the MT and MF were disrupted by combined treatment with the two drugs, colchicine and cytochalasin B; the keratin networks turned into a star-like lattice rapidly within 1-2h. Neither colchicine nor cytochalasin B alone elicited significant organizational change in the keratin networks of the two cell lines.

Switches and latches: a biochemical tug-of-war between the kinases and phosphatases that control mitosis.

PubMed

Domingo-Sananes, Maria Rosa; Kapuy, Orsolya; Hunt, Tim; Novak, Bela

2011-12-27

Activation of the cyclin-dependent kinase (Cdk1) cyclin B (CycB) complex (Cdk1:CycB) in mitosis brings about a remarkable extent of protein phosphorylation. Cdk1:CycB activation is switch-like, controlled by two auto-amplification loops--Cdk1:CycB activates its activating phosphatase, Cdc25, and inhibits its inhibiting kinase, Wee1. Recent experimental evidence suggests that parallel to Cdk1:CycB activation during mitosis, there is inhibition of its counteracting phosphatase activity. We argue that the downregulation of the phosphatase is not just a simple latch that suppresses futile cycles of phosphorylation/dephosphorylation during mitosis. Instead, we propose that phosphatase regulation creates coherent feed-forward loops and adds extra amplification loops to the Cdk1:CycB regulatory network, thus forming an integral part of the mitotic switch. These network motifs further strengthen the bistable characteristic of the mitotic switch, which is based on the antagonistic interaction of two groups of proteins: M-phase promoting factors (Cdk1:CycB, Cdc25, Greatwall and Endosulfine/Arpp19) and interphase promoting factors (Wee1, PP2A-B55 and a Greatwall counteracting phosphatase, probably PP1). The bistable character of the switch implies the existence of a CycB threshold for entry into mitosis. The end of G2 phase is determined by the point where CycB level crosses the CycB threshold for Cdk1 activation.

FANCA safeguards interphase and mitosis during hematopoiesis in vivo

PubMed Central

Abdul-Sater, Zahi; Cerabona, Donna; Sierra Potchanant, Elizabeth; Sun, Zejin; Enzor, Rikki; He, Ying; Robertson, Kent; Goebel, W. Scott; Nalepa, Grzegorz

2015-01-01

Fanconi anemia (FA/BRCA) signaling network controls multiple genome-housekeeping checkpoints, from interphase DNA repair to mitosis. The in vivo role of abnormal cell division in FA remains unknown. Here, we quantified the origins of genomic instability in FA patients and mice in vivo and ex vivo. We found that both mitotic errors and interphase DNA damage significantly contribute to genomic instability during FA-deficient hematopoiesis and in non-hematopoietic human and murine FA primary cells. Super-resolution microscopy coupled with functional assays revealed that FANCA shuttles to the pericentriolar material (PCM) to regulate spindle assembly at mitotic entry. Loss of FA signaling rendered cells hypersensitive to spindle chemotherapeutics and allowed escape from the chemotherapy-induced spindle assembly checkpoint. In support of these findings, direct comparison of DNA cross-linking and antimitotic chemotherapeutics in primary FANCA−/− cells revealed genomic instability originating through divergent cell cycle checkpoint aberrations. Our data indicate that the FA/BRCA signaling functions as an in vivo gatekeeper of genomic integrity throughout interphase and mitosis, which may have implications for future targeted therapies in FA and FA-deficient cancers. PMID:26366677

A bHLH-Based Feedback Loop Restricts Vascular Cell Proliferation in Plants.

PubMed

Vera-Sirera, Francisco; De Rybel, Bert; Úrbez, Cristina; Kouklas, Evangelos; Pesquera, Marta; Álvarez-Mahecha, Juan Camilo; Minguet, Eugenio G; Tuominen, Hannele; Carbonell, Juan; Borst, Jan Willem; Weijers, Dolf; Blázquez, Miguel A

2015-11-23

Control of tissue dimensions in multicellular organisms requires the precise quantitative regulation of mitotic activity. In plants, where cells are immobile, tissue size is achieved through control of both cell division orientation and mitotic rate. The bHLH transcription factor heterodimer formed by target of monopteros5 (TMO5) and lonesome highway (LHW) is a central regulator of vascular width-increasing divisions. An important unanswered question is how its activity is limited to specify vascular tissue dimensions. Here we identify a regulatory network that restricts TMO5/LHW activity. We show that thermospermine synthase ACAULIS5 antagonizes TMO5/LHW activity by promoting the accumulation of SAC51-LIKE (SACL) bHLH transcription factors. SACL proteins heterodimerize with LHW-therefore likely competing with TMO5/LHW interactions-prevent activation of TMO5/LHW target genes, and suppress the over-proliferation caused by excess TMO5/LHW activity. These findings connect two thus-far disparate pathways and provide a mechanistic understanding of the quantitative control of vascular tissue growth. Copyright © 2015 Elsevier Inc. All rights reserved.

A case of metastatic haemangiopericytoma to the thyroid gland: Case report and literature review

PubMed Central

PROIETTI, AGNESE; SARTORI, CHIARA; TORREGROSSA, LIBORIO; VITTI, PAOLO; AGHABABYAN, ALEKSANDR; FREGOLI, LORENZO; MICCOLI, PAOLO; BASOLO, FULVIO

2012-01-01

Haemangiopericytoma (HPC) is a mesenchymal neoplasm accounting for a minority of all vascular tumours. HPC mostly arises in the lower extremities and the retroperitoneum, while the head and neck area is the third most common site. The majority of HPCs are histologically benign. However, a small percentage possess atypical features, such as a high mitotic rate, high cellularity and foci of necrosis. We report a case of classical abdominal HPC that presented 7 years after the first surgical resection with thyroid metastases of malignant HPC. Microscopic examination revealed multiple hypercellular nodules with an infiltrative growth pattern. These nodules consisted of tightly packed fusiform or spindle-shaped cells with nuclear polymorphism and an increased mitotic rate. The tumour cells exhibited a marked expression of CD34. Cells were arranged around a prominent vascular network, occasionally with a ‘staghorn’ configuration. The results of this study support and confirm the theory that HPC is a rare neoplasm with unpredictable behaviour, as largely debated in the international literature. Therefore, this study emphasized the importance of applying strict diagnostic criteria in making the most appropriate diagnosis. PMID:22783428

Mitotic cells generate protrusive extracellular forces to divide in three-dimensional microenvironments

NASA Astrophysics Data System (ADS)

Nam, Sungmin; Chaudhuri, Ovijit

2018-06-01

During mitosis, or cell division, mammalian cells undergo extensive morphological changes, including elongation along the mitotic axis, which is perpendicular to the plane that bisects the two divided cells. Although much is known about the intracellular dynamics of mitosis, it is unclear how cells are able to divide in tissues, where the changes required for mitosis are mechanically constrained by surrounding cells and extracellular matrix. Here, by confining cells three dimensionally in hydrogels, we show that dividing cells generate substantial protrusive forces that deform their surroundings along the mitotic axis, clearing space for mitotic elongation. When forces are insufficient to create space for mitotic elongation, mitosis fails. We identify one source of protrusive force as the elongation of the interpolar spindle, an assembly of microtubules aligned with the mitotic axis. Another source of protrusive force is shown to be contraction of the cytokinetic ring, the polymeric structure that cleaves a dividing cell at its equator, which drives expansion along the mitotic axis. These findings reveal key functions for the interpolar spindle and cytokinetic ring in protrusive extracellular force generation, and explain how dividing cells overcome mechanical constraints in confining microenvironments, including some types of tumour.

Repeated furrow formation from a single mitotic apparatus in cylindrical sand dollar eggs.

PubMed

Rappaport, R

1985-04-01

The methods used previously to demonstrate the ability of a single mitotic apparatus to elicit multiple furrows involved considerable cell distortion and did not permit the investigator to control the positioning of the parts or to observe satisfactorily the early stages of furrow development. In this investigation, Echinarachnius parma eggs were confined in 82 microns i.d. transparent, silicone rubber-walled capillaries, and the mitotic apparatus was moved by pushing the poles inward with 55-microns-diameter glass balls. When the mitotic apparatus was shifted immediately after the furrow first appeared, a new furrow appeared in the normal relation to the new position in 1-2 minutes. The same mitotic apparatus could elicit up to 13 furrows as it was shifted back and forth by alternately pushing in the poles. The previous furrow regressed as the new furrow developed. The operations protracted the furrow establishment period to as long as 24.5 minutes after establishment of the first furrow. The characteristics of furrow regression were related to the distance the mitotic apparatus was moved. It is unlikely that regression was caused either by stress imposed on the surface or the removal of the mitotic apparatus from the vicinity of the furrow.

Simple method for culture of peripheral blood lymphocytes of Testudinidae.

PubMed

Silva, T L; Silva, M I A; Venancio, L P R; Zago, C E S; Moscheta, V A G; Lima, A V B; Vizotto, L D; Santos, J R; Bonini-Domingos, C R; Azeredo-Oliveira, M T V

2011-12-06

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.

[The effect of pemolin on the mitotic activity of Vicia faba L (author's transl)].

PubMed

Brabec, F; Röper, W

1976-02-01

The effect of diverse concentrations of 5-phenyl-2-imino-4-oxazolidone (PIO, pemolin, Tradon) on the mitotic activity in lateral roots of Vicia faba L. was studied by aerated and non-aerated hydrocultivation with and without mineral nutrition, respectively. With optimal conditions (aerated nutrient solution) weak PIO-concentrations, most significantly 10(-6) g/ml, effected a marked increase of the mitotic index. Contrarily, strong PIO-concentrations (10(-4) and 3 X 10(-4) g/ml = saturated solution) significantly decreased the mitotic index though simultaneously preserving the mitotic activity in long-term experiments, when on account of nutrient deficiency it had already collapsed in weak PIO-concentrations and the controls. The activating effect of weak PIO-concentrations compared with the controls is more significant in stress situations (nutrient deficiency, O2-deficiency) than under optimal conditions. Furthermore a slight acceleration of mid-mitotic phases (metaphase--anaphase) recognized by a marked decrease in percentage of these phases, can be stated with weak PIO-concentrations, again particularly so with 10(-6) g/ml. In total, dependent on concentration, pemolin presumably may either activate or suppress cell metabolism and particularly the mitotic cycle. The exact site of action of the substance is still unknown.

Twitter-Delivered Behavioral Weight-Loss Interventions: A Pilot Series.

PubMed

Pagoto, Sherry L; Waring, Molly E; Schneider, Kristin L; Oleski, Jessica L; Olendzki, Effie; Hayes, Rashelle B; Appelhans, Bradley M; Whited, Matthew C; Busch, Andrew M; Lemon, Stephenie C

2015-10-23

Lifestyle interventions are efficacious at reducing risk for diabetes and cardiovascular disease but have not had a significant public health impact given high cost and patient and provider burden. Online social networks may reduce the burden of lifestyle interventions to the extent that they displace in-person visits and may enhance opportunities for social support for weight loss. We conducted an iterative series of pilot studies to evaluate the feasibility and acceptability of using online social networks to deliver a lifestyle intervention. In Study 1 (n=10), obese participants with depression received lifestyle counseling via 12 weekly group visits and a private group formed using the online social network, Twitter. Mean weight loss was 2.3 pounds (SD 7.7; range -19.2 to 8.2) or 1.2% (SD 3.6) of baseline weight. A total of 67% (6/9) of participants completing exit interviews found the support of the Twitter group at least somewhat useful. In Study 2 (n=11), participants were not depressed and were required to be regular users of social media. Participants lost, on average, 5.6 pounds (SD 6.3; range -15 to 0) or 3.0% (SD 3.4) of baseline weight, and 100% (9/9) completing exit interviews found the support of the Twitter group at least somewhat useful. To explore the feasibility of eliminating in-person visits, in Study 3 (n=12), we delivered a 12-week lifestyle intervention almost entirely via Twitter by limiting the number of group visits to one, while using the same inclusion criteria as that used in Study 2. Participants lost, on average, 5.4 pounds (SD 6.4; range -14.2 to 3.9) or 3.0% (SD 3.1) of baseline weight, and 90% (9/10) completing exit interviews found the support of the Twitter group at least somewhat useful. Findings revealed that a private Twitter weight-loss group was both feasible and acceptable for many patients, particularly among regular users of social media. Future research should evaluate the efficacy and cost-effectiveness of online social network-delivered lifestyle interventions relative to traditional modalities.

Twitter-Delivered Behavioral Weight-Loss Interventions: A Pilot Series

PubMed Central

Waring, Molly E; Schneider, Kristin L; Oleski, Jessica L; Olendzki, Effie; Hayes, Rashelle B; Appelhans, Bradley M; Whited, Matthew C; Busch, Andrew M; Lemon, Stephenie C

2015-01-01

Background Lifestyle interventions are efficacious at reducing risk for diabetes and cardiovascular disease but have not had a significant public health impact given high cost and patient and provider burden. Objective Online social networks may reduce the burden of lifestyle interventions to the extent that they displace in-person visits and may enhance opportunities for social support for weight loss. Methods We conducted an iterative series of pilot studies to evaluate the feasibility and acceptability of using online social networks to deliver a lifestyle intervention. Results In Study 1 (n=10), obese participants with depression received lifestyle counseling via 12 weekly group visits and a private group formed using the online social network, Twitter. Mean weight loss was 2.3 pounds (SD 7.7; range -19.2 to 8.2) or 1.2% (SD 3.6) of baseline weight. A total of 67% (6/9) of participants completing exit interviews found the support of the Twitter group at least somewhat useful. In Study 2 (n=11), participants were not depressed and were required to be regular users of social media. Participants lost, on average, 5.6 pounds (SD 6.3; range -15 to 0) or 3.0% (SD 3.4) of baseline weight, and 100% (9/9) completing exit interviews found the support of the Twitter group at least somewhat useful. To explore the feasibility of eliminating in-person visits, in Study 3 (n=12), we delivered a 12-week lifestyle intervention almost entirely via Twitter by limiting the number of group visits to one, while using the same inclusion criteria as that used in Study 2. Participants lost, on average, 5.4 pounds (SD 6.4; range -14.2 to 3.9) or 3.0% (SD 3.1) of baseline weight, and 90% (9/10) completing exit interviews found the support of the Twitter group at least somewhat useful. Findings revealed that a private Twitter weight-loss group was both feasible and acceptable for many patients, particularly among regular users of social media. Conclusions Future research should evaluate the efficacy and cost-effectiveness of online social network-delivered lifestyle interventions relative to traditional modalities. PMID:26500186

D1-Asn-298 in photosystem II is involved in a hydrogen-bond network near the redox-active tyrosine YZ for proton exit during water oxidation.

PubMed

Nagao, Ryo; Ueoka-Nakanishi, Hanayo; Noguchi, Takumi

2017-12-08

In photosynthetic water oxidation, two water molecules are converted into one oxygen molecule and four protons at the Mn 4 CaO 5 cluster in photosystem II (PSII) via the S-state cycle. Efficient proton exit from the catalytic site to the lumen is essential for this process. However, the exit pathways of individual protons through the PSII proteins remain to be identified. In this study, we examined the involvement of a hydrogen-bond network near the redox-active tyrosine Y Z in proton transfer during the S-state cycle. We focused on spectroscopic analyses of a site-directed variant of D1-Asn-298, a residue involved in a hydrogen-bond network near Y Z We found that the D1-N298A mutant of Synechocystis sp. PCC 6803 exhibits an O 2 evolution activity of ∼10% of the wild-type. D1-N298A and the wild-type D1 had very similar features of thermoluminescence glow curves and of an FTIR difference spectrum upon Y Z oxidation, suggesting that the hydrogen-bonded structure of Y Z and electron transfer from the Mn 4 CaO 5 cluster to Y Z were little affected by substitution. In the D1-N298A mutant, however, the flash-number dependence of delayed luminescence showed a monotonic increase without oscillation, and FTIR difference spectra of the S-state cycle indicated partial and significant inhibition of the S 2 → S 3 and S 3 → S 0 transitions, respectively. These results suggest that the D1-N298A substitution inhibits the proton transfer processes in the S 2 → S 3 and S 3 → S 0 transitions. This in turn indicates that the hydrogen-bond network near Y Z can be functional as a proton transfer pathway during photosynthetic water oxidation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitotic phosphorylation of SUN1 loosens its connection with the nuclear lamina while the LINC complex remains intact.

PubMed

Patel, Jennifer T; Bottrill, Andrew; Prosser, Suzanna L; Jayaraman, Sangeetha; Straatman, Kees; Fry, Andrew M; Shackleton, Sue

2014-01-01

At the onset mitosis in higher eukaryotes, the nuclear envelope (NE) undergoes dramatic deconstruction to allow separation of duplicated chromosomes. Studies have shown that during this process of nuclear envelope breakdown (NEBD), the extensive protein networks of the nuclear lamina are disassembled through phosphorylation of lamins and several inner nuclear membrane (INM) proteins. The LINC complex, composed of SUN and nesprin proteins, is involved in multiple interactions at the NE and plays vital roles in nuclear and cellular mechanics by connecting the nucleus to the cytoskeleton. Here, we show that SUN1, located in the INM, undergoes mitosis-specific phosphorylation on at least 3 sites within its nucleoplasmic N-terminus. We further identify Cdk1 as the kinase responsible for serine 48 and 333 phosphorylation, while serine 138 is phosphorylated by Plk1. In mitotic cells, SUN1 loses its interaction with N-terminal domain binding partners lamin A/C, emerin, and short nesprin-2 isoforms. Furthermore, a triple phosphomimetic SUN1 mutant displays increased solubility and reduced retention at the NE. In contrast, the central LINC complex interaction between the SUN1 C-terminus and the KASH domain of nesprin-2 is maintained during mitosis. Together, these data support a model whereby mitotic phosphorylation of SUN1 disrupts interactions with nucleoplasmic binding partners, promoting disassembly of the nuclear lamina and, potentially, its chromatin interactions. At the same time, our data add to an emerging picture that the core LINC complex plays an active role in NEBD.

The structure of the mitotic spindle and nucleolus during mitosis in the amebo-flagellate Naegleria.

PubMed

Walsh, Charles J

2012-01-01

Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division.

The Utilization during Mitotic Cell Division of Loci Controlling Meiotic Recombination and Disjunction in DROSOPHILA MELANOGASTER

PubMed Central

Baker, Bruce S.; Carpenter, Adelaide T. C.; Ripoll, P.

1978-01-01

To inquire whether the loci identified by recombination-defective and disjunction-defective meiotic mutants in Drosophila are also utilized during mitotic cell division, the effects of 18 meiotic mutants (representing 13 loci) on mitotic chromosome stability have been examined genetically. To do this, meiotic-mutant-bearing flies heterozygous for recessive somatic cell markers were examined for the frequencies and types of spontaneous clones expressing the cell markers. In such flies, marked clones can arise via mitotic recombination, mutation, chromosome breakage, nondisjunction or chromosome loss, and clones from these different origins can be distinguished. In addition, meiotic mutants at nine loci have been examined for their effects on sensitivity to killing by UV and X rays.—Mutants at six of the seven recombination-defective loci examined (mei-9, mei-41, c(3)G, mei-W68, mei-S282, mei-352, mei-218) cause mitotic chromosome instability in both sexes, whereas mutants at one locus (mei-218) do not affect mitotic chromosome stability. Thus many of the loci utilized during meiotic recombination also function in the chromosomal economy of mitotic cells.—The chromosome instability produced by mei-41 alleles is the consequence of chromosome breakage, that of mei-9 alleles is primarily due to chromosome breakage and, to a lesser extent, to an elevated frequency of mitotic recombination, whereas no predominant mechanism responsible for the instability caused by c(3)G alleles is discernible. Since these three loci are defective in their responses to mutagen damage, their effects on chromosome stability in nonmutagenized cells are interpreted as resulting from an inability to repair spontaneous lesions. Both mei-W68 and mei-S282 increase mitotic recombination (and in mei-W68, to a lesser extent, chromosome loss) in the abdomen but not the wing. In the abdomen, the primary effect on chromosome stability occurs during the larval period when the abdominal histoblasts are in a nondividing (G2) state.—Mitotic recombination is at or above control levels in the presence of each of the recombination-defective meiotic mutants examined, suggesting that meiotic and mitotic recombination are under separate genetic control in Drosophila.—Of the six mutants examined that are defective in processes required for regular meiotic chromosome segregation, four (l(1)TW-6cs, cand, mei-S332, ord) affect mitotic chromosome behavior. At semi-restrictive temperatures, the cold sensitive lethal l(1)TW-6cs causes very frequent somatic spots, a substantial proportion of which are attributable to nondisjunction or loss. Thus, this locus specifies a function essential for chromosome segregation at mitosis as well as at the first meiotic division in females. The patterns of mitotic effects caused by cand, mei-S332, and ord suggest that they may be leaky alleles at essential loci that specify functions common to meiosis and mitosis. Mutants at the two remaining loci (nod, pal) do not affect mitotic chromosome stability. PMID:17248870

The influence of serotonin on the mitotic rate in the colonic crypt epithelium and in colonic adenocarcinoma in rats.

PubMed

Tutton, P J; Barkla, D H

1978-01-01

1. The mitotic rate in the crypts of Lieberkühn of the descending colon and in dimethylhydrazine-induced adenocarcinomata of the descending colon of rat was measured using a stathmokinetic technique. 2. Intraperitoneal injection of a small dose (10 microgram/kg) of serotonin resulted in an increase in the tumour cell mitotic rate. 3. Blockade of serotonin receptors by 2-bromolysergic acid diethylamide and depletion of tissue serotonin levels following injection of DL-6-fluorotryptophan both result in a decrease in the tumour cell mitotic rate. 4. Treatment with serotonin, 2-bromolysergic acid diethylamide and DL-6-fluorotryptophan were all without effect on the colonic crypt cell mitotic rate.

Cheating or Cheated? Surviving Secondary Exit Exams in a Neoliberal Era

ERIC Educational Resources Information Center

Buckner, Elizabeth; Hodges, Rebecca

2016-01-01

Cheating on exams is a rampant and highly developed practice among youth in the Arab world, often involving elaborate networks, advanced technology and adult authorities. Rather than viewing cheating as mere laziness or immorality, this article interrogates the social meanings of cheating by comparing the practices and discourses of cheating on…

A Novel College Network Resource Management Method using Cloud Computing

NASA Astrophysics Data System (ADS)

Lin, Chen

At present information construction of college mainly has construction of college networks and management information system; there are many problems during the process of information. Cloud computing is development of distributed processing, parallel processing and grid computing, which make data stored on the cloud, make software and services placed in the cloud and build on top of various standards and protocols, you can get it through all kinds of equipments. This article introduces cloud computing and function of cloud computing, then analyzes the exiting problems of college network resource management, the cloud computing technology and methods are applied in the construction of college information sharing platform.

Mitosis can drive cell cannibalism through entosis

PubMed Central

Durgan, Joanne; Tseng, Yun-Yu; Hamann, Jens C; Domart, Marie-Charlotte; Collinson, Lucy; Overholtzer, Michael; Florey, Oliver

2017-01-01

Entosis is a form of epithelial cell cannibalism that is prevalent in human cancer, typically triggered by loss of matrix adhesion. Here, we report an alternative mechanism for entosis in human epithelial cells, driven by mitosis. Mitotic entosis is regulated by Cdc42, which controls mitotic morphology. Cdc42 depletion enhances mitotic deadhesion and rounding, and these biophysical changes, which depend on RhoA activation and are phenocopied by Rap1 inhibition, permit subsequent entosis. Mitotic entosis occurs constitutively in some human cancer cell lines and mitotic index correlates with cell cannibalism in primary human breast tumours. Adherent, wild-type cells can act efficiently as entotic hosts, suggesting that normal epithelia may engulf and kill aberrantly dividing neighbours. Finally, we report that Paclitaxel/taxol promotes mitotic rounding and subsequent entosis, revealing an unconventional activity of this drug. Together, our data uncover an intriguing link between cell division and cannibalism, of significance to both cancer and chemotherapy. DOI: http://dx.doi.org/10.7554/eLife.27134.001 PMID:28693721

A dynamic mode of mitotic bookmarking by transcription factors

PubMed Central

Teves, Sheila S; An, Luye; Hansen, Anders S; Xie, Liangqi; Darzacq, Xavier; Tjian, Robert

2016-01-01

During mitosis, transcription is shut off, chromatin condenses, and most transcription factors (TFs) are reported to be excluded from chromosomes. How do daughter cells re-establish the original transcription program? Recent discoveries that a select set of TFs remain bound on mitotic chromosomes suggest a potential mechanism for maintaining transcriptional programs through the cell cycle termed mitotic bookmarking. Here we report instead that many TFs remain associated with chromosomes in mouse embryonic stem cells, and that the exclusion previously described is largely a fixation artifact. In particular, most TFs we tested are significantly enriched on mitotic chromosomes. Studies with Sox2 reveal that this mitotic interaction is more dynamic than in interphase and is facilitated by both DNA binding and nuclear import. Furthermore, this dynamic mode results from lack of transcriptional activation rather than decreased accessibility of underlying DNA sequences in mitosis. The nature of the cross-linking artifact prompts careful re-examination of the role of TFs in mitotic bookmarking. DOI: http://dx.doi.org/10.7554/eLife.22280.001 PMID:27855781

Loading of PAX3 to Mitotic Chromosomes Is Mediated by Arginine Methylation and Associated with Waardenburg Syndrome*

PubMed Central

Wu, Tsu-Fang; Yao, Ya-Li; Lai, I-Lu; Lai, Chien-Chen; Lin, Pei-Lun; Yang, Wen-Ming

2015-01-01

PAX3 is a transcription factor critical to gene regulation in mammalian development. Mutations in PAX3 are associated with Waardenburg syndrome (WS), but the mechanism of how mutant PAX3 proteins cause WS remains unclear. Here, we found that PAX3 loads on mitotic chromosomes using its homeodomain. PAX3 WS mutants with mutations in homeodomain lose the ability to bind mitotic chromosomes. Moreover, loading of PAX3 on mitotic chromosomes requires arginine methylation, which is regulated by methyltransferase PRMT5 and demethylase JMJD6. Mutant PAX3 proteins that lose mitotic chromosome localization block cell proliferation and normal development of zebrafish. These results reveal the molecular mechanism of PAX3s loading on mitotic chromosomes and the importance of this localization pattern in normal development. Our findings suggest that PAX3 WS mutants interfere with the normal functions of PAX3 in a dominant negative manner, which is important to the understanding of the pathogenesis of Waardenburg syndrome. PMID:26149688

Alzheimer Aβ disrupts the mitotic spindle and directly inhibits mitotic microtubule motors

PubMed Central

Borysov, Sergiy I; Granic, Antoneta; Padmanabhan, Jaya; Walczak, Claire E

2011-01-01

Chromosome mis-segregation and aneuploidy are greatly induced in Alzheimer disease and models thereof by mutant forms of the APP and PS proteins and by their product, the Aβ peptide. Here we employ human somatic cells and Xenopus egg extracts to show that Aβ impairs the assembly and maintenance of the mitotic spindle. Mechanistically, these defects result from Aβ's inhibition of mitotic motor kinesins, including Eg5, KIF4A and MCAK. In vitro studies show that oligomeric Aβ directly inhibits recombinant MCAK by a noncompetitive mechanism. In contrast, inhibition of Eg5 and KIF4A is competitive with respect to both ATP and microtubules, indicating that Aβ interferes with their interactions with the microtubules of the mitotic spindle. Consistently, increased levels of polymerized microtubules or of the microtubule stabilizing protein Tau significantly decrease the inhibitory effect of Aβ on Eg5 and KIF4A. Together, these results indicate that by disrupting the interaction between specific kinesins and microtubules and by exerting a direct inhibitory effect on the motor activity, excess Aβ deregulates the mechanical forces that govern the spindle and thereby leads to the generation of defective mitotic structures. The resulting defect in neurogenesis can account for the over 30% aneuploid/hyperploid, degeneration-prone neurons observed in Alzheimer disease brain. The finding of mitotic motors including Eg5 in mature post-mitotic neurons implies that their inhibition by Aβ may also disrupt neuronal function and plasticity. PMID:21566458

HCdc14A is involved in cell cycle regulation of human brain vascular endothelial cells following injury induced by high glucose, free fatty acids and hypoxia.

PubMed

Su, Jingjing; Zhou, Houguang; Tao, Yinghong; Guo, Zhuangli; Zhang, Shuo; Zhang, Yu; Huang, Yanyan; Tang, Yuping; Hu, Renming; Dong, Qiang

2015-01-01

Cell cycle processes play a vital role in vascular endothelial proliferation and dysfunction. Cell division cycle protein 14 (Cdc14) is an important cell cycle regulatory phosphatase. Previous studies in budding yeast demonstrated that Cdc14 could trigger the inactivation of mitotic cyclin-dependent kinases (Cdks), which are required for mitotic exit and cytokinesis. However, the exact function of human Cdc14 (hCdc14) in cell cycle regulation during vascular diseases is yet to be elucidated. There are two HCdc14 homologs: hCdc14A and hCdc14B. In the current study, we investigated the potential role of hCdc14A in high glucose-, free fatty acids (FFAs)-, and hypoxia-induced injury in cultured human brain vascular endothelial cells (HBVECs). Data revealed that high glucose, FFA, and hypoxia down-regulated hCdc14A expression remarkably, and also affected the expression of other cell cycle-related proteins such as cyclin B, cyclin D, cyclin E, and p53. Furthermore, the combined addition of the three stimuli largely blocked cell cycle progression, decreased cell proliferation, and increased apoptosis. We also determined that hCdc14A was localized mainly to centrosomes during interphase and spindles during mitosis using confocal microscopy, and that it could affect the expression of other cycle-related proteins. More importantly, the overexpression of hCdc14A accelerated cell cycle progression, enhanced cell proliferation, and promoted neoplastic transformation, whereas the knockdown of hCdc14A using small interfering RNA produced the opposite effects. Therefore, these findings provide novel evidence that hCdc14A might be involved in cell cycle regulation in cultured HBVECs during high glucose-, FFA-, and hypoxia-induced injury. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins.

PubMed

Ohta, Shinya; Kimura, Michiko; Takagi, Shunsuke; Toramoto, Iyo; Ishihama, Yasushi

2016-09-02

During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset.

Fe-S cluster coordination of the chromokinesin KIF4A alters its sub-cellular localization during mitosis.

PubMed

Ben-Shimon, Lilach; Paul, Viktoria D; David-Kadoch, Galit; Volpe, Marina; Stümpfig, Martin; Bill, Eckhard; Mühlenhoff, Ulrich; Lill, Roland; Ben-Aroya, Shay

2018-05-30

Fe-S clusters act as co-factors of proteins with diverse functions, e.g. in DNA repair. Down-regulation of the cytosolic iron-sulfur protein assembly (CIA) machinery promotes genomic instability by the inactivation of multiple DNA repair pathways. Furthermore, CIA deficiencies are associated with so far unexplained mitotic defects. Here, we show that CIA2B and MMS19, constituents of the CIA targeting complex involved in facilitating Fe-S cluster insertion into cytosolic and nuclear target proteins, co-localize with components of the mitotic machinery. Down-regulation of CIA2B and MMS19 impairs the mitotic cycle. We identify the chromokinesin KIF4A as a mitotic component involved in these effects. KIF4A binds a Fe-S cluster in vitro through its conserved cysteine-rich domain. We demonstrate in vivo that this domain is required for the mitosis-related KIF4A localization and for the mitotic defects associated with KIF4A knockout. KIF4A is the first identified mitotic component carrying such a post-translational modification. These findings suggest that the lack of Fe-S clusters in KIF4A upon down-regulation of the CIA targeting complex contributes to the mitotic defects. © 2018. Published by The Company of Biologists Ltd.

Immunodetection of phosphohistone H3 as a surrogate of mitotic figure count and clinical outcome in cutaneous melanoma.

PubMed

Tetzlaff, Michael T; Curry, Jonathan L; Ivan, Doina; Wang, Wei-Lien; Torres-Cabala, Carlos A; Bassett, Roland L; Valencia, Karla M; McLemore, Michael S; Ross, Merrick I; Prieto, Victor G

2013-09-01

In the American Joint Committee on Cancer (AJCC)-TNM (2009) staging system, the key prognostic factor in cutaneous melanoma is the depth of dermal invasion (Breslow thickness) with further refinement according to the presence of epidermal ulceration or dermal mitoses. Immunodetection of phosphohistone H3 has been shown to facilitate the identification of mitotic figures in various neoplasms. We selected 120 cases of primary cutaneous melanoma with completely annotated histopathologic parameters and clinical outcomes and performed double immunohistochemical staining for MLANA (Mart-1/Melan-A) and phosphohistone H3. One hundred and thirteen cases were amenable to antiphosphohistone H3 staining from 66 men and 47 women, with mean age of 64 years (9-93), including 61 superficial spreading type, 24 nodular, 6 lentigo maligna, 8 acral lentiginous, and 14 unclassified. The mean Breslow thickness was 2.53 mm (0.20-25), ulceration was present in 25/113 (22%) and the mean mitotic count was 3.2/mm(2) (<1-29/mm(2)). In 27/113 (24%) of the cases, antiphosphohistone H3 failed to highlight mitotic figures anywhere in the tissue (normal or tumor cell), whereas in 86/113 (76%) antiphosphohistone H3 detected at least one mitotic figure. Among the latter, antiphosphohistone H3 did not detect mitotic figures in dermal tumor cells in 37/86 cases (43%), whereas anti-PHH3 identified at least one melanocytic mitotic figure in the other 49/86 cases (57%; range: 1-66/mm(2)). The relationship between phosphohistone H3 and manual mitotic count was statistically significant (Pearson correlation=0.59, P<0.0001). Logistic regression analyses demonstrated an association between the development of subsequent metastatic disease and the following variables: mitotic figures (odds ratio (OR)=5.7; P=0.0001); phosphohistone H3-positive mitotic figures (OR=3.0; P=0.008); Breslow thickness (OR=4.0 per mm; P=0.0002); ulceration (OR=3.94; P=0.008). The application of phosphohistone H3 immunohistochemistry to the description of primary cutaneous melanoma is useful in identifying mitotic figures, improves upon the specificity of this designation when used together with MLANA, and correlates with an increased risk for metastasis in univariate analyses.

G1 arrest and differentiation can occur independently of Rb family function

PubMed Central

Wirt, Stacey E.; Adler, Adam S.; Gebala, Véronique; Weimann, James M.; Schaffer, Bethany E.; Saddic, Louis A.; Viatour, Patrick; Vogel, Hannes; Chang, Howard Y.; Meissner, Alex

2010-01-01

The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9–11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway. PMID:21059851

Theoretical evaluation of a V/STOL fighter model utilizing the PAN AIR code

NASA Technical Reports Server (NTRS)

Howell, G. A.; Bhateley, I. C.

1982-01-01

The PAN AIR computer code was investigated as a tool for predicting closely coupled aerodynamic and propulsive flowfields of arbitrary configurations. The NASA/Ames V/STOL fighter model, a configuration of complex geometry, was analyzed with the PAN AIR code. A successful solution for this configuration was obtained when the nozzle exit was treated as an impermeable surface and no wakes were included around the nozzle exit. When separated flow was simulated from the end of the nacelle, requiring the use of wake networks emanating from the nozzle exit, a number of problems were encountered. A circular body nacelle model was used to investigate various techniques for simulating the exhaust plume in PAN AIR. Several approaches were tested and eliminated because they could not correctly simulate the interference effects. Only one plume modeling technique gave good results. A PAN AIR computation that used a plume shape and inflow velocities obtained from the Navier-Stokes solution for the plume produced results for the effects of power that compared well with experimental data.

AN INDIRECT METHOD TO ASSAY FOR MITOTIC CENTERS IN SAND DOLLAR (DENDRASTER EXCENTRICUS) EGGS

PubMed Central

Went, Hans A.

1966-01-01

It is possible consistently to induce sea urchin and sand dollar eggs to cleave directly from one cell into four cells. This is done by exposing the fertilized eggs to benzimidazole for 20 to 30 min beginning about early metaphase. The mitotic apparatus regresses, the cells do not cleave, and shortly after they are returned to normal sea water an early-prophase-appearing nucleus is present in each cell. Each cell then organizes a tetrapolar tetrahedral mitotic apparatus de novo, instead of transforming a bipolar mitotic apparatus into a tetrapolar figure, and cleaves one-to-four. In another type of experiment, it appears that sand dollar eggs exposed to mercaptoethanol during the first period of mitotic center duplication have only half as many centers by first cleavage metaphase as the normal controls. This is consistent with an earlier report by Mazia et al (1960). Using this same experimental technique, it was demonstrated that benzimidazole, on the contrary, does not interfere with mitotic center duplication in sand dollar eggs. A labeling experiment demonstrated that benzimidazole does not interfere markedly with the normal pattern of incorporation of C14-thymidine into the DNA of sea urchin eggs. The data reported here suggest that judicious treatment of sand dollar eggs (and probably sea urchin eggs, too) with benzimidazole can induce the eggs to cleave into as many cells as there were mitotic centers sometime earlier, for example at early metaphase of the first cleavage division. This provides a very useful tool for studies on the process of mitotic center duplication. PMID:6008198

An indirect method to assay for mitotic centers in sand dollar (Dendraster excentricus) eggs.

PubMed

Went, H A

1966-09-01

It is possible consistently to induce sea urchin and sand dollar eggs to cleave directly from one cell into four cells. This is done by exposing the fertilized eggs to benzimidazole for 20 to 30 min beginning about early metaphase. The mitotic apparatus regresses, the cells do not cleave, and shortly after they are returned to normal sea water an early-prophase-appearing nucleus is present in each cell. Each cell then organizes a tetrapolar tetrahedral mitotic apparatus de novo, instead of transforming a bipolar mitotic apparatus into a tetrapolar figure, and cleaves one-to-four. In another type of experiment, it appears that sand dollar eggs exposed to mercaptoethanol during the first period of mitotic center duplication have only half as many centers by first cleavage metaphase as the normal controls. This is consistent with an earlier report by Mazia et al (1960). Using this same experimental technique, it was demonstrated that benzimidazole, on the contrary, does not interfere with mitotic center duplication in sand dollar eggs. A labeling experiment demonstrated that benzimidazole does not interfere markedly with the normal pattern of incorporation of C(14)-thymidine into the DNA of sea urchin eggs. The data reported here suggest that judicious treatment of sand dollar eggs (and probably sea urchin eggs, too) with benzimidazole can induce the eggs to cleave into as many cells as there were mitotic centers sometime earlier, for example at early metaphase of the first cleavage division. This provides a very useful tool for studies on the process of mitotic center duplication.

Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor, B. Frazier; McNeely, Samuel C.; Miller, Heather L.

2008-07-15

Arsenite, a known mitotic disruptor, causes cell cycle arrest and cell death at anaphase. The mechanism causing mitotic arrest is highly disputed. We compared arsenite to the spindle poisons nocodazole and paclitaxel. Immunofluorescence analysis of {alpha}-tubulin in interphase cells demonstrated that, while nocodazole and paclitaxel disrupt microtubule polymerization through destabilization and hyperpolymerization, respectively, microtubules in arsenite-treated cells remain comparable to untreated cells even at supra-therapeutic concentrations. Immunofluorescence analysis of {alpha}-tubulin in mitotic cells showed spindle formation in arsenite- and paclitaxel-treated cells but not in nocodazole-treated cells. Spindle formation in arsenite-treated cells appeared irregular and multi-polar. {gamma}-tubulin staining showed that cellsmore » treated with nocodazole and therapeutic concentrations of paclitaxel contained two centrosomes. In contrast, most arsenite-treated mitotic cells contained more than two centrosomes, similar to centrosome abnormalities induced by heat shock. Of the three drugs tested, only arsenite treatment increased expression of the inducible isoform of heat shock protein 70 (HSP70i). HSP70 and HSP90 proteins are intimately involved in centrosome regulation and mitotic spindle formation. HSP90 inhibitor 17-DMAG sensitized cells to arsenite treatment and increased arsenite-induced centrosome abnormalities. Combined treatment of 17-DMAG and arsenite resulted in a supra-additive effect on viability, mitotic arrest, and centrosome abnormalities. Thus, arsenite-induced abnormal centrosome amplification and subsequent mitotic arrest is independent of effects on tubulin polymerization and may be due to specific stresses that are protected against by HSP90 and HSP70.« less

The MiAge Calculator: a DNA methylation-based mitotic age calculator of human tissue types.

PubMed

Youn, Ahrim; Wang, Shuang

2018-01-01

Cell division is important in human aging and cancer. The estimation of the number of cell divisions (mitotic age) of a given tissue type in individuals is of great interest as it allows not only the study of biological aging (using a new molecular aging target) but also the stratification of prospective cancer risk. Here, we introduce the MiAge Calculator, a mitotic age calculator based on a novel statistical framework, the MiAge model. MiAge is designed to quantitatively estimate mitotic age (total number of lifetime cell divisions) of a tissue using the stochastic replication errors accumulated in the epigenetic inheritance process during cell divisions. With the MiAge model, the MiAge Calculator was built using the training data of DNA methylation measures of 4,020 tumor and adjacent normal tissue samples from eight TCGA cancer types and was tested using the testing data of DNA methylation measures of 2,221 tumor and adjacent normal tissue samples of five other TCGA cancer types. We showed that within each of the thirteen cancer types studied, the estimated mitotic age is universally accelerated in tumor tissues compared to adjacent normal tissues. Across the thirteen cancer types, we showed that worse cancer survivals are associated with more accelerated mitotic age in tumor tissues. Importantly, we demonstrated the utility of mitotic age by showing that the integration of mitotic age and clinical information leads to improved survival prediction in six out of the thirteen cancer types studied. The MiAge Calculator is available at http://www.columbia.edu/∼sw2206/softwares.htm .

DOE Office of Scientific and Technical Information (OSTI.GOV)

Era, Saho; Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501; Abe, Takuya

Highlights: Black-Right-Pointing-Pointer SENP1 knockout chicken DT40 cells are hypersensitive to spindle poisons. Black-Right-Pointing-Pointer Spindle poison treatment of SENP1{sup -/-} cells leads to increased mitotic slippage. Black-Right-Pointing-Pointer Mitotic slippage in SENP1{sup -/-} cells associates with apoptosis and endoreplication. Black-Right-Pointing-Pointer SENP1 counteracts sister chromatid separation during mitotic arrest. Black-Right-Pointing-Pointer Plk1-mediated cohesion down-regulation is involved in colcemid cytotoxicity. -- Abstract: SUMO conjugation is a reversible posttranslational modification that regulates protein function. SENP1 is one of the six SUMO-specific proteases present in vertebrate cells and its altered expression is observed in several carcinomas. To characterize SENP1 role in genome integrity, we generated Senp1 knockoutmore » chicken DT40 cells. SENP1{sup -/-} cells show normal proliferation, but are sensitive to spindle poisons. This hypersensitivity correlates with increased sister chromatid separation, mitotic slippage, and apoptosis. To test whether the cohesion defect had a causal relationship with the observed mitotic events, we restored the cohesive status of sister chromatids by introducing the TOP2{alpha}{sup +/-} mutation, which leads to increased catenation, or by inhibiting Plk1 and Aurora B kinases that promote cohesin release from chromosomes during prolonged mitotic arrest. Although TOP2{alpha} is SUMOylated during mitosis, the TOP2{alpha}{sup +/-} mutation had no obvious effect. By contrast, inhibition of Plk1 or Aurora B rescued the hypersensitivity of SENP1{sup -/-} cells to colcemid. In conclusion, we identify SENP1 as a novel factor required for mitotic arrest and cohesion maintenance during prolonged mitotic arrest induced by spindle poisons.« less

User experiences with clinical social franchising: qualitative insights from providers and clients in Ghana and Kenya.

PubMed

Sieverding, Maia; Briegleb, Christina; Montagu, Dominic

2015-02-01

Clinical social franchising is a rapidly growing delivery model in private healthcare markets in low- and middle-income countries. Despite this growth, little is known about providers' perceptions of the benefits and challenges of social franchising or clients' reasons for choosing franchised facilities over other healthcare options. We examine these questions in the context of three social franchise networks in Ghana and Kenya. We conducted in-depth interviews with a purposive sample of providers from the BlueStar Ghana, and Amua and Tunza networks in Kenya. We also conducted qualitative exit interviews with female clients who were leaving franchised facilities after a visit for a reproductive or child health reason. The total sample consists of 47 providers and 47 clients across the three networks. Providers perceived the main benefits of participation in a social franchise network to be training opportunities and access to a consistent supply of low-cost family planning commodities; few providers mentioned branding as a benefit of participation. Although most providers said that client flows for franchised services increased after joining the network, they did not associate this with improved finances for their facility. Clients overwhelmingly cited the quality of the client-provider relationship as their main motivation for attending the franchise facility. Recognition of the franchise brand was low among clients who were exiting a franchised facility. The most important benefit of social franchise programs to both providers and their clients may have more to do with training on business practices, patient counseling and customer service, than with subsidies, technical input, branding or clinical support. This finding may lead to a reconsideration of how franchise programs interact with both their member clinics and the larger health-seeking communities they serve.

Mechanical control of mitotic progression in single animal cells

PubMed Central

Cattin, Cedric J.; Düggelin, Marcel; Martinez-Martin, David; Gerber, Christoph; Müller, Daniel J.; Stewart, Martin P.

2015-01-01

Despite the importance of mitotic cell rounding in tissue development and cell proliferation, there remains a paucity of approaches to investigate the mechanical robustness of cell rounding. Here we introduce ion beam-sculpted microcantilevers that enable precise force-feedback–controlled confinement of single cells while characterizing their progression through mitosis. We identify three force regimes according to the cell response: small forces (∼5 nN) that accelerate mitotic progression, intermediate forces where cells resist confinement (50–100 nN), and yield forces (>100 nN) where a significant decline in cell height impinges on microtubule spindle function, thereby inhibiting mitotic progression. Yield forces are coincident with a nonlinear drop in cell height potentiated by persistent blebbing and loss of cortical F-actin homogeneity. Our results suggest that a buildup of actomyosin-dependent cortical tension and intracellular pressure precedes mechanical failure, or herniation, of the cell cortex at the yield force. Thus, we reveal how the mechanical properties of mitotic cells and their response to external forces are linked to mitotic progression under conditions of mechanical confinement. PMID:26305930

Proteasome inhibition enhances the efficacy of volasertib-induced mitotic arrest in AML in vitro and prolongs survival in vivo.

PubMed

Schnerch, Dominik; Schüler, Julia; Follo, Marie; Felthaus, Julia; Wider, Dagmar; Klingner, Kathrin; Greil, Christine; Duyster, Justus; Engelhardt, Monika; Wäsch, Ralph

2017-03-28

Elderly and frail patients, diagnosed with acute myeloid leukemia (AML) and ineligible to undergo intensive treatment, have a dismal prognosis. The small molecule inhibitor volasertib induces a mitotic block via inhibition of polo-like kinase 1 and has shown remarkable anti-leukemic activity when combined with low-dose cytarabine. We have demonstrated that AML cells are highly vulnerable to cell death in mitosis yet manage to escape a mitotic block through mitotic slippage by sustained proteasome-dependent slow degradation of cyclin B. Therefore, we tested whether interfering with mitotic slippage through proteasome inhibition arrests and kills AML cells more efficiently during mitosis. We show that therapeutic doses of bortezomib block the slow degradation of cyclin B during a volasertib-induced mitotic arrest in AML cell lines and patient-derived primary AML cells. In a xenotransplant mouse model of human AML, mice receiving volasertib in combination with bortezomib showed superior disease control compared to mice receiving volasertib alone, highlighting the potential therapeutic impact of this drug combination.

A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells

PubMed Central

Hehnly, Heidi; Canton, David; Bucko, Paula; Langeberg, Lorene K; Ogier, Leah; Gelman, Irwin; Santana, L Fernando; Wordeman, Linda; Scott, John D

2015-01-01

Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin−/− mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division. DOI: http://dx.doi.org/10.7554/eLife.09384.001 PMID:26406118

Loading of PAX3 to Mitotic Chromosomes Is Mediated by Arginine Methylation and Associated with Waardenburg Syndrome.

PubMed

Wu, Tsu-Fang; Yao, Ya-Li; Lai, I-Lu; Lai, Chien-Chen; Lin, Pei-Lun; Yang, Wen-Ming

2015-08-14

PAX3 is a transcription factor critical to gene regulation in mammalian development. Mutations in PAX3 are associated with Waardenburg syndrome (WS), but the mechanism of how mutant PAX3 proteins cause WS remains unclear. Here, we found that PAX3 loads on mitotic chromosomes using its homeodomain. PAX3 WS mutants with mutations in homeodomain lose the ability to bind mitotic chromosomes. Moreover, loading of PAX3 on mitotic chromosomes requires arginine methylation, which is regulated by methyltransferase PRMT5 and demethylase JMJD6. Mutant PAX3 proteins that lose mitotic chromosome localization block cell proliferation and normal development of zebrafish. These results reveal the molecular mechanism of PAX3s loading on mitotic chromosomes and the importance of this localization pattern in normal development. Our findings suggest that PAX3 WS mutants interfere with the normal functions of PAX3 in a dominant negative manner, which is important to the understanding of the pathogenesis of Waardenburg syndrome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

[Mechanism of mutant induction in the ade2 gene of diploid Saccharomyces cerevisiae yeasts by ultraviolet rays].

PubMed

Gordenin, D A; Inge-Vechtomov, S G

1981-01-01

Ultraviolet light (UV) at 3000 ergs/mm-2 induces ade2 mutants with a frequency about 10(-4) in wild-type haploid strains of yeast and about 10(-5) in diploid wild-type strains. UV irradiation effectively induced mitotic segregation of ade2 in the heterozygous diploid (the frequency of segregation is 6%). Interallelic complementation and localization spectra are similar for mutations induced both in haploids and diploids. The occurrence of ade2 mutants in diploids correlated with mitotic segregation of the marker his8 which is situated in the same arm of XY chromosome as ade2 is, distal to the centromere. Our data about the frequency of ade2 mutants in diploids and haploids, the frequency of ade2 mitotic segregation, mitotic segregation of other markers and genetic characteristics of ade2 mutations confirm the suggestion that the major mechanism of diploid ade2 mutants appearance is mutation in one of the two ADE2 alleles and consequent mitotic homozygotisation of mutation as a result of mitotic crossingover between ade2 and the centromere.

Regulation of spindle integrity and mitotic fidelity by BCCIP

PubMed Central

Huhn, S C; Liu, J; Ye, C; Lu, H; Jiang, X; Feng, X; Ganesan, S; White, E; Shen, Z

2017-01-01

Centrosomes together with the mitotic spindle ensure the faithful distribution of chromosomes between daughter cells, and spindle orientation is a major determinant of cell fate during tissue regeneration. Spindle defects are not only an impetus of chromosome instability but are also a cause of developmental disorders involving defective asymmetric cell division. In this work, we demonstrate BCCIP, especially BCCIPα, as a previously unidentified component of the mitotic spindle pole and the centrosome. We demonstrate that BCCIP localizes proximal to the mother centriole and participates in microtubule organization and then redistributes to the spindle pole to ensure faithful spindle architecture. We find that BCCIP depletion leads to morphological defects, disoriented mitotic spindles, chromosome congression defects and delayed mitotic progression. Our study identifies BCCIP as a novel factor critical for microtubule regulation and explicates a mechanism utilized by BCCIP in tumor suppression. PMID:28394342

Comparative analysis of mitotic aberrations induced by diethyl sulphate (DES) and sodium azide (SA) in Vicia faba L. (Fabaceae).

PubMed

Bhat, Tariq Ahmad; Sharma, Monika; Anis, M

2007-03-01

The present investigation provides a comparative account of different concentrations (0.01, 0.02, 0.03, 0.04, 0.05 and 0.06%) of diethylsulphate (DES) and Sodium Azide (SA) on mitotic aberrations, seed germination, seedling survival, plant height and mitotic index in Vicia faba L. variety major. The control plants were normal while as treated ones showed significant alterations. The mutagens caused dose dependent decrease in seed germination, seedling survival, plant height and mitotic index. All the parameters were found negatively affected and were positively correlated with mutagenic concentrations. The cytological study revealed various types of mitotic aberrations, among them the dominant were fragments, stickiness, precocious separation, c-metaphase, ring chromosomes, unequal separation, laggards, bridges, micronuclei, disturbed anaphase etc. Stickiness and fragments were more frequent as compared to other types.

Capacity-constrained traffic assignment in networks with residual queues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lam, W.H.K.; Zhang, Y.

2000-04-01

This paper proposes a capacity-constrained traffic assignment model for strategic transport planning in which the steady-state user equilibrium principle is extended for road networks with residual queues. Therefore, the road-exit capacity and the queuing effects can be incorporated into the strategic transport model for traffic forecasting. The proposed model is applicable to the congested network particularly when the traffic demands exceeds the capacity of the network during the peak period. An efficient solution method is proposed for solving the steady-state traffic assignment problem with residual queues. Then a simple numerical example is employed to demonstrate the application of the proposedmore » model and solution method, while an example of a medium-sized arterial highway network in Sioux Falls, South Dakota, is used to test the applicability of the proposed solution to real problems.« less

Sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-Golgi network of AtT20 cells.

PubMed

Tooze, J; Tooze, S A; Fuller, S D

1987-09-01

Murine hepatitis virus (strain A59), (MHV-A59) is a coronavirus that buds into pre-Golgi compartments and then exploits the exocytic pathway of the host cell to reach the exterior. The fibroblastic cells in which replication of this virus is usually studied have only a constitutive exocytic pathway that the virus uses. MHV-A59 also infects, albeit inefficiently, AtT20 cells, murine pituitary tumor cells with a regulated as well as a constitutive exocytic pathway. Here we examine AtT20 cells at early times after the infection, when the Golgi apparatus retains its morphological and biochemical integrity. We observe that progeny coronavirus and secretory protein destined for the secretory granules of the regulated exocytic pathway traverse the same Golgi stacks and accumulate in the trans-Golgi network. Their pathways diverge at this site, the condensed secretory proteins including the ACTH going to the secretory granules and the coronavirus to post-Golgi transport vesicles devoid of ACTH. On very rare occasions there is missorting such that aggregates of condensed secretory proteins and viruses occur together in post-Golgi vesicles. We conclude that the constitutive and regulated exocytic pathways, identified respectively by the progeny virions and the secretory protein ACTH, diverge at the exit from the trans-Golgi network.

Candidate genes and pathogenesis investigation for sepsis-related acute respiratory distress syndrome based on gene expression profile.

PubMed

Wang, Min; Yan, Jingjun; He, Xingxing; Zhong, Qiang; Zhan, Chengye; Li, Shusheng

2016-04-18

Acute respiratory distress syndrome (ARDS) is a potentially devastating form of acute inflammatory lung injury as well as a major cause of acute respiratory failure. Although researchers have made significant progresses in elucidating the pathophysiology of this complex syndrome over the years, the absence of a universal detail disease mechanism up until now has led to a series of practical problems for a definitive treatment. This study aimed to predict some genes or pathways associated with sepsis-related ARDS based on a public microarray dataset and to further explore the molecular mechanism of ARDS. A total of 122 up-regulated DEGs and 91 down-regulated differentially expressed genes (DEGs) were obtained. The up- and down-regulated DEGs were mainly involved in functions like mitotic cell cycle and pathway like cell cycle. Protein-protein interaction network of ARDS analysis revealed 20 hub genes including cyclin B1 (CCNB1), cyclin B2 (CCNB2) and topoisomerase II alpha (TOP2A). A total of seven transcription factors including forkhead box protein M1 (FOXM1) and 30 target genes were revealed in the transcription factor-target gene regulation network. Furthermore, co-cited genes including CCNB2-CCNB1 were revealed in literature mining for the relations ARDS related genes. Pathways like mitotic cell cycle were closed related with the development of ARDS. Genes including CCNB1, CCNB2 and TOP2A, as well as transcription factors like FOXM1 might be used as the novel gene therapy targets for sepsis related ARDS.

Regulation of mitosis by the NIMA kinase involves TINA and its newly discovered partner, An-WDR8, at spindle pole bodies

PubMed Central

Shen, Kuo-Fang; Osmani, Stephen A.

2013-01-01

The NIMA kinase is required for mitotic nuclear pore complex disassembly and potentially controls other mitotic-specific events. To investigate this possibility, we imaged NIMA–green fluorescent protein (GFP) using four-dimensional spinning disk confocal microscopy. At mitosis NIMA-GFP locates to spindle pole bodies (SPBs), which contain Cdk1/cyclin B, followed by Aurora, TINA, and the BimC kinesin. NIMA promotes NPC disassembly in a spatially regulated manner starting near SPBs. NIMA is also required for TINA, a NIMA-interacting protein, to locate to SPBs during initiation of mitosis, and TINA is then necessary for locating NIMA back to SPBs during mitotic progression. To help expand the NIMA-TINA pathway, we affinity purified TINA and found it to uniquely copurify with An-WDR8, a WD40-domain protein conserved from humans to plants. Like TINA, An-WDR8 accumulates within nuclei during G2 but disperses from nuclei before locating to mitotic SPBs. Without An-WDR8, TINA levels are greatly reduced, whereas TINA is necessary for mitotic targeting of An-WDR8. Finally, we show that TINA is required to anchor mitotic microtubules to SPBs and, in combination with An-WDR8, for successful mitosis. The findings provide new insights into SPB targeting and indicate that the mitotic microtubule-anchoring system at SPBs involves WDR8 in complex with TINA. PMID:24152731

Effect of HIV-1 Tat on the formation of the mitotic spindle by interaction with ribosomal protein S3.

PubMed

Kim, Jiyoung; Kim, Yeon-Soo

2018-06-06

Human immunodeficiency virus type 1 (HIV-1) Tat, an important regulator of viral transcription, interacts with diverse cellular proteins and promotes or inhibits cell proliferation. Here, we show that ribosomal protein S3 (RPS3) plays an important role in mitosis through an interaction with α-tubulin and that Tat binds to and inhibits the localization of RPS3 in the mitotic spindle during mitosis. RPS3 colocalized with α-tubulin around chromosomes in the mitotic spindle. Depletion of RPS3 promoted α-tubulin assembly, while overexpression of RPS3 impaired α-tubulin assembly. Depletion of RPS3 resulted in aberrant mitotic spindle formation, segregation failure, and defective abscission. Moreover, ectopic expression of RPS3 rescued the cell proliferation defect in RPS3-knockdown cells. HIV-1 Tat interacted with RPS3 through its basic domain and increased the level of RPS3 in the nucleus. Expression of Tat caused defects in mitotic spindle formation and chromosome assembly in mitosis. Moreover, the localization of RPS3 in the mitotic spindle was disrupted when HIV-1 Tat was expressed in HeLa and Jurkat cells. These results suggest that Tat inhibits cell proliferation via an interaction with RPS3 and thereby disrupts mitotic spindle formation during HIV-1 infection. These results might provide insight into the mechanism underlying lymphocyte pathogenesis during HIV-1 infection.

PIASy Mediates SUMO-2/3 Conjugation of Poly(ADP-ribose) Polymerase 1 (PARP1) on Mitotic Chromosomes*

PubMed Central

Ryu, Hyunju; Al-Ani, Gada; Deckert, Katelyn; Kirkpatrick, Donald; Gygi, Steven P.; Dasso, Mary; Azuma, Yoshiaki

2010-01-01

PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins. PMID:20228053

Energy star compliant voice over internet protocol (VoIP) telecommunications network including energy star compliant VoIP devices

DOEpatents

Kouchri, Farrokh Mohammadzadeh

2012-11-06

A Voice over Internet Protocol (VoIP) communications system, a method of managing a communications network in such a system and a program product therefore. The system/network includes an ENERGY STAR (E-star) aware softswitch and E-star compliant communications devices at system endpoints. The E-star aware softswitch allows E-star compliant communications devices to enter and remain in power saving mode. The E-star aware softswitch spools messages and forwards only selected messages (e.g., calls) to the devices in power saving mode. When the E-star compliant communications devices exit power saving mode, the E-star aware softswitch forwards spooled messages.

Translocation of the papillomavirus L2/vDNA complex across the limiting membrane requires the onset of mitosis.

PubMed

Calton, Christine M; Bronnimann, Matthew P; Manson, Ariana R; Li, Shuaizhi; Chapman, Janice A; Suarez-Berumen, Marcela; Williamson, Tatum R; Molugu, Sudheer K; Bernal, Ricardo A; Campos, Samuel K

2017-05-01

The human papillomavirus type 16 (HPV16) L2 protein acts as a chaperone to ensure that the viral genome (vDNA) traffics from endosomes to the trans-Golgi network (TGN) and eventually the nucleus, where HPV replication occurs. En route to the nucleus, the L2/vDNA complex must translocate across limiting intracellular membranes. The details of this critical process remain poorly characterized. We have developed a system based on subcellular compartmentalization of the enzyme BirA and its cognate substrate to detect membrane translocation of L2-BirA from incoming virions. We find that L2 translocation requires transport to the TGN and is strictly dependent on entry into mitosis, coinciding with mitotic entry in synchronized cells. Cell cycle arrest causes retention of L2/vDNA at the TGN; only release and progression past G2/M enables translocation across the limiting membrane and subsequent infection. Microscopy of EdU-labeled vDNA reveals a rapid and dramatic shift in vDNA localization during early mitosis. At late G2/early prophase vDNA egresses from the TGN to a pericentriolar location, accumulating there through prometaphase where it begins to associate with condensed chromosomes. By metaphase and throughout anaphase the vDNA is seen bound to the mitotic chromosomes, ensuring distribution into both daughter nuclei. Mutations in a newly defined chromatin binding region of L2 potently blocked translocation, suggesting that translocation is dependent on chromatin binding during prometaphase. This represents the first time a virus has been shown to functionally couple the penetration of limiting membranes to cellular mitosis, explaining in part the tropism of HPV for mitotic basal keratinocytes.

Curcumin-induced mitotic arrest is characterized by spindle abnormalities, defects in chromosomal congression and DNA damage

PubMed Central

Manson, Margaret M.

2013-01-01

The chemopreventive agent curcumin has anti-proliferative effects in many tumour types, but characterization of cell cycle arrest, particularly with physiologically relevant concentrations, is still incomplete. Following oral ingestion, the highest concentrations of curcumin are achievable in the gut. Although it has been established that curcumin induces arrest at the G2/M stage of the cell cycle in colorectal cancer lines, it is not clear whether arrest occurs at the G2/M transition or in mitosis. To elucidate the precise stage of arrest, we performed a direct comparison of the levels of curcumin-induced G2/M boundary and mitotic arrest in eight colorectal cancer lines (Caco-2, DLD-1, HCA-7, HCT116p53+/+, HCT116p53–/–, HCT116p21–/–, HT-29 and SW480). Flow cytometry confirmed that these lines underwent G2/M arrest following treatment for 12h with clinically relevant concentrations of curcumin (5–10 μM). In all eight lines, the majority of this arrest occurred at the G2/M transition, with a proportion of cells arresting in mitosis. Examination of the mitotic index using fluorescence microscopy showed that the HCT116 and Caco-2 lines exhibited the highest levels of curcumin-induced mitotic arrest. Image analysis revealed impaired mitotic progression in all lines, exemplified by mitotic spindle abnormalities and defects in chromosomal congression. Pre-treatment with inhibitors of the DNA damage signalling pathway abrogated curcumin-induced mitotic arrest, but had little effect at the G2/M boundary. Moreover, pH2A.X staining seen in mitotic, but not interphase, cells suggests that this aberrant mitosis results in DNA damage. PMID:23125222

Automation of Some Operations of a Wind Tunnel Using Artificial Neural Networks

NASA Technical Reports Server (NTRS)

Decker, Arthur J.; Buggele, Alvin E.

1996-01-01

Artificial neural networks were used successfully to sequence operations in a small, recently modernized, supersonic wind tunnel at NASA-Lewis Research Center. The neural nets generated correct estimates of shadowgraph patterns, pressure sensor readings and mach numbers for conditions occurring shortly after startup and extending to fully developed flow. Artificial neural networks were trained and tested for estimating: sensor readings from shadowgraph patterns, shadowgraph patterns from shadowgraph patterns and sensor readings from sensor readings. The 3.81 by 10 in. (0.0968 by 0.254 m) tunnel was operated with its mach 2.0 nozzle, and shadowgraph was recorded near the nozzle exit. These results support the thesis that artificial neural networks can be combined with current workstation technology to automate wind tunnel operations.

Integrity of the Pericentriolar Material Is Essential for Maintaining Centriole Association during M Phase

PubMed Central

Rhee, Kunsoo

2015-01-01

A procentriole is assembled next to the mother centriole during S phase and remains associated until M phase. After functioning as a spindle pole during mitosis, the mother centriole and procentriole are separated at the end of mitosis. A close association of the centriole pair is regarded as an intrinsic block to the centriole reduplication. Therefore, deregulation of this process may cause a problem in the centriole number control, resulting in increased genomic instability. Despite its importance for faithful centriole duplication, the mechanism of centriole separation is not fully understood yet. Here, we report that centriole pairs are prematurely separated in cells whose cell cycle is arrested at M phase by STLC. Dispersal of the pericentriolar material (PCM) was accompanied. This phenomenon was independent of the separase activity but needed the PLK1 activity. Nocodazole effectively inhibited centriole scattering in STLC-treated cells, possibly by reducing the microtubule pulling force around centrosomes. Inhibition of PLK1 also reduced the premature separation of centrioles and the PCM dispersal as well. These results revealed the importance of PCM integrity in centriole association. Therefore, we propose that PCM disassembly is one of the driving forces for centriole separation during mitotic exit. PMID:26407333

A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts.

PubMed

Sambasivan, Ramkumar; Pavlath, Grace K; Dhawan, Jyotsna

2008-03-01

Cellular quiescence is characterized not only by reduced mitotic and metabolic activity but also by altered gene expression. Growing evidence suggests that quiescence is not merely a basal state but is regulated by active mechanisms. To understand the molecular programme that governs reversible cell cycle exit, we focused on quiescence-related gene expression in a culture model of myogenic cell arrest and activation. Here we report the identification of quiescence-induced genes using a gene-trap strategy. Using a retroviral vector, we generated a library of gene traps in C2C12 myoblasts that were screened for arrest-induced insertions by live cell sorting (FACS-gal). Several independent gene- trap lines revealed arrest-dependent induction of betagal activity, confirming the efficacy of the FACS screen. The locus of integration was identified in 15 lines. In three lines,insertion occurred in genes previously implicated in the control of quiescence, i.e. EMSY - a BRCA2--interacting protein, p8/com1 - a p300HAT -- binding protein and MLL5 - a SET domain protein. Our results demonstrate that expression of chromatin modulatory genes is induced in G0, providing support to the notion that this reversibly arrested state is actively regulated.

Molecular cloning, developmental expression, and cellular localization of the 70-kDa RPA-1 subunit of Drosophila melanogaster.

PubMed

Perdigão, J; Logarinho, E; Avides, M C; Sunkel, C E

1999-12-01

Replication protein A (RPA) is a highly conserved multifunctional heterotrimeric complex, involved in DNA replication, repair, recombination, and possibly transcription. Here, we report the cloning of the gene that codes for the largest subunit of the Drosophila melanogaster RPA homolog, dmRPA70. In situ hybridization showed that dmRPA70 RNA is present in developing embryos during the first 16 cycles. After this point, dm-RPA70 expression is downregulated in cells that enter a G1 phase and exit the mitotic cycle, becoming restricted to brief bursts of accumulation from late G1 to S phase. This pattern of regulated expression is also observed in the developing eye imaginal disc. In addition, we have shown that the presence of cyclin E is necessary and sufficient to drive the expression of dmRPA70 in embryonic cells arrested in G1 but is not required in tissues undergoing endoreduplication. Immunolocalization showed that in early developing embryos, the dmRPA70 protein associates with chromatin from the end of mitosis until the beginning of the next prophase in a dynamic speckled pattern that is strongly suggestive of its association with replication foci.

Role of Inn1 and its interactions with Hof1 and Cyk3 in promoting cleavage furrow and septum formation in S. cerevisiae

PubMed Central

Nishihama, Ryuichi; Schreiter, Jennifer H.; Onishi, Masayuki; Vallen, Elizabeth A.; Hanna, Julia; Moravcevic, Katarina; Lippincott, Margaret F.; Han, Haesun; Lemmon, Mark A.; Pringle, John R.

2009-01-01

Cytokinesis requires coordination of actomyosin ring (AMR) contraction with rearrangements of the plasma membrane and extracellular matrix. In Saccharomyces cerevisiae, new membrane, the chitin synthase Chs2 (which forms the primary septum [PS]), and the protein Inn1 are all delivered to the division site upon mitotic exit even when the AMR is absent. Inn1 is essential for PS formation but not for Chs2 localization. The Inn1 C-terminal region is necessary for localization, and distinct PXXP motifs in this region mediate functionally important interactions with SH3 domains in the cytokinesis proteins Hof1 (an F-BAR protein) and Cyk3 (whose overexpression can restore PS formation in inn1Δ cells). The Inn1 N terminus resembles C2 domains but does not appear to bind phospholipids; nonetheless, when overexpressed or fused to Hof1, it can provide Inn1 function even in the absence of the AMR. Thus, Inn1 and Cyk3 appear to cooperate in activating Chs2 for PS formation, which allows coordination of AMR contraction with ingression of the cleavage furrow. PMID:19528296

Cyclin B in mouse oocytes and embryos: importance for human reproduction and aneuploidy.

PubMed

Polański, Zbigniew; Homer, Hayden; Kubiak, Jacek Z

2012-01-01

Oocyte maturation and early embryo development require precise coordination between cell cycle progression and the developmental programme. Cyclin B plays a major role in this process: its accumulation and degradation is critical for driving the cell cycle through activation and inactivation of the major cell cycle kinase, CDK1. CDK1 activation is required for M-phase entry whereas its inactivation leads to exit from M-phase. The tempo of oocyte meiotic and embryonic mitotic divisions is set by the rate of cyclin B accumulation and the timing of its destruction. By controlling when cyclin B destruction is triggered and by co-ordinating this with the completion of chromosome alignment, the spindle assembly checkpoint (SAC) is a critical quality control system important for averting aneuploidy and for building in the flexibility required to better integrate cell cycle progression with development. In this review we focus on cyclin B metabolism in mouse oocytes and embryos and illustrate how the cell cycle-powered clock (in fact cyclin B-powered clock) controls oocyte maturation and early embryo development, thereby providing important insight into human reproduction and potential causes of Down syndrome.

Regulation of Plant Cellular and Organismal Development by SUMO.

PubMed

Elrouby, Nabil

2017-01-01

This chapter clearly demonstrates the breadth and spectrum of the processes that SUMO regulates during plant development. The gross phenotypes observed in mutants of the SUMO conjugation and deconjugation enzymes reflect these essential roles, and detailed analyses of these mutants under different growth conditions revealed roles in biotic and abiotic stress responses, phosphate starvation, nitrate and sulphur metabolism, freezing and drought tolerance and response to excess copper. SUMO functions also intersect with those regulated by several hormones such as salicylic acid , abscisic acid , gibberellins and auxin, and detailed studies provide mechanistic clues of how sumoylation may regulate these processes. The regulation of COP1 and PhyB functions by sumoylation provides very strong evidence that SUMO is heavily involved in the regulation of light signaling in plants. At the cellular and subcellular levels, SUMO regulates meristem architecture, the switch from the mitotic cycle into the endocycle, meiosis, centromere decondensation and exit from mitosis, transcriptional control, and release from transcriptional silencing. Most of these advances in our understanding of SUMO functions during plant development emerged over the past 6-7 years, and they may only predict a prominent rise of SUMO as a major regulator of eukaryotic cellular and organismal growth and development.

Effects of caffeine on radiation-induced phenomena associated with cell- cycle traverse of mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walters, R.A.; Gurley, L.R.; Tobby, R.A.

1974-02-01

Caffeine induced a state of G/sub 1/ arrest when added to an exponentially growing culture of Chinese hamster cells (line CHO). In addition to its effect on cell-cycle traverse, caffeine ameliorated a number of the responses of cells to ionizing radiation. The duration of the division delay period following x-irradiation of caffeine-treated cells was reduced, and the magnitude of reduction was dependent on caffeine concentration. Cells irradiated during the DNA synthetic phase in the presence of caffeine were delayed less in their exit from S, measured autoradiographically, and the radiation-induced reduction of radioactive thymidine incorporation into DNA was lessened. Cellsmore » synchronized by isoleucine deprivation, while being generally less sensitive to the effects of ionizing radiation than mitotically synchronized cells, were equally responsive to the effects of caffeine. The x-rayinduced reduction of phosphorylation of lysine-rich histone F1 was less in caffeine-treated cells than in untreated cells. Finally, survival after irradiation was only slightly reduced in caffeinetreated cells. A possible role of cyclic AMP in cell-cycle traverse of irradiated cells is discussed. (auth)« less

Mitotic Recombination and Genetic Changes in Saccharomyces cerevisiae during Wine Fermentation

PubMed Central

Puig, Sergi; Querol, Amparo; Barrio, Eladio; Pérez-Ortín, José E.

2000-01-01

Natural strains of Saccharomyces cerevisiae are prototrophic homothallic yeasts that sporulate poorly, are often heterozygous, and may be aneuploid. This genomic constitution may confer selective advantages in some environments. Different mechanisms of recombination, such as meiosis or mitotic rearrangement of chromosomes, have been proposed for wine strains. We studied the stability of the URA3 locus of a URA3/ura3 wine yeast in consecutive grape must fermentations. ura3/ura3 homozygotes were detected at a rate of 1 × 10−5 to 3 × 10−5 per generation, and mitotic rearrangements for chromosomes VIII and XII appeared after 30 mitotic divisions. We used the karyotype as a meiotic marker and determined that sporulation was not involved in this process. Thus, we propose a hypothesis for the genome changes in wine yeasts during vinification. This putative mechanism involves mitotic recombination between homologous sequences and does not necessarily imply meiosis. PMID:10788381

Calibrated mitotic oscillator drives motile ciliogenesis.

PubMed

Al Jord, Adel; Shihavuddin, Asm; Servignat d'Aout, Raphaël; Faucourt, Marion; Genovesio, Auguste; Karaiskou, Anthi; Sobczak-Thépot, Joëlle; Spassky, Nathalie; Meunier, Alice

2017-11-10

Cell division and differentiation depend on massive and rapid organelle remodeling. The mitotic oscillator, centered on the cyclin-dependent kinase 1-anaphase-promoting complex/cyclosome (CDK1-APC/C) axis, spatiotemporally coordinates this reorganization in dividing cells. Here we discovered that nondividing cells could also implement this mitotic clocklike regulatory circuit to orchestrate subcellular reorganization associated with differentiation. We probed centriole amplification in differentiating mouse-brain multiciliated cells. These postmitotic progenitors fine-tuned mitotic oscillator activity to drive the orderly progression of centriole production, maturation, and motile ciliation while avoiding the mitosis commitment threshold. Insufficient CDK1 activity hindered differentiation, whereas excessive activity accelerated differentiation yet drove postmitotic progenitors into mitosis. Thus, postmitotic cells can redeploy and calibrate the mitotic oscillator to uncouple cytoplasmic from nuclear dynamics for organelle remodeling associated with differentiation. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

The bipolar assembly domain of the mitotic motor kinesin-5

PubMed Central

Acar, Seyda; Carlson, David B.; Budamagunta, Madhu S.; Yarov-Yarovoy, Vladimir; Correia, John J.; Niñonuevo, Milady R.; Jia, Weitao; Tao, Li; Leary, Julie A.; Voss, John C.; Evans, James E.; Scholey, Jonathan M.

2013-01-01

An outstanding unresolved question is how does the mitotic spindle utilize microtubules and mitotic motors to coordinate accurate chromosome segregation during mitosis? This process depends upon the mitotic motor, kinesin-5, whose unique bipolar architecture, with pairs of motor domains lying at opposite ends of a central rod, allows it to crosslink microtubules within the mitotic spindle and to coordinate their relative sliding during spindle assembly, maintenance and elongation. The structural basis of kinesin-5’s bipolarity is, however, unknown, as protein asymmetry has so far precluded its crystallization. Here we use electron microscopy of single molecules of kinesin-5 and its subfragments, combined with hydrodynamic analysis plus mass spectrometry, circular dichroism and site-directed spin label electron paramagnetic resonance spectroscopy, to show how a staggered antiparallel coiled-coil ‘BASS’ (bipolar assembly) domain directs the assembly of four kinesin-5 polypeptides into bipolar minifilaments. PMID:23299893

Screening for Key Pathways Associated with the Development of Osteoporosis by Bioinformatics Analysis

PubMed Central

Liu, Yanqing; Wang, Yueqiu; Zhang, Yanxia; Liu, Zhiyong; Xiang, Hongfei; Peng, Xianbo

2017-01-01

Objectives. We aimed to find the key pathways associated with the development of osteoporosis. Methods. We downloaded expression profile data of GSE35959 and analyzed the differentially expressed genes (DEGs) in 3 comparison groups (old_op versus middle, old_op versus old, and old_op versus senescent). KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses were carried out. Besides, Venn diagram analysis and gene functional interaction (FI) network analysis were performed. Results. Totally 520 DEGs, 966 DEGs, and 709 DEGs were obtained in old_op versus middle, old_op versus old, and old_op versus senescent groups, respectively. Lysosome pathway was the significantly enriched pathways enriched by intersection genes. The pathways enriched by subnetwork modules suggested that mitotic metaphase and anaphase and signaling by Rho GTPases in module 1 had more proteins from module. Conclusions. Lysosome pathway, mitotic metaphase and anaphase, and signaling by Rho GTPases may be involved in the development of osteoporosis. Furthermore, Rho GTPases may regulate the balance of bone resorption and bone formation via controlling osteoclast and osteoblast. These 3 pathways may be regarded as the treatment targets for osteoporosis. PMID:28466021

Caspase 2 in mitotic catastrophe: The terminator of aneuploid and tetraploid cells.

PubMed

Vitale, Ilio; Manic, Gwenola; Castedo, Maria; Kroemer, Guido

2017-01-01

Mitotic catastrophe is an oncosuppressive mechanism that targets cells experiencing defective mitoses via the activation of specific cell cycle checkpoints, regulated cell death pathways and/or cell senescence. This prevents the accumulation of karyotypic aberrations, which otherwise may drive oncogenesis and tumor progression. Here, we summarize experimental evidence confirming the role of caspase 2 (CASP2) as the main executor of mitotic catastrophe, and we discuss the signals that activate CASP2 in the presence of mitotic aberrations. In addition, we summarize the main p53-dependent and -independent effector pathways through which CASP2 limits chromosomal instability and non-diploidy, hence mediating robust oncosuppressive functions.

Phospho-Regulation of DDA3 Function in Mitosis

PubMed Central

Jang, Chang-Young; Coppinger, Judith A.; Yates, John R.; Fang, Guowei

2010-01-01

DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. Through a mass spectrometry analysis, we found that DDA3 is phosphorylated on Ser225 during mitosis. Phosphorylation of this residue is important for the mitotic function of DDA3, as the phospho-mimicking DDA3-S225D variant, but not the nonphosphorable DDA3-S225A mutant, rescues the DDA3-knockdown phenotype. We conclude that the mitotic function of DDA3 is regulated by phosphorylation on the Ser225 residue. PMID:20117088

Robust mitotic entry is ensured by a latching switch.

PubMed

Tuck, Chloe; Zhang, Tongli; Potapova, Tamara; Malumbres, Marcos; Novák, Béla

2013-01-01

Cell cycle events are driven by Cyclin dependent kinases (CDKs) and by their counter-acting phosphatases. Activation of the Cdk1:Cyclin B complex during mitotic entry is controlled by the Wee1/Myt1 inhibitory kinases and by Cdc25 activatory phosphatase, which are themselves regulated by Cdk1:Cyclin B within two positive circuits. Impairing these two feedbacks with chemical inhibitors induces a transient entry into M phase referred to as mitotic collapse. The pathology of mitotic collapse reveals that the positive circuits play a significant role in maintaining the M phase state. To better understand the function of these feedback loops during G2/M transition, we propose a simple model for mitotic entry in mammalian cells including spatial control over Greatwall kinase phosphorylation. After parameter calibration, the model is able to recapture the complex and non-intuitive molecular dynamics reported by Potapova et al. (Potapova et al., 2011). Moreover, it predicts the temporal patterns of other mitotic regulators which have not yet been experimentally tested and suggests a general design principle of cell cycle control: latching switches buffer the cellular stresses which accompany cell cycle processes to ensure that the transitions are smooth and robust.

A Brief History of Research on Mitotic Mechanisms.

PubMed

McIntosh, J Richard; Hays, Thomas

2016-12-21

This chapter describes in summary form some of the most important research on chromosome segregation, from the discovery and naming of mitosis in the nineteenth century until around 1990. It gives both historical and scientific background for the nine chapters that follow, each of which provides an up-to-date review of a specific aspect of mitotic mechanism. Here, we trace the fruits of each new technology that allowed a deeper understanding of mitosis and its underlying mechanisms. We describe how light microscopy, including phase, polarization, and fluorescence optics, provided descriptive information about mitotic events and also enabled important experimentation on mitotic functions, such as the dynamics of spindle fibers and the forces generated for chromosome movement. We describe studies by electron microscopy, including quantitative work with serial section reconstructions. We review early results from spindle biochemistry and genetics, coupled to molecular biology, as these methods allowed scholars to identify key molecular components of mitotic mechanisms. We also review hypotheses about mitotic mechanisms whose testing led to a deeper understanding of this fundamental biological event. Our goal is to provide modern scientists with an appreciation of the work that has laid the foundations for their current work and interests.

Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos

PubMed Central

Tavernier, Nicolas; Noatynska, Anna; Panbianco, Costanza; Martino, Lisa; Van Hove, Lucie; Schwager, Françoise; Léger, Thibaut

2015-01-01

The molecular mechanisms governing mitotic entry during animal development are incompletely understood. Here, we show that the mitotic kinase CDK-1 phosphorylates Suppressor of Par-Two 1 (SPAT-1)/Bora to regulate its interaction with PLK-1 and to trigger mitotic entry in early Caenorhabditis elegans embryos. Embryos expressing a SPAT-1 version that is nonphosphorylatable by CDK-1 and that is defective in PLK-1 binding in vitro present delays in mitotic entry, mimicking embryos lacking SPAT-1 or PLK-1 functions. We further show that phospho–SPAT-1 activates PLK-1 by triggering phosphorylation on its activator T loop in vitro by Aurora A. Likewise, we show that phosphorylation of human Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A, suggesting that this mechanism is conserved in humans. Our results suggest that CDK-1 activates PLK-1 via SPAT-1 phosphorylation to promote entry into mitosis. We propose the existence of a positive feedback loop that connects Cdk1 and Plk1 activation to ensure a robust control of mitotic entry and cell division timing. PMID:25753036

A Brief History of Research on Mitotic Mechanisms

PubMed Central

McIntosh, J. Richard; Hays, Thomas

2016-01-01

This chapter describes in summary form some of the most important research on chromosome segregation, from the discovery and naming of mitosis in the nineteenth century until around 1990. It gives both historical and scientific background for the nine chapters that follow, each of which provides an up-to-date review of a specific aspect of mitotic mechanism. Here, we trace the fruits of each new technology that allowed a deeper understanding of mitosis and its underlying mechanisms. We describe how light microscopy, including phase, polarization, and fluorescence optics, provided descriptive information about mitotic events and also enabled important experimentation on mitotic functions, such as the dynamics of spindle fibers and the forces generated for chromosome movement. We describe studies by electron microscopy, including quantitative work with serial section reconstructions. We review early results from spindle biochemistry and genetics, coupled to molecular biology, as these methods allowed scholars to identify key molecular components of mitotic mechanisms. We also review hypotheses about mitotic mechanisms whose testing led to a deeper understanding of this fundamental biological event. Our goal is to provide modern scientists with an appreciation of the work that has laid the foundations for their current work and interests. PMID:28009830

UV-C irradiation delays mitotic progression by recruiting Mps1 to kinetochores.

PubMed

Zhang, Xiaojuan; Ling, Youguo; Wang, Wenjun; Zhang, Yanhong; Ma, Qingjun; Tan, Pingping; Song, Ting; Wei, Congwen; Li, Ping; Liu, Xuedong; Ma, Runlin Z; Zhong, Hui; Cao, Cheng; Xu, Quanbin

2013-04-15

The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well defined. Herein, we found that UV-C irradiation (254 nm) increases recruitment of the spindle checkpoint proteins Mps1 and Mad2 to the kinetochore during metaphase, suggesting that the spindle assembly checkpoint (SAC) is reactivated. In accordance with this, cells exposed to UV-C showed delayed mitotic progression, characterized by a prolonged chromosomal alignment during metaphase. UV-C irradiation also induced the DNA damage response and caused a significant accumulation of γ-H2AX on mitotic chromosomes. Unexpectedly, the mitotic delay upon UV-C irradiation is not due to the DNA damage response but to the relocation of Mps1 to the kinetochore. Further, we found that UV-C irradiation activates Aurora B kinase. Importantly, the kinase activity of Aurora B is indispensable for full recruitment of Mps1 to the kinetochore during both prometaphase and metaphase. Taking these findings together, we propose that UV irradiation delays mitotic progression by evoking the Aurora B-Mps1 signaling cascade, which exerts its role through promoting the association of Mps1 with the kinetochore in metaphase.

"Couch Surfing" of Latino Foster Care Alumni: Reliance on Peers as Social Capital

ERIC Educational Resources Information Center

Perez, Beatrix F.; Romo, Harriett D.

2011-01-01

Youth exiting foster care often experience difficulties transitioning into adulthood. This paper focuses on Latino foster care youth in a major southwestern U.S. city and addresses the importance of peer networks as a crucial form of social capital as youth leave foster care. Case studies illustrate experiences of foster care alumni ranging in age…

Morphoproteomics, E6/E7 in-situ hybridization, and biomedical analytics define the etiopathogenesis of HPV-associated oropharyngeal carcinoma and provide targeted therapeutic options.

PubMed

Brown, Robert E; Naqvi, Syed; McGuire, Mary F; Buryanek, Jamie; Karni, Ron J

2017-08-17

Human papillomavirus (HPV) has been identified as an etiopathogenetic factor in oropharyngeal squamous cell carcinoma. The HPV E6 and E7 oncogenes are instrumental in promoting proliferation and blocking differentiation leading to tumorigenesis. Although surgical intervention can remove such tumors, the potential for an etiologic field effect with recurrent disease is real. A downstream effector of E7 oncoprotein, enhancer of zeste homolog 2 (EZH2), is known to promote proliferation and to pose a block in differentiation and in turn, could lead to HPV-induced malignant transformation. However, the EZH2 pathway is amenable to low toxicity therapies designed to promote differentiation to a more benign state and prevent recurrent disease by inhibiting the incorporation of HPV into the genome. This is the first study using clinical specimens to demonstrate EZH2 protein expression in oropharyngeal carcinoma (OPC). The study included eight patients with oropharyngeal carcinoma, confirmed p16INK4a- positive by immunohistochemistry (IHC). The tissue expression of E6/E7 messenger RNA (mRNA) was measured by RNAscope® in-situ hybridization technology. Expression of EZH2, Ki-67, and mitotic indices were assessed by morphoproteomic analysis. Biomedical analytics expanded the results with data from Ingenuity Pathway Analysis (IPA) and KEGG databases to construct a molecular network pathway for further insights. Expression of E6 and E7 oncogenes in p16INK4a- positive oropharyngeal carcinoma was confirmed. EZH2 and its correlates, including elevated proliferation index (Ki-67) and mitotic progression were also present. Biomedical analytics validated the relationship between HPV- E6 and E7 and the expression of the EZH2 pathway. There is morphoproteomic and mRNA evidence of the association of p16INK4a-HPV infection with the E6 and E7 oncogenes and the expression of EZH2, Ki-67 and mitotic progression in oropharyngeal carcinoma. The molecular network biology was confirmed by biomedical analytics as consistent with published literature. This is significant because the biology lends itself to targeted therapeutic options using metformin, curcumin, celecoxib and sulforaphane as therapeutic strategies to prevent progression or recurrence of disease.

Study of Double-Weighted Graph Model and Optimal Path Planning for Tourist Scenic Area Oriented Intelligent Tour Guide

NASA Astrophysics Data System (ADS)

Shi, Y.; Long, Y.; Wi, X. L.

2014-04-01

When tourists visiting multiple tourist scenic spots, the travel line is usually the most effective road network according to the actual tour process, and maybe the travel line is different from planned travel line. For in the field of navigation, a proposed travel line is normally generated automatically by path planning algorithm, considering the scenic spots' positions and road networks. But when a scenic spot have a certain area and have multiple entrances or exits, the traditional described mechanism of single point coordinates is difficult to reflect these own structural features. In order to solve this problem, this paper focuses on the influence on the process of path planning caused by scenic spots' own structural features such as multiple entrances or exits, and then proposes a doubleweighted Graph Model, for the weight of both vertexes and edges of proposed Model can be selected dynamically. And then discusses the model building method, and the optimal path planning algorithm based on Dijkstra algorithm and Prim algorithm. Experimental results show that the optimal planned travel line derived from the proposed model and algorithm is more reasonable, and the travelling order and distance would be further optimized.

Image analysis assisted study of mitotic figures in oral epithelial dysplasia and squamous cell carcinoma using differential stains.

PubMed

Tandon, Ankita; Singh, Narendra Nath; Brave, V R; Sreedhar, Gadiputi

2016-11-01

Mitosis is a process of cell division resulting in two genetically equivalent daughter cells. Excessive proliferation of cells due to mitosis is the hallmark in pre cancer and cancer. This study was conducted to count the number of mitotic figures in normal oral mucosa, oral epithelial dysplasia and squamous cell carcinoma in both Hematoxylin and Eosin (H&E) and Crystal Violet stained sections. Also the overall number of mitotic figures with both stains were compared along with the evaluation of staining efficacy of both the stains. The present study was conducted on 20 specimens each of the three categories. These were further divided into two groups for staining with H&E and with 1% Crystal Violet respectively. Images were captured and analyzed using image analysis software Dewinter Biowizard 4.1. Comparison of mitotic figure count in three categories in sections stained with both stains showed statistically significant difference ( p  < 0.001). The mean number of mitotic figures seen in Crystal Violet reagent were significantly higher as seen in H&E stain ( p  < 0.001). The overall diagnostic efficacy of Crystal Violet was 87.6%. Crystal Violet scored over H&E stain and also helped to better appreciate metaphases in Squamous cell carcinoma and telophases in dysplasia. Number of mitotic figures progressively increase with the advancement of the pathology. Use of 1% Crystal Violet provides better appreciation of mitotic figures and can be employed as a selective stain in routine histopathology.

Grading system for blood vessel tumor emboli of invasive ductal carcinoma of the breast.

PubMed

Sugiyama, Michiko; Hasebe, Takahiro; Shimada, Hiroko; Takeuchi, Hideki; Shimizu, Kyoko; Shimizu, Michio; Yasuda, Masanori; Ueda, Shigeto; Shigekawa, Takashi; Osaki, Akihiko; Saeki, Toshiaki

2015-06-01

We previously reported that the number of mitotic and apoptotic figures in tumor cells in blood vessel tumor emboli had the greatest significant power for the accurate prediction of the outcome of patients with invasive ductal carcinoma of the breast. The purpose of the present study was to devise a grading system for blood vessel tumor emboli based on the mitotic and apoptotic figures of tumor cells in blood vessel tumor emboli, enabling accurate prediction of the outcome of patients with invasive ductal carcinoma of the breast. We classified 263 invasive ductal carcinomas into the following 3 grades according to the numbers of mitotic and apoptotic figures in tumor cells located in blood vessels within 1 high-power field: grade 0, no blood vessel invasion; grade 1, absence of mitotic figures and presence of any number of apoptotic figures, or 1 mitotic figure and 0 to 2 apoptotic figures; and grade 2, 1 mitotic figure and 3 or more apoptotic figures, or 2 or more mitotic figures and 1 or more apoptotic figures. Multivariate analyses with well-known prognostic factors demonstrated that grade 2 blood vessel tumor emboli significantly increased the hazard ratios for tumor recurrence independent of the nodal status, pathological TNM stage, hormone receptor status, or HER2 status. The presently reported grading system for blood vessel tumor emboli is the strongest histologic factor for accurate prediction of the outcome of patients with invasive ductal carcinoma of the breast. Copyright © 2015 Elsevier Inc. All rights reserved.

2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1) induces G2/M arrest and mitotic catastrophe in human leukemia HL-60 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, Mei-Hua; Liu, Chin-Yu; Lin, Chiao-Min

2012-03-01

2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1), a 2-phenyl-1,8-naphthyridin-4-one (2-PN) derivative, was synthesized and evaluated as an effective antimitotic agent in our laboratory. However, the molecular mechanisms are uncertain. In this study, HKL-1 was demonstrated to induce multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human leukemia HL-60 cells. Western blotting showed that HKL-1 induces mitotic catastrophe in HL-60 cells through regulating mitotic phase-specific kinases (down-regulating CDK1, cyclin B1, CENP-E, and aurora B) and regulating the expression of Bcl-2 family proteins (down-regulating Bcl-2 and up-regulating Bax and Bak), followed by caspase-9/-3 cleavage. These findings suggest that HKL-1more » appears to exert its cytotoxicity toward HL-60 cells in culture by inducing mitotic catastrophe. Highlights: ► HKL-1 is a potential antimitotic agent against HL-60 cells. ► HKL-1 induces spindle disruption and sustained resulted in mitotic catastrophe. ► CENP-E and aurora B protein expressions significantly reduced. ► Bcl-2 family protein expressions altered and caspase-9/-3 activation. ► HKL-1 is an attractive candidate for possible use as a novel antimitotic agent.« less

Checkpoints couple transcription network oscillator dynamics to cell-cycle progression.

PubMed

Bristow, Sara L; Leman, Adam R; Simmons Kovacs, Laura A; Deckard, Anastasia; Harer, John; Haase, Steven B

2014-09-05

The coupling of cyclin dependent kinases (CDKs) to an intrinsically oscillating network of transcription factors has been proposed to control progression through the cell cycle in budding yeast, Saccharomyces cerevisiae. The transcription network regulates the temporal expression of many genes, including cyclins, and drives cell-cycle progression, in part, by generating successive waves of distinct CDK activities that trigger the ordered program of cell-cycle events. Network oscillations continue autonomously in mutant cells arrested by depletion of CDK activities, suggesting the oscillator can be uncoupled from cell-cycle progression. It is not clear what mechanisms, if any, ensure that the network oscillator is restrained when progression in normal cells is delayed or arrested. A recent proposal suggests CDK acts as a master regulator of cell-cycle processes that have the potential for autonomous oscillatory behavior. Here we find that mitotic CDK is not sufficient for fully inhibiting transcript oscillations in arrested cells. We do find that activation of the DNA replication and spindle assembly checkpoints can fully arrest the network oscillator via overlapping but distinct mechanisms. Further, we demonstrate that the DNA replication checkpoint effector protein, Rad53, acts to arrest a portion of transcript oscillations in addition to its role in halting cell-cycle progression. Our findings indicate that checkpoint mechanisms, likely via phosphorylation of network transcription factors, maintain coupling of the network oscillator to progression during cell-cycle arrest.

Can health insurance improve access to quality care for the Indian poor?

PubMed

Michielsen, Joris; Criel, Bart; Devadasan, Narayanan; Soors, Werner; Wouters, Edwin; Meulemans, Herman

2011-08-01

Recently, the Indian government launched health insurance schemes for the poor both to protect them from high health spending and to improve access to high-quality health services. This article aims to review the potentials of health insurance interventions in order to improve access to quality care in India based on experiences of community health insurance schemes. PubMed, Ovid MEDLINE (R), All EBM Reviews, CSA Sociological Abstracts, CSA Social Service Abstracts, EconLit, Science Direct, the ISI Web of Knowledge, Social Science Research Network and databases of research centers were searched up to September 2010. An Internet search was executed. One thousand hundred and thirty-three papers were assessed for inclusion and exclusion criteria. Twenty-five papers were selected providing information on eight schemes. A realist review was performed using Hirschman's exit-voice theory: mechanisms to improve exit strategies (financial assets and infrastructure) and strengthen patient's long voice route (quality management) and short voice route (patient pressure). All schemes use a mix of measures to improve exit strategies and the long voice route. Most mechanisms are not effective in reality. Schemes that focus on the patients' bargaining position at the patient-provider interface seem to improve access to quality care. Top-down health insurance interventions with focus on exit strategies will not work out fully in the Indian context. Government must actively facilitate the potential of CHI schemes to emancipate the target group so that they may transform from mere passive beneficiaries into active participants in their health.

Hurricanes Katrina and Rita: role of individuals and collaborative networks in mobilizing/coordinating societal and professional resources for major disasters

PubMed Central

Mattox, Kenneth L

2006-01-01

The medical support for the coordinated effort for Harris County Texas (Houston) to rescue evacuees from New Orleans following Hurricane Katrina was part of an integrated collaborative network. Both public health and operational health care was structured to custom meet the needs of the evacuees and to create an exit strategy for the clinic and shelter. Integrating local hospital and physician resources into the Joint Incident Command was essential. Outside assistance, including federal and national resources must be coordinated through the local incident command. PMID:16420647

Predicting re-victimization of battered women 3 years after exiting a shelter program.

PubMed

Bybee, Deborah; Sullivan, Cris M

2005-09-01

This study examined interpersonal and ecological predictors of re-victimization of a sample of women with abusive partners. All women (N = 124) had sought refuge from a battered women's shelter 3 years earlier, and half the sample had been randomly assigned to receive free, short-term advocacy services immediately upon exit from the shelter. Results 2 years post-intervention revealed positive change in the lives of participants (C. M. Sullivan & D. Bybee, 1999), including a decrease in abuse for women who had worked with advocates. The current study examined intervention effects 3 years after the program ended, as well as other predictors of re-abuse. Nineteen percent of the original sample had experienced domestic violence between 2 and 3 years after shelter exit (65% by current partners, 35% by ex-partners). The advocacy program's effect on risk of re-victimization did not continue 3 years post-intervention. However, having worked with an advocate 3 years prior continued to have a positive impact on women's quality of life and level of social support. The risk of being abused 3 years post-shelter stay was exacerbated by a number of factors present 1 year prior, including women's (1) having experienced abuse in the 6 months before that point; (2) having difficulties accessing resources; (3) having problems with the state welfare system; and (4) having people in their social networks who made their lives difficult. Women were at less risk of abuse if, 1 year earlier, they (1) were employed; (2) reported higher quality of life; and (3) had people in their networks who provided practical help and/or were available to talk about personal matters. These findings support the hypothesis that access to resources and social support serve as protective factors against continued abuse.

Zeb2 Regulates Cell Fate at the Exit from Epiblast State in Mouse Embryonic Stem Cells

PubMed Central

Stryjewska, Agata; Dries, Ruben; Pieters, Tim; Verstappen, Griet; Conidi, Andrea; Coddens, Kathleen; Francis, Annick; Umans, Lieve; van IJcken, Wilfred F. J.; Berx, Geert; van Grunsven, Leo A.; Grosveld, Frank G.; Goossens, Steven; Haigh, Jody J.

2016-01-01

Abstract In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm versus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulatory network in these cell‐fate decisions. We generated Zeb2 knockout (KO) mouse ESCs, subjected them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal RNA‐sequencing (RNA‐seq) and reduced representation bisulfite sequencing (RRBS) analysis in neural differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression and that Zeb2 KO ESCs can exit from their naïve state. However, most cells in these EBs stall in an early epiblast‐like state and are impaired in both neural and mesendodermal differentiation. Genes involved in pluripotency, epithelial‐to‐mesenchymal transition (EMT), and DNA‐(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1‐binding sites correlate with loss‐of‐methylation in neural‐stimulating conditions, however, after the cells initially acquired the correct DNA‐methyl marks. Interestingly, cells from such Zeb2 KO EBs maintain the ability to re‐adapt to 2i + LIF conditions even after prolonged differentiation, while knockdown of Tet1 partially rescues their impaired differentiation. Hence, in addition to its role in EMT, Zeb2 is critical in ESCs for exit from the epiblast state, and links the pluripotency network and DNA‐methylation with irreversible commitment to differentiation. Stem Cells 2017;35:611–625 PMID:27739137

Zeb2 Regulates Cell Fate at the Exit from Epiblast State in Mouse Embryonic Stem Cells.

PubMed

Stryjewska, Agata; Dries, Ruben; Pieters, Tim; Verstappen, Griet; Conidi, Andrea; Coddens, Kathleen; Francis, Annick; Umans, Lieve; van IJcken, Wilfred F J; Berx, Geert; van Grunsven, Leo A; Grosveld, Frank G; Goossens, Steven; Haigh, Jody J; Huylebroeck, Danny

2017-03-01

In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm versus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulatory network in these cell-fate decisions. We generated Zeb2 knockout (KO) mouse ESCs, subjected them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal RNA-sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS) analysis in neural differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression and that Zeb2 KO ESCs can exit from their naïve state. However, most cells in these EBs stall in an early epiblast-like state and are impaired in both neural and mesendodermal differentiation. Genes involved in pluripotency, epithelial-to-mesenchymal transition (EMT), and DNA-(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1-binding sites correlate with loss-of-methylation in neural-stimulating conditions, however, after the cells initially acquired the correct DNA-methyl marks. Interestingly, cells from such Zeb2 KO EBs maintain the ability to re-adapt to 2i + LIF conditions even after prolonged differentiation, while knockdown of Tet1 partially rescues their impaired differentiation. Hence, in addition to its role in EMT, Zeb2 is critical in ESCs for exit from the epiblast state, and links the pluripotency network and DNA-methylation with irreversible commitment to differentiation. Stem Cells 2017;35:611-625. © 2016 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Comparison of staining of mitotic figures by haematoxylin and eosin-and crystal violet stains, in oral epithelial dysplasia and squamous cell carcinoma.

PubMed

Ankle, Madhuri R; Kale, Alka D; Charantimath, Seema

2007-01-01

Mitosis of cells gives rise to tissue integrity. Defects during mitosis bring about abnormalities. Excessive proliferation of cells due to increased mitosis is one such outcome, which is the hallmark in precancer and cancer. The localization of proliferating cells or their precursors may not be obvious and easy. Establishing an easy way to distinguish these mitotic cells will help in grading and understanding their biological potential. Although immunohistochemistry is an advanced method in use, the cost and time factor makes it less feasible for many laboratories. Selective histochemical stains like toluidine blue, giemsa and crystal violet have been used in tissues including the developing brain, neural tissue and skin. 1) To compare the staining of mitotic cells in haematoxylin and eosin with that in crystal violet. 2) To compare the number of mitotic figures present in normal oral mucosa, epithelial dysplasia and oral squamous cell carcinoma in crystal violet-stained sections with that in H and E-stained sections. Ten tissues of normal oral mucosa and 15 tissues each of oral epithelial dysplasia seen in tobacco-associated leukoplakia and squamous cell carcinoma were studied to evaluate the selectivity of 1% crystal violet for mitotic figures. The staining was compared with standard H and E staining. Statistical analysis was done using Mann-Whitney U test. A statistically significant increase in the mean mitotic count was observed in crystal violet-stained sections of epithelial dysplasia as compared to the H and E-stained sections (p=0.0327). A similar increase in the mitotic counts was noted in crystal violet-stained sections of oral squamous cell carcinoma as compared to the H and E-stained sections.(p=0.0443). No significant difference was found in the mitotic counts determined in dysplasia or carcinoma by either the crystal violet (p=0.4429) or the H and E-staining techniques (p=0.2717). One per cent crystal violet provides a definite advantage over the H and E-stained sections in selectively staining the mitotic figures.

Targeting Alp7/TACC to the spindle pole body is essential for mitotic spindle assembly in fission yeast

PubMed Central

Tang, Ngang Heok; Okada, Naoyuki; Fong, Chii Shyang; Arai, Kunio; Sato, Masamitsu; Toda, Takashi

2014-01-01

The conserved TACC protein family localises to the centrosome (the spindle pole body, SPB in fungi) and mitotic spindles, thereby playing a crucial role in bipolar spindle assembly. However, it remains elusive how TACC proteins are recruited to the centrosome/SPB. Here, using fission yeast Alp7/TACC, we have determined clustered five amino acid residues within the TACC domain required for SPB localisation. Critically, these sequences are essential for the functions of Alp7, including proper spindle formation and mitotic progression. Moreover, we have identified pericentrin-like Pcp1 as a loading factor to the mitotic SPB, although Pcp1 is not a sole platform. PMID:24937146

Mio depletion links mTOR regulation to Aurora A and Plk1 activation at mitotic centrosomes

PubMed Central

Trinkle-Mulcahy, Laura; Porter, Michael; Jeyaprakash, A. Arockia

2015-01-01

Coordination of cell growth and proliferation in response to nutrient supply is mediated by mammalian target of rapamycin (mTOR) signaling. In this study, we report that Mio, a highly conserved member of the SEACAT/GATOR2 complex necessary for the activation of mTORC1 kinase, plays a critical role in mitotic spindle formation and subsequent chromosome segregation by regulating the proper concentration of active key mitotic kinases Plk1 and Aurora A at centrosomes and spindle poles. Mio-depleted cells showed reduced activation of Plk1 and Aurora A kinase at spindle poles and an impaired localization of MCAK and HURP, two key regulators of mitotic spindle formation and known substrates of Aurora A kinase, resulting in spindle assembly and cytokinesis defects. Our results indicate that a major function of Mio in mitosis is to regulate the activation/deactivation of Plk1 and Aurora A, possibly by linking them to mTOR signaling in a pathway to promote faithful mitotic progression. PMID:26124292

Physical Limits on the Precision of Mitotic Spindle Positioning by Microtubule Pushing forces: Mechanics of mitotic spindle positioning.

PubMed

Howard, Jonathon; Garzon-Coral, Carlos

2017-11-01

Tissues are shaped and patterned by mechanical and chemical processes. A key mechanical process is the positioning of the mitotic spindle, which determines the size and location of the daughter cells within the tissue. Recent force and position-fluctuation measurements indicate that pushing forces, mediated by the polymerization of astral microtubules against- the cell cortex, maintain the mitotic spindle at the cell center in Caenorhabditis elegans embryos. The magnitude of the centering forces suggests that the physical limit on the accuracy and precision of this centering mechanism is determined by the number of pushing microtubules rather than by thermally driven fluctuations. In cells that divide asymmetrically, anti-centering, pulling forces generated by cortically located dyneins, in conjunction with microtubule depolymerization, oppose the pushing forces to drive spindle displacements away from the center. Thus, a balance of centering pushing forces and anti-centering pulling forces localize the mitotic spindles within dividing C. elegans cells. © 2017 The Authors. BioEssays published by Wiley Periodicals, Inc.

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

PubMed Central

Whigham, Arlene; Clarke, Rosemary; Brenes-Murillo, Alejandro J; Estes, Brett; Madhessian, Diana; Lundberg, Emma; Wadsworth, Patricia

2017-01-01

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed ‘early risers’. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase. PMID:29052541

SLK-dependent activation of ERMs controls LGN–NuMA localization and spindle orientation

PubMed Central

Machicoane, Mickael; de Frutos, Cristina A.; Fink, Jenny; Rocancourt, Murielle; Lombardi, Yannis; Garel, Sonia; Piel, Matthieu

2014-01-01

Mitotic spindle orientation relies on a complex dialog between the spindle microtubules and the cell cortex, in which F-actin has been recently implicated. Here, we report that the membrane–actin linkers ezrin/radixin/moesin (ERMs) are strongly and directly activated by the Ste20-like kinase at mitotic entry in mammalian cells. Using microfabricated adhesive substrates to control the axis of cell division, we found that the activation of ERMs plays a key role in guiding the orientation of the mitotic spindle. Accordingly, impairing ERM activation in apical progenitors of the mouse embryonic neocortex severely disturbed spindle orientation in vivo. At the molecular level, ERM activation promotes the polarized association at the mitotic cortex of leucine-glycine-asparagine repeat protein (LGN) and nuclear mitotic apparatus (NuMA) protein, two essential factors for spindle orientation. We propose that activated ERMs, together with Gαi, are critical for the correct localization of LGN–NuMA force generator complexes and hence for proper spindle orientation. PMID:24958772

Optical volume and mass measurements show that mammalian cells swell during mitosis

PubMed Central

Zlotek-Zlotkiewicz, Ewa; Monnier, Sylvain; Cappello, Giovanni; Le Berre, Mael

2015-01-01

The extent, mechanism, and function of cell volume changes during specific cellular events, such as cell migration and cell division, have been poorly studied, mostly because of a lack of adequate techniques. Here we unambiguously report that a large range of mammalian cell types display a significant increase in volume during mitosis (up to 30%). We further show that this increase in volume is tightly linked to the mitotic state of the cell and not to its spread or rounded shape and is independent of the presence of an intact actomyosin cortex. Importantly, this volume increase is not accompanied by an increase in dry mass and thus corresponds to a decrease in cell density. This mitotic swelling might have important consequences for mitotic progression: it might contribute to produce strong pushing forces, allowing mitotic cells to round up; it might also, by lowering cytoplasmic density, contribute to the large change of physicochemical properties observed in mitotic cells. PMID:26598614

O-Linked N-Acetylglucosamine Cycling Regulates Mitotic Spindle Organization*

PubMed Central

Tan, Ee Phie; Caro, Sarah; Potnis, Anish; Lanza, Christopher; Slawson, Chad

2013-01-01

Any defects in the correct formation of the mitotic spindle will lead to chromosomal segregation errors, mitotic arrest, or aneuploidy. We demonstrate that O-linked N-acetylglucosamine (O-GlcNAc), a post-translational modification of serine and threonine residues in nuclear and cytoplasmic proteins, regulates spindle function. In O-GlcNAc transferase or O-GlcNAcase gain of function cells, the mitotic spindle is incorrectly assembled. Chromosome condensation and centrosome assembly is impaired in these cells. The disruption in spindle architecture is due to a reduction in histone H3 phosphorylation by Aurora kinase B. However, gain of function cells treated with the O-GlcNAcase inhibitor Thiamet-G restored the assembly of the spindle and partially rescued histone phosphorylation. Together, these data suggest that the coordinated addition and removal of O-GlcNAc, termed O-GlcNAc cycling, regulates mitotic spindle organization and provides a potential new perspective on how O-GlcNAc regulates cellular events. PMID:23946484

Anti-mitotic agents: Are they emerging molecules for cancer treatment?

PubMed

Penna, Larissa Siqueira; Henriques, João Antonio Pêgas; Bonatto, Diego

2017-05-01

Mutations in cancer cells frequently result in cell cycle alterations that lead to unrestricted growth compared to normal cells. Considering this phenomenon, many drugs have been developed to inhibit different cell-cycle phases. Mitotic phase targeting disturbs mitosis in tumor cells, triggers the spindle assembly checkpoint and frequently results in cell death. The first anti-mitotics to enter clinical trials aimed to target tubulin. Although these drugs improved the treatment of certain cancers, and many anti-microtubule compounds are already approved for clinical use, severe adverse events such as neuropathies were observed. Since then, efforts have been focused on the development of drugs that also target kinases, motor proteins and multi-protein complexes involved in mitosis. In this review, we summarize the major proteins involved in the mitotic phase that can also be targeted for cancer treatment. Finally, we address the activity of anti-mitotic drugs tested in clinical trials in recent years. Copyright © 2017 Elsevier Inc. All rights reserved.

Human papillomavirus type 16 E7 oncoprotein engages but does not abrogate the mitotic spindle assembly checkpoint

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Yueyang; Munger, Karl, E-mail: kmunger@rics.bwh.harvard.edu

2012-10-10

The mitotic spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during mitosis by censoring kinetochore-microtubule interactions. It is frequently rendered dysfunctional during carcinogenesis causing chromosome missegregation and genomic instability. There are conflicting reports whether the HPV16 E7 oncoprotein drives chromosomal instability by abolishing the SAC. Here we report that degradation of mitotic cyclins is impaired in cells with HPV16 E7 expression. RNAi-mediated depletion of Mad2 or BubR1 indicated the involvement of the SAC, suggesting that HPV16 E7 expression causes sustained SAC engagement. Mutational analyses revealed that HPV16 E7 sequences that are necessary for retinoblastoma tumor suppressor protein binding as wellmore » as sequences previously implicated in binding the nuclear and mitotic apparatus (NuMA) protein and in delocalizing dynein from the mitotic spindle contribute to SAC engagement. Importantly, however, HPV16 E7 does not markedly compromise the SAC response to microtubule poisons.« less

A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends

PubMed Central

Kern, David M.; Nicholls, Peter K.; Page, David C.

2016-01-01

The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP is expressed as two distinct isoforms in mammals: a longer, testis-specific isoform that was used for the previous studies in mitotic cells and a novel, shorter mitotic isoform. Unlike the long isoform, short SKAP rescues SKAP depletion in mitosis and displays robust microtubule plus-end tracking, including localization to astral microtubules. Eliminating SKAP microtubule binding results in severe chromosome segregation defects. In contrast, SKAP mutants specifically defective for plus-end tracking facilitate proper chromosome segregation but display spindle positioning defects. Cells lacking SKAP plus-end tracking have reduced Clasp1 localization at microtubule plus ends and display increased lateral microtubule contacts with the cell cortex, which we propose results in unbalanced dynein-dependent cortical pulling forces. Our work reveals an unappreciated role for the Astrin/SKAP complex as an astral microtubule mediator of mitotic spindle positioning. PMID:27138257

Suspension of Mitotic Activity in Dentate Gyrus of the Hibernating Ground Squirrel

PubMed Central

Popov, Victor I.; Kraev, Igor V.; Ignat'ev, Dmitri A.; Stewart, Michael G.

2011-01-01

Neurogenesis occurs in the adult mammalian hippocampus, a region of the brain important for learning and memory. Hibernation in Siberian ground squirrels provides a natural model to study mitosis as the rapid fall in body temperature in 24 h (from 35-36°C to +4–6°C) permits accumulation of mitotic cells at different stages of the cell cycle. Histological methods used to study adult neurogenesis are limited largely to fixed tissue, and the mitotic state elucidated depends on the specific phase of mitosis at the time of day. However, using an immunohistochemical study of doublecortin (DCX) and BrdU-labelled neurons, we demonstrate that the dentate gyrus of the ground squirrel hippocampus contains a population of immature cells which appear to possess mitotic activity. Our data suggest that doublecortin-labelled immature cells exist in a mitotic state and may represent a renewable pool for generation of new neurons within the dentate gyrus. PMID:21773054

Heat shock protein inhibitors, 17-DMAG and KNK437, enhance arsenic trioxide-induced mitotic apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu Yichen; Yen Wenyen; Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan

2009-04-15

Arsenic trioxide (ATO) has recently emerged as a promising therapeutic agent in leukemia because of its ability to induce apoptosis. However, there is no sufficient evidence to support its therapeutic use for other types of cancers. In this study, we investigated if, and how, 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG), an antagonist of heat shock protein 90 (HSP90), and KNK437, a HSP synthesis inhibitor, potentiated the cytotoxic effect of ATO. Our results showed that cotreatment with ATO and either 17-DMAG or KNK437 significantly increased ATO-induced cell death and apoptosis. siRNA-mediated attenuation of the expression of the inducible isoform of HSP70 (HSP70i) or HSP90{alpha}/{beta} alsomore » enhanced ATO-induced apoptosis. In addition, cotreatment with ATO and 17-DMAG or KNK437 significantly increased ATO-induced mitotic arrest and ATO-induced BUBR1 phosphorylation and PDS1 accumulation. Cotreatment also significantly increased the percentage of mitotic cells with abnormal mitotic spindles and promoted metaphase arrest as compared to ATO treatment alone. These results indicated that 17-DMAG or KNK437 may enhance ATO cytotoxicity by potentiating mitotic arrest and mitotic apoptosis possibly through increased activation of the spindle checkpoint.« less

Relationship between DNA ploidy and proliferative cell nuclear antigen index in canine hemangiopericytoma.

PubMed

Kang, Seong-Kwi; Park, Nam-Yong; Cho, Ho-Sung; Shin, Sung-Shik; Kang, Mun-Il; Kim, Sang-Ki; Hyun, Changbaig; Park, In-Chul; Kim, Jong-Tack; Jeong, Cheol; Park, Sung-Hee; Park, Su-Jin; Jeong, Jae-Ho; Kim, You-Jung; Ochiai, Kenji; Umemura, Takashi; Cho, Kyoung-Oh

2006-03-01

The mitotic index is reported to be correlated with recurrence, mean patient survival, and metastasis of canine hemangiopericytoma (CHP). However, to the authors' knowledge, studies investigating the parameters that can predict recurrence or metastasis of CHP with low mitotic index have not been done. To evaluate growth kinetics of CHP with low mitotic index, a retrospective analysis of the proliferative activity by antiproliferative cell nuclear antigen monoclonal antibody and DNA contents by flow cytometry (FCM) was performed with 21 formalin-fixed and paraffin-embedded CHP samples. Of the 21 tumors evaluated by FCM, 6 (26.6%) were aneuploid tumors, and 15 (71.4%) were diploid tumors. There was significant correlation between the PCNA index and ploidy pattern. The diploid group had 39.1 +/- 9.2 PCNA index, whereas the aneuploid group's proliferative cell nuclear antigen (PCNA) index was 63.1 +/- 8.2. The diploid group had mean mitotic index value of 1.140 +/- 0.855, and the aneuploid group had a mean value of 1.067 +/- 0.767. From these results, the CHP samples with low mitotic index were classified into either the aneuploid group with higher PCNA index or the diploid group with lower PCNA index, suggesting that DNA ploidy and proliferative activity may give an indication about malignancy of CHPs with a low mitotic index.

Wnt activation followed by Notch inhibition promotes mitotic hair cell regeneration in the postnatal mouse cochlea

PubMed Central

Li, Wenyan; Chen, Yan; Zhang, Shasha; Tang, Mingliang; Sun, Shan; Chai, Renjie; Li, Huawei

2016-01-01

Hair cell (HC) loss is the main cause of permanent hearing loss in mammals. Previous studies have reported that in neonatal mice cochleae, Wnt activation promotes supporting cell (SC) proliferation and Notch inhibition promotes the trans-differentiation of SCs into HCs. However, Wnt activation alone fails to regenerate significant amounts of new HCs, Notch inhibition alone regenerates the HCs at the cost of exhausting the SC population, which leads to the death of the newly regenerated HCs. Mitotic HC regeneration might preserve the SC number while regenerating the HCs, which could be a better approach for long-term HC regeneration. We present a two-step gene manipulation, Wnt activation followed by Notch inhibition, to accomplish mitotic regeneration of HCs while partially preserving the SC number. We show that Wnt activation followed by Notch inhibition strongly promotes the mitotic regeneration of new HCs in both normal and neomycin-damaged cochleae while partially preserving the SC number. Lineage tracing shows that the majority of the mitotically regenerated HCs are derived specifically from the Lgr5+ progenitors with or without HC damage. Our findings suggest that the co-regulation of Wnt and Notch signaling might provide a better approach to mitotically regenerate HCs from Lgr5+ progenitor cells. PMID:27564256

Sulforaphane induces reactive oxygen species-mediated mitotic arrest and subsequent apoptosis in human bladder cancer 5637 cells.

PubMed

Park, Hyun Soo; Han, Min Ho; Kim, Gi-Young; Moon, Sung-Kwon; Kim, Wun-Jae; Hwang, Hye Jin; Park, Kun Young; Choi, Yung Hyun

2014-02-01

The present study was undertaken to determine whether sulforaphane-derived reactive oxygen species (ROS) might cause growth arrest and apoptosis in human bladder cancer 5637 cells. Our results show that the reduced viability of 5637 cells by sulforaphane is due to mitotic arrest, but not the G2 phase. The sulforaphane-induced mitotic arrest correlated with an induction of cyclin B1 and phosphorylation of Cdk1, as well as a concomitant increased complex between cyclin B1 and Cdk1. Sulforaphane-induced apoptosis was associated with the activation of caspase-8 and -9, the initiators caspases of the extrinsic and intrinsic apoptotic pathways, respectively, and activation of effector caspase-3 and cleavage of poly (ADP-ribose) polymerase. However, blockage of caspase activation inhibited apoptosis and abrogated growth inhibition in sulforaphane-treated 5637 cells. This study further investigated the roles of ROS with respect to mitotic arrest and the apoptotic effect of sulforaphane, and the maximum level of ROS accumulation was observed 3h after sulforaphane treatment. However, a ROS scavenger, N-acetyl-L-cysteine, notably attenuated sulforaphane-mediated apoptosis as well as mitotic arrest. Overall, these results suggest that sulforaphane induces mitotic arrest and apoptosis of 5637 cells via a ROS-dependent pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

UV-C irradiation delays mitotic progression by recruiting Mps1 to kinetochores

PubMed Central

Zhang, Xiaojuan; Ling, Youguo; Wang, Wenjun; Zhang, Yanhong; Ma, Qingjun; Tan, Pingping; Song, Ting; Wei, Congwen; Li, Ping; Liu, Xuedong; Ma, Runlin Z.; Zhong, Hui; Cao, Cheng; Xu, Quanbin

2013-01-01

The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well defined. Herein, we found that UV-C irradiation (254 nm) increases recruitment of the spindle checkpoint proteins Mps1 and Mad2 to the kinetochore during metaphase, suggesting that the spindle assembly checkpoint (SAC) is reactivated. In accordance with this, cells exposed to UV-C showed delayed mitotic progression, characterized by a prolonged chromosomal alignment during metaphase. UV-C irradiation also induced the DNA damage response and caused a significant accumulation of γ-H2AX on mitotic chromosomes. Unexpectedly, the mitotic delay upon UV-C irradiation is not due to the DNA damage response but to the relocation of Mps1 to the kinetochore. Further, we found that UV-C irradiation activates Aurora B kinase. Importantly, the kinase activity of Aurora B is indispensable for full recruitment of Mps1 to the kinetochore during both prometaphase and metaphase. Taking these findings together, we propose that UV irradiation delays mitotic progression by evoking the Aurora B-Mps1 signaling cascade, which exerts its role through promoting the association of Mps1 with the kinetochore in metaphase. PMID:23531678

Loops determine the mechanical properties of mitotic chromosomes

NASA Astrophysics Data System (ADS)

Zhang, Yang; Heermann, Dieter W.

2013-03-01

In mitosis, chromosomes undergo a condensation into highly compacted, rod-like objects. Many models have been put forward for the higher-order organization of mitotic chromosomes including radial loop and hierarchical folding models. Additionally, mechanical properties of mitotic chromosomes under different conditions were measured. However, the internal organization of mitotic chromosomes still remains unclear. Here we present a polymer model for mitotic chromosomes and show how chromatin loops play a major role for their mechanical properties. The key assumption of the model is the ability of the chromatin fibre to dynamically form loops with the help of binding proteins. Our results show that looping leads to a tight compaction and significantly increases the bending rigidity of chromosomes. Moreover, our qualitative prediction of the force elongation behaviour is close to experimental findings. This indicates that the internal structure of mitotic chromosomes is based on self-organization of the chromatin fibre. We also demonstrate how number and size of loops have a strong influence on the mechanical properties. We suggest that changes in the mechanical characteristics of chromosomes can be explained by an altered internal loop structure. YZ gratefully appreciates funding by the German National Academic Foundation (Studienstiftung des deutschen Volkes) and support by the Heidelberg Graduate School for Mathematical and Computational Methods in the Sciences (HGS MathComp).

Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity

PubMed Central

Dewey, Evan B.; Johnston, Christopher A.

2017-01-01

Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila. Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial–mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation. PMID:28747439

Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rahayu, Retno; Ohsaki, Eriko; Omori, Hiroko

In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy–electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres,more » and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division. - Highlights: • This is the first report showing LANA dots on mitotic chromosomes by fluorescent microscopy followed by electron microscopy. • LANA dots localized randomly on condensed chromosomes other than centromere/pericentromere and telomere/peritelomre. • Cellular mitotic checkpoint should not be always involved in the segregation of KSHV genomes in the latency.« less

Protective effects of mito-TEMPO against doxorubicin cardiotoxicity in mice.

PubMed

Rocha, Viviane Costa Junqueira; França, Luciana Souza de Aragão; de Araújo, Cintia Figueiredo; Ng, Ayling Martins; de Andrade, Candace Machado; Andrade, André Cronemberger; Santos, Emanuelle de Souza; Borges-Silva, Mariana da Cruz; Macambira, Simone Garcia; Noronha-Dutra, Alberto Augusto; Pontes-de-Carvalho, Lain Carlos

2016-03-01

Doxorubicin (DOX) is a chemotherapeutic that is widely used for the treatment of many human tumors. However, the development of cardiotoxicity has limited its use. The aim of the present study was to evaluate the possible efficacy of mito-TEMPO (mito-T) as a protective agent against DOX-induced cardiotoxicity in mice. C57BL/6 mice were treated twice with mito-T at low (5 mg/kg body weight) or high (20 mg/kg body weight) dose and once with DOX (24 mg/kg body weight) or saline (0.1 mL/20 g body weight) by means of intraperitoneal injections. The levels of malondialdehyde (MLDA), a marker of lipid peroxidation, and serum levels of creatine kinase were evaluated 48 h after the injection of DOX. DOX induced lipid peroxidation in heart mitochondria (p < 0.001), and DOX-treated mice receiving mito-T at low dose had levels of MLDA significantly lower than the mice that received only DOX (p < 0.01). Furthermore, administration of mito-T alone did not cause any significant changes from control values. Additionally, DOX-treated mice treated with mito-T at high dose showed decrease in serum levels of total CK compared to mice treated with DOX alone (p < 0.05). Our results indicate that mito-T protects mice against DOX-induced cardiotoxicity.

Division of constricted and urethane-treated sand dollar eggs: a test of the polar stimulation hypothesis.

PubMed

Rappaport, R; Rappaport, B N

1984-07-01

In spherical cells with a central mitotic apparatus, the centers of the asters are closer to the poles than to the equator. This circumstance is basic to several hypothetical explanations of the way in which the mitotic apparatus establishes the division mechanism. This investigation was designed to determine whether that geometrical relationship is necessary for division. Fertilized, mechanically denuded sand dollar eggs were inserted into glass loops, which reduced the diameter in the constriction plane from the normal 142 to 78-80 microns and partly constricted the cell into equal parts. The mitotic apparatus straddled the constriction, and its length was not significantly changed. The manipulation increased the distance from the astral centers to the poles and decreased the distance from the astral centers to the equator to a degree that reversed the normal distance relations. These cells divided normally. Ethyl urethane (0.06 M) reduces the size of the mitotic apparatus and blocks cleavage in spherical cells. When treated cells are confined in 80-microns i.d. capillaries, they divide. Treated cells also divide when they are constricted by an 80-microns i.d. glass loop if the mitotic apparatus straddles the constriction. An equal degree of constriction in the subfurrow and subpolar areas did not reverse the effect of urethane. The results demonstrate that cleavage does not depend on the normal distance relation between the mitotic apparatus and the poles, and that the urethane effect can be remedied only by reducing the distance between the mitotic apparatus and the equatorial surface. Both findings are inconsistent with the polar stimulation hypothesis.

Phosphohistone-H3 (PHH3) is prognostic relevant in Merkel cell carcinomas but Merkel cell polyomavirus is a more powerful prognostic factor than AJCC clinical stage, PHH3, Ki-67 or mitotic indices.

PubMed

Iwasaki, Takeshi; Matsushita, Michiko; Nonaka, Daisuke; Kato, Masako; Nagata, Keiko; Murakami, Ichiro; Hayashi, Kazuhiko

2015-08-01

Merkel cell carcinomas (MCCs) associated with Merkel cell polyomavirus (MCPyV) have better prognosis than those without MCPyV. The relationship between mitotic index (MI) and MCC outcome has remained elusive because of the difficulty in differentiating mitotic cells from apoptotic ones. We evaluated the role of phosphohistone-H3 (PHH3) (Ser10), a new mitotic count biomarker, in MCPyV-positive or -negative MCC patients, and assessed its prognostic value in comparison to Ki-67 labeling index or MI using hematoxylin and eosin (HE) staining. We compared the prognostic value of PHH3 mitotic index with that of MI by HE in 19 MCPyV-positive and 9 MCPyV-negative MCC patients. PHH3-positive immunoreactivity was mostly observed in mitotic figures. Multivariate analysis significantly showed that MCPyV status (HR, 0.004; 95% CI 0.0003-0.058) and the American Joint Committee of Cancer (AJCC) stage (HR, 5.02; 95% CI 1.23-20.51) were observed as significantly independent prognostic factors for OS. PHH3-positive cell counts/10 HPF was a slightly significant independent prognostic factor for OS (HR, 4.96; 95% CI 0.93-26.55). PHH3-positive MI and MCPyV status in MCC patients are useful in prognostication, although MCPyV-infection is a more powerful prognostic factor in MCCs than the AJCC scheme on proliferation or mitotic indices. © 2015 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

Mitotic figure counts are significantly overestimated in resection specimens of invasive breast carcinomas.

PubMed

Lehr, Hans-Anton; Rochat, Candice; Schaper, Cornelia; Nobile, Antoine; Shanouda, Sherien; Vijgen, Sandrine; Gauthier, Arnaud; Obermann, Ellen; Leuba, Susana; Schmidt, Marcus; C, Curzio Ruegg; Delaloye, Jean-Francois; Simiantonaki, Nectaria; Schaefer, Stephan C

2013-03-01

Several authors have demonstrated an increased number of mitotic figures in breast cancer resection specimen when compared with biopsy material. This has been ascribed to a sampling artifact where biopsies are (i) either too small to allow formal mitotic figure counting or (ii) not necessarily taken form the proliferating tumor periphery. Herein, we propose a different explanation for this phenomenon. Biopsy and resection material of 52 invasive ductal carcinomas was studied. We counted mitotic figures in 10 representative high power fields and quantified MIB-1 immunohistochemistry by visual estimation, counting and image analysis. We found that mitotic figures were elevated by more than three-fold on average in resection specimen over biopsy material from the same tumors (20±6 vs 6±2 mitoses per 10 high power fields, P=0.008), and that this resulted in a relative diminution of post-metaphase figures (anaphase/telophase), which made up 7% of all mitotic figures in biopsies but only 3% in resection specimen (P<0.005). At the same time, the percentages of MIB-1 immunostained tumor cells among total tumor cells were comparable in biopsy and resection material, irrespective of the mode of MIB-1 quantification. Finally, we found no association between the size of the biopsy material and the relative increase of mitotic figures in resection specimen. We propose that the increase in mitotic figures in resection specimen and the significant shift towards metaphase figures is not due to a sampling artifact, but reflects ongoing cell cycle activity in the resected tumor tissue due to fixation delay. The dwindling energy supply will eventually arrest tumor cells in metaphase, where they are readily identified by the diagnostic pathologist. Taken together, we suggest that the rapidly fixed biopsy material better represents true tumor biology and should be privileged as predictive marker of putative response to cytotoxic chemotherapy.

Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development

PubMed Central

McCoy, Rajiv C.; Demko, Zachary P.; Ryan, Allison; Banjevic, Milena; Hill, Matthew; Sigurjonsson, Styrmir; Rabinowitz, Matthew; Petrov, Dmitri A.

2015-01-01

Whole-chromosome imbalances affect over half of early human embryos and are the leading cause of pregnancy loss. While these errors frequently arise in oocyte meiosis, many such whole-chromosome abnormalities affecting cleavage-stage embryos are the result of chromosome missegregation occurring during the initial mitotic cell divisions. The first wave of zygotic genome activation at the 4–8 cell stage results in the arrest of a large proportion of embryos, the vast majority of which contain whole-chromosome abnormalities. Thus, the full spectrum of meiotic and mitotic errors can only be detected by sampling after the initial cell divisions, but prior to this selective filter. Here, we apply 24-chromosome preimplantation genetic screening (PGS) to 28,052 single-cell day-3 blastomere biopsies and 18,387 multi-cell day-5 trophectoderm biopsies from 6,366 in vitro fertilization (IVF) cycles. We precisely characterize the rates and patterns of whole-chromosome abnormalities at each developmental stage and distinguish errors of meiotic and mitotic origin without embryo disaggregation, based on informative chromosomal signatures. We show that mitotic errors frequently involve multiple chromosome losses that are not biased toward maternal or paternal homologs. This outcome is characteristic of spindle abnormalities and chaotic cell division detected in previous studies. In contrast to meiotic errors, our data also show that mitotic errors are not significantly associated with maternal age. PGS patients referred due to previous IVF failure had elevated rates of mitotic error, while patients referred due to recurrent pregnancy loss had elevated rates of meiotic error, controlling for maternal age. These results support the conclusion that mitotic error is the predominant mechanism contributing to pregnancy losses occurring prior to blastocyst formation. This high-resolution view of the full spectrum of whole-chromosome abnormalities affecting early embryos provides insight into the cytogenetic mechanisms underlying their formation and the consequences for human fertility. PMID:26491874

Research and realization implementation of monitor technology on illegal external link of classified computer

NASA Astrophysics Data System (ADS)

Zhang, Hong

2017-06-01

In recent years, with the continuous development and application of network technology, network security has gradually entered people's field of vision. The host computer network external network of violations is an important reason for the threat of network security. At present, most of the work units have a certain degree of attention to network security, has taken a lot of means and methods to prevent network security problems such as the physical isolation of the internal network, install the firewall at the exit. However, these measures and methods to improve network security are often not comply with the safety rules of human behavior damage. For example, the host to wireless Internet access and dual-network card to access the Internet, inadvertently formed a two-way network of external networks and computer connections [1]. As a result, it is possible to cause some important documents and confidentiality leak even in the the circumstances of user unaware completely. Secrecy Computer Violation Out-of-band monitoring technology can largely prevent the violation by monitoring the behavior of the offending connection. In this paper, we mainly research and discuss the technology of secret computer monitoring.

Cell cycle gene expression networks discovered using systems biology: Significance in carcinogenesis

PubMed Central

Scott, RE; Ghule, PN; Stein, JL; Stein, GS

2015-01-01

The early stages of carcinogenesis are linked to defects in the cell cycle. A series of cell cycle checkpoints are involved in this process. The G1/S checkpoint that serves to integrate the control of cell proliferation and differentiation is linked to carcinogenesis and the mitotic spindle checkpoint with the development of chromosomal instability. This paper presents the outcome of systems biology studies designed to evaluate if networks of covariate cell cycle gene transcripts exist in proliferative mammalian tissues including mice, rats and humans. The GeneNetwork website that contains numerous gene expression datasets from different species, sexes and tissues represents the foundational resource for these studies (www.genenetwork.org). In addition, WebGestalt, a gene ontology tool, facilitated the identification of expression networks of genes that co-vary with key cell cycle targets, especially Cdc20 and Plk1 (www.bioinfo.vanderbilt.edu/webgestalt). Cell cycle expression networks of such covariate mRNAs exist in multiple proliferative tissues including liver, lung, pituitary, adipose and lymphoid tissues among others but not in brain or retina that have low proliferative potential. Sixty-three covariate cell cycle gene transcripts (mRNAs) compose the average cell cycle network with p = e−13 to e−36. Cell cycle expression networks show species, sex and tissue variability and they are enriched in mRNA transcripts associated with mitosis many of which are associated with chromosomal instability. PMID:25808367

Elucidating cdc25’s Oncogenic Mechanism in Breast Cancer Using Pin1, a Negative Mitotic Regulator

DTIC Science & Technology

2000-07-01

inhibitor aphidicolin. This defect in replication checkpoint function was reversed after addition of recombinant wild type Pin 1, but not an isomerase... inhibitor , aphidicolin. Mock-depleted extracts effectively postponed mitotic entry in response to replication inhibition, while depletion of Pin 1 from...fail to haft mitotic entry in response to the DNA polymerase inhibitor , aphidicolin. The addition of recombinant Pin1 restores the appropriate G2

Inhibition of intra-Golgi transport in vitro by mitotic kinase.

PubMed

Stuart, R A; Mackay, D; Adamczewski, J; Warren, G

1993-02-25

It has previously been shown that exocytic and endocytic membrane traffic are inhibited in mitotic mammalian cells. Here we have used a cell-free intra-Golgi transport assay supplemented with heterologous cytosols to mimic this effect in vitro. Cytosols with high histone kinase activity, made either from mitotic cells or by cyclin A treatment of interphase cells, inhibited intra-Golgi transport by up to 75%. Inhibition of transport was reversed by the kinase inhibitor staurosporine or by reduction in ATP levels leading to inactivation of histone kinase. The data indicate that cell cycle control of intra-Golgi transport is due to a reversible modification of cytosol, and this assay system may be used to study the molecular mechanism of mitotic transport inhibition in mammalian cells.

Effects of caffeine on mitotic index, mitotic aberrations and bimitosis with and without aeration.

PubMed

Röper, W

1977-07-01

The effects of 1 to 3 h 0.2% caffeine treatment on mitosis in lateral roots of Vicia faba with and without aeration have been investigated. During the treatment a marked decrease of the mitotic index followed by strong deviations and changing phase indices can be stated. By means of aeration the number of mitotic aberrations increases with time of treatment, while it decreases without aeration until 3 h treatment. Tetraploid cells are supposed to be formed by spindle aberrations at early anaphase. The number of binucleate and tetraploid cells is affected by aeration during caffeine treatment. During division of the binucleate cells tetraploid nuclei are formed by fusions, so the population of binucleate cells may become smaller.

Sensitivity and usefulness of anti-phosphohistone-H3 antibody immunostaining for counting mitotic figures in meningioma cases.

PubMed

Fukushima, Shintaro; Terasaki, Mizuhiko; Sakata, Kiyohiko; Miyagi, Naohisa; Kato, Seiya; Sugita, Yasuo; Shigemori, Minoru

2009-01-01

According to current World Health Organization (WHO) criteria, counting mitotic figures (MF), which is equal to the mitotic index (MI), on paraffin sections stained with hematoxylin and eosin (HE) is one of the recognized classification methods for meningiomas. However, it is not always easy to find the area of highest mitotic activity, and there are different perspectives among pathologists with regard to differentiating MF from non-MF, i.e., which are apoptotic figures and which are crushed or distorted cells. Moreover, there is an issue of overgrading in meningiomas with preoperative feeder embolization. Recently, anti-phosphohistone-H3 (PHH3) antibody has been reported as a mitosis-specific marker for meningioma grading. In this study, we attempted PHH3 immunostaining for our meningioma cases and verified not only the sensitivity of PHH3 immunostaining but also that of its usefulness in grading meningiomas. Forty-five initial histologically confirmed meningiomas (37 benign, 7 atypical, and 1 anaplastic) were reviewed according to current WHO criteria based on counting MF on HE-stained slides. PHH3-immunostained MF were counted in the same way, and the MIB-1 labeling index (LI) was calculated for each sample. PHH3-labeled MF were easily identified and permitted rapid recognition of the areas of highest mitotic activity. As a result, significant increase of PHH3 mitotic index (PHH3-MI) in comparison with HE mitotic index (HE-MI) and strong correlations with HE-MI to PHH3-MI as well as PHH3-MI to MIB-1 LI were demonstrated. Furthermore, no significant differences of PHH3-MI between cases with and without feeder embolization were demonstrated. As such, PHH3 may be a sensitive and useful marker for meningioma grading as based on the MF.

Live-cell imaging visualizes frequent mitotic skipping during senescence-like growth arrest in mammary carcinoma cells exposed to ionizing radiation.

PubMed

Suzuki, Masatoshi; Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi

2012-06-01

Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO(2)-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ß-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation. Copyright © 2012 Elsevier Inc. All rights reserved.

[Genetic control of mitotic crossing-over in yeasts. III. Induction by 8-methoxypsoralen and long-wave UV irradiation (lambda=365 nm)].

PubMed

Fedorova, I V; Marfin, S V

1982-02-01

The lethal effect of 8-methoxypsoralen (8-MOP) plus 365 nm light has been studied in haploid radiosensitive strains of Saccharomyces cerevisiae. The diploid of wild type and the diploid homozygous for the rad2 mutation (this mutation blocks the excision of UV-induced pyrimidine dimers) were more resistant to the lethal effect of 8-MOP plus 365 nm light than the haploid of wild type and rad2 haploid, respectively. The diploid homozygous for rad54 mutation (the mutation blocks the repair of double-strand breaks in DNA) was more sensitive than haploid rad54. The method of repeated irradiation allowed to study the capacity of radiosensitive diploids to remove monoadducts induced by 8-MOP in DNA. This process was very effective in diploids of wild type and in the rad54 rad54 diploid, while the rad2 rad2 diploid was characterized by nearly complete absence of monoadduct excision. The study of mitotic crossing over and mitotic segregation in yeast diploids, containing a pair of complementing alleles of the ade2 gene (red/pink) has shown a very high recombinogenic effect of 8-MOP plus 365 nm light. The rad2 mutation slightly increased the frequency of mitotic segregation and mitotic crossing over. The rad54 mutation decreased the frequency of mitotic segregation and entirely suppressed mitotic crossing over. The method of repeated irradiation showed that the cross-links, but not monoadducts, are the main cause of high recombinogenic effect of 8-MOP plus 365 nm light. The possible participation of different repair systems in recombinational processes induced by 8-MOP in yeast cells is discussed.

Evaluation of efficacy of 1% Crystal Violet & Nuclear Fast Red stain compared to Haematoxyline & Eosin stain for assessing mitotic figures in oral premalignant and malignant lesions.

PubMed

Motiwale, Gauri; Jaiswal, Shradha; Vikey, Ashok; Motiwale, Tejas; Bagulkar, Bhupesh; Bhat, Atul; Kapoor, Prakhar

2016-07-01

Various chromosomal arrangements in cells undergoing division are referred to as Mitotic figure (MF). The abnormal excess of mitotic figures is commonly seen in oral epithelial dysplasia (ED) and oral squamous cell carcinoma (OSCC). In present study, we compared the number of mitotic figures in normal oral mucosa, epithelial dysplasia & OSCC sections with haematoxyline & eosine (H&E) and 1%Crystal Violet & Nuclear Fast Red (CV&NFR) stain, also the efficacy of the CV&NFR stain as compared to H & E stain. We investigated the correlation between the number of mitotic figures & grades of OSCC. Study sample comprised of two serial sections of archival blocks of normal oral mucosa & diagnosed cases of epithelial dysplasia & OSCC. One slide stained with H& E & the other one with 1% CV & NFR. Mitotic figures were counted with the grid eyepiece. There was significant increase in number of MFs in oral ED and OSCC in comparison with normal oral mucosa. There was a highly significant increase in number of MFs in CV&NFR stained tissue sections when compared with H & E stain. Metaphase is the most commonly observed phase of mitosis. In summary, our study proposes the use of Crystal violet & Nuclear fast red stain as a selective stain for better contrast & easy identification MFs. © 2016 Old City Publishing, Inc.

Less understood issues: p21(Cip1) in mitosis and its therapeutic potential.

PubMed

Kreis, N-N; Louwen, F; Yuan, J

2015-04-02

p21(Cip1) is a multifunctional protein and a key player in regulating different cellular processes. The transcription of p21 is regulated by p53-dependent and -independent pathways. The expression of p21 is increased in response to various cellular stresses to arrest the cell cycle and ensure genomic stability. p21 has been shown to be a tumor suppressor and an oncogene as well. The function of p21 in mitosis has been proposed but not systematically studied. We have recently shown that p21 binds to and inhibits the activity of Cdk1/cyclin B1, and is important for a fine-tuned mitotic progression. Loss of p21 prolongs the duration of mitosis and results in severe mitotic defects like chromosome segregation and cytokinesis failures promoting consequently genomic instability. Moreover, p21 is dramatically stabilized in mitotic tumor cells upon treatment with mitotic agents like paclitaxel or mitotic kinase inhibitors. Increased p21 is mainly localized in the cytoplasm and associates with cell survival indicating a crucial role of p21 in susceptibility to mitotic agents in tumor cells. In this review we will briefly summarize the structure and general physiological functions as well as regulation of p21, discuss in detail its role in mitosis and its potential to serve as a therapeutic target.

Partitioning of the Golgi Apparatus during Mitosis in Living HeLa Cells

PubMed Central

Shima, David T.; Haldar, Kasturi; Pepperkok, Rainer; Watson, Rose; Warren, Graham

1997-01-01

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH2-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I–GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus. PMID:9182657

Immunolocalization of dually phosphorylated MAPKs in dividing root meristem cells of Vicia faba, Pisum sativum, Lupinus luteus and Lycopersicon esculentum.

PubMed

Winnicki, Konrad; Żabka, Aneta; Bernasińska, Joanna; Matczak, Karolina; Maszewski, Janusz

2015-06-01

In plants, phosphorylated MAPKs display constitutive nuclear localization; however, not all studied plant species show co-localization of activated MAPKs to mitotic microtubules. The mitogen-activated protein kinase (MAPK) signaling pathway is involved not only in the cellular response to biotic and abiotic stress but also in the regulation of cell cycle and plant development. The role of MAPKs in the formation of a mitotic spindle has been widely studied and the MAPK signaling pathway was found to be indispensable for the unperturbed course of cell division. Here we show cellular localization of activated MAPKs (dually phosphorylated at their TXY motifs) in both interphase and mitotic root meristem cells of Lupinus luteus, Pisum sativum, Vicia faba (Fabaceae) and Lycopersicon esculentum (Solanaceae). Nuclear localization of activated MAPKs has been found in all species. Co-localization of these kinases to mitotic microtubules was most evident in L. esculentum, while only about 50% of mitotic cells in the root meristems of P. sativum and V. faba displayed activated MAPKs localized to microtubules during mitosis. Unexpectedly, no evident immunofluorescence signals at spindle microtubules and phragmoplast were noted in L. luteus. Considering immunocytochemical analyses and studies on the impact of FR180204 (an inhibitor of animal ERK1/2) on mitotic cells, we hypothesize that MAPKs may not play prominent role in the regulation of microtubule dynamics in all plant species.

LIS1 controls mitosis and mitotic spindle organization via the LIS1–NDEL1–dynein complex

PubMed Central

Moon, Hyang Mi; Youn, Yong Ha; Pemble, Hayley; Yingling, Jessica; Wittmann, Torsten; Wynshaw-Boris, Anthony

2014-01-01

Heterozygous LIS1 mutations are responsible for the human neuronal migration disorder lissencephaly. Mitotic functions of LIS1 have been suggested from many organisms throughout evolution. However, the cellular functions of LIS1 at distinct intracellular compartments such as the centrosome and the cell cortex have not been well defined especially during mitotic cell division. Here, we used detailed cellular approaches and time-lapse live cell imaging of mitosis from Lis1 mutant mouse embryonic fibroblasts to reveal critical roles of LIS1 in mitotic spindle regulation. We found that LIS1 is required for the tight control of chromosome congression and segregation to dictate kinetochore–microtubule (MT) interactions and anaphase progression. In addition, LIS1 is essential for the establishment of mitotic spindle pole integrity by maintaining normal centrosome number. Moreover, LIS1 plays crucial roles in mitotic spindle orientation by increasing the density of astral MT plus-end movements toward the cell cortex, which enhances cortical targeting of LIS1–dynein complex. Overexpression of NDEL1–dynein and MT stabilization rescues spindle orientation defects in Lis1 mutants, demonstrating that mouse LIS1 acts via the LIS1–NDEL1–dynein complex to regulate astral MT plus-ends dynamics and establish proper contacts of MTs with the cell cortex to ensure precise cell division. PMID:24030547

High-Resolution Mapping of Two Types of Spontaneous Mitotic Gene Conversion Events in Saccharomyces cerevisiae

PubMed Central

Yim, Eunice; O’Connell, Karen E.; St. Charles, Jordan; Petes, Thomas D.

2014-01-01

Gene conversions and crossovers are related products of the repair of double-stranded DNA breaks by homologous recombination. Most previous studies of mitotic gene conversion events have been restricted to measuring conversion tracts that are <5 kb. Using a genetic assay in which the lengths of very long gene conversion tracts can be measured, we detected two types of conversions: those with a median size of ∼6 kb and those with a median size of >50 kb. The unusually long tracts are initiated at a naturally occurring recombination hotspot formed by two inverted Ty elements. We suggest that these long gene conversion events may be generated by a mechanism (break-induced replication or repair of a double-stranded DNA gap) different from the short conversion tracts that likely reflect heteroduplex formation followed by DNA mismatch repair. Both the short and long mitotic conversion tracts are considerably longer than those observed in meiosis. Since mitotic crossovers in a diploid can result in a heterozygous recessive deleterious mutation becoming homozygous, it has been suggested that the repair of DNA breaks by mitotic recombination involves gene conversion events that are unassociated with crossing over. In contrast to this prediction, we found that ∼40% of the conversion tracts are associated with crossovers. Spontaneous mitotic crossover events in yeast are frequent enough to be an important factor in genome evolution. PMID:24990991

Ki-67 acts as a biological surfactant to disperse mitotic chromosomes

PubMed Central

Cuylen, Sara; Blaukopf, Claudia; Politi, Antonio Z.; Müller-Reichert, Thomas; Neumann, Beate; Poser, Ina; Ellenberg, Jan; Hyman, Anthony A.; Gerlich, Daniel W.

2016-01-01

Summary Eukaryotic genomes are partitioned into chromosomes, which during mitosis form compact and spatially well-separated mechanical bodies1–3.This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes4 and the discovery of proteins at the chromosome surface3,5,6, the molecular and biophysical basis of mitotic chromosome individuality have remained unclear. We report that Ki-67, a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of Ki-67 is not confined within a specific protein domain but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrical barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-color labeling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic for polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery and suggests that natural proteins can function as surfactants in intracellular compartmentalization. PMID:27362226

The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae.

PubMed

Lettier, Gaëlle; Feng, Qi; de Mayolo, Adriana Antúnez; Erdeniz, Naz; Reid, Robert J D; Lisby, Michael; Mortensen, Uffe H; Rothstein, Rodney

2006-11-10

Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells.

Elevating the frequency of chromosome mis-segregation as a strategy to kill tumor cells

PubMed Central

Janssen, Aniek; Kops, Geert J. P. L.; Medema, René H.

2009-01-01

The mitotic checkpoint has evolved to prevent chromosome mis-segregations by delaying mitosis when unattached chromosomes are present. Inducing severe chromosome segregation errors by ablating the mitotic checkpoint causes cell death. Here we have analyzed the consequences of gradual increases in chromosome segregation errors on the viability of tumor cells and normal human fibroblasts. Partial reduction of essential mitotic checkpoint components in four tumor cell lines caused mild chromosome mis-segregations, but no lethality. These cells were, however, remarkably more sensitive to low doses of taxol, which enhanced the amount and severity of chromosome segregation errors. Sensitization to taxol was achieved by reducing levels of Mps1 or BubR1, proteins having dual roles in checkpoint activation and chromosome alignment, but not by reducing Mad2, functioning solely in the mitotic checkpoint. Moreover, we find that untransformed human fibroblasts with reduced Mps1 levels could not be sensitized to sublethal doses of taxol. Thus, targeting the mitotic checkpoint and chromosome alignment simultaneously may selectively kill tumor cells by enhancing chromosome mis-segregations. PMID:19855003

A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division

PubMed Central

Li, Yubing; Liu, Dianyi; López-Paz, Cristina; Olson, Bradley JSC; Umen, James G

2016-01-01

Proliferating cells actively control their size by mechanisms that are poorly understood. The unicellular green alga Chlamydomonas reinhardtii divides by multiple fission, wherein a ‘counting’ mechanism couples mother cell-size to cell division number allowing production of uniform-sized daughters. We identified a sizer protein, CDKG1, that acts through the retinoblastoma (RB) tumor suppressor pathway as a D-cyclin-dependent RB kinase to regulate mitotic counting. Loss of CDKG1 leads to fewer mitotic divisions and large daughters, while mis-expression of CDKG1 causes supernumerous mitotic divisions and small daughters. The concentration of nuclear-localized CDKG1 in pre-mitotic cells is set by mother cell size, and its progressive dilution and degradation with each round of cell division may provide a link between mother cell-size and mitotic division number. Cell-size-dependent accumulation of limiting cell cycle regulators such as CDKG1 is a potentially general mechanism for size control. DOI: http://dx.doi.org/10.7554/eLife.10767.001 PMID:27015111

Localization of phosphorylated forms of Bcl-2 in mitosis: co-localization with Ki-67 and nucleolin in nuclear structures and on mitotic chromosomes.

PubMed

Barboule, Nadia; Truchet, Isabelle; Valette, Annie

2005-04-01

Bcl-2 phosphorylation is a normal physiological process occurring at mitosis or during mitotic arrest induced by microtubule damaging agents. The consequences of Bcl-2 phosphorylation on its function are still controversial. To better understand the role of Bcl-2 phosphorylation in mitosis, we studied the subcellular localization of phosphorylated forms of Bcl-2. Immunofluorescence experiments performed in synchronized HeLa cells indicate for the first time that mitotic phosphorylated forms of Bcl-2 can be detected in nuclear structures in prophase cells together with nucleolin and Ki-67. In later mitotic stages, as previously described, phosphorylated forms of Bcl-2 are localized on mitotic chromosomes. In addition, we demonstrate that Bcl-2 in these structures is at least in part phosphorylated on the T56 residue. Then, coimmunoprecipitation experiments reveal that, in cells synchronized at the onset of mitosis, Bcl-2 is present in a complex with nucleolin, cdc2 kinase and PP1 phosphatase. Taken together, these data further support the idea that Bcl-2 could have a new function at mitosis.

Developmental alterations in centrosome integrity contribute to the post-mitotic state of mammalian cardiomyocytes

PubMed Central

Zebrowski, David C; Vergarajauregui, Silvia; Wu, Chi-Chung; Piatkowski, Tanja; Becker, Robert; Leone, Marina; Hirth, Sofia; Ricciardi, Filomena; Falk, Nathalie; Giessl, Andreas; Just, Steffen; Braun, Thomas; Weidinger, Gilbert; Engel, Felix B

2015-01-01

Mammalian cardiomyocytes become post-mitotic shortly after birth. Understanding how this occurs is highly relevant to cardiac regenerative therapy. Yet, how cardiomyocytes achieve and maintain a post-mitotic state is unknown. Here, we show that cardiomyocyte centrosome integrity is lost shortly after birth. This is coupled with relocalization of various centrosome proteins to the nuclear envelope. Consequently, postnatal cardiomyocytes are unable to undergo ciliogenesis and the nuclear envelope adopts the function as cellular microtubule organizing center. Loss of centrosome integrity is associated with, and can promote, cardiomyocyte G0/G1 cell cycle arrest suggesting that centrosome disassembly is developmentally utilized to achieve the post-mitotic state in mammalian cardiomyocytes. Adult cardiomyocytes of zebrafish and newt, which are able to proliferate, maintain centrosome integrity. Collectively, our data provide a novel mechanism underlying the post-mitotic state of mammalian cardiomyocytes as well as a potential explanation for why zebrafish and newts, but not mammals, can regenerate their heart. DOI: http://dx.doi.org/10.7554/eLife.05563.001 PMID:26247711

Breast cancer mitosis detection in histopathological images with spatial feature extraction

NASA Astrophysics Data System (ADS)

Albayrak, Abdülkadir; Bilgin, Gökhan

2013-12-01

In this work, cellular mitosis detection in histopathological images has been investigated. Mitosis detection is very expensive and time consuming process. Development of digital imaging in pathology has enabled reasonable and effective solution to this problem. Segmentation of digital images provides easier analysis of cell structures in histopathological data. To differentiate normal and mitotic cells in histopathological images, feature extraction step is very crucial step for the system accuracy. A mitotic cell has more distinctive textural dissimilarities than the other normal cells. Hence, it is important to incorporate spatial information in feature extraction or in post-processing steps. As a main part of this study, Haralick texture descriptor has been proposed with different spatial window sizes in RGB and La*b* color spaces. So, spatial dependencies of normal and mitotic cellular pixels can be evaluated within different pixel neighborhoods. Extracted features are compared with various sample sizes by Support Vector Machines using k-fold cross validation method. According to the represented results, it has been shown that separation accuracy on mitotic and non-mitotic cellular pixels gets better with the increasing size of spatial window.

Anti-mitotic potential of 7-diethylamino-3(2 Prime -benzoxazolyl)-coumarin in 5-fluorouracil-resistant human gastric cancer cell line SNU620/5-FU

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Nam Hyun; Kim, Su-Nam; Oh, Joa Sub

2012-02-24

Highlights: Black-Right-Pointing-Pointer DBC exerts antiproliferative potential against 5FU-resistant human gastric cancer cells. Black-Right-Pointing-Pointer This effect is mediated by destabilization of microtubules and subsequent mitotic arrest. Black-Right-Pointing-Pointer DBC enhances apoptosis via caspase activation and downregulation of antiapoptotic genes. -- Abstract: In this study, we investigate an anti-mitotic potential of the novel synthetic coumarin-based compound, 7-diethylamino-3(2 Prime -benzoxazolyl)-coumarin, in 5-fluorouracil-resistant human gastric cancer cell line SNU-620-5FU and its parental cell SNU-620. It exerts the anti-proliferative effects with similar potencies against both cancer cells, which is mediated by destabilization of microtubules and subsequent mitotic arrest. Furthermore, this compound enhances caspase-dependent apoptotic cell deathmore » via decreased expression of anti-apoptotic genes. Taken together, our data strongly support anti-mitotic potential of 7-diethylamino-3(2 Prime -benzoxazolyl)-coumarin against drug-resistant cancer cells which will prompt us to further develop as a novel microtubule inhibitor for drug-resistant cancer chemotherapy.« less

Multilane Traffic Flow Modeling Using Cellular Automata Theory

NASA Astrophysics Data System (ADS)

Chechina, Antonina; Churbanova, Natalia; Trapeznikova, Marina

2018-02-01

The paper deals with the mathematical modeling of traffic flows on urban road networks using microscopic approach. The model is based on the cellular automata theory and presents a generalization of the Nagel-Schreckenberg model to a multilane case. The created program package allows to simulate traffic on various types of road fragments (T or X type intersection, strait road elements, etc.) and on road networks that consist of these elements. Besides that, it allows to predict the consequences of various decisions regarding road infrastructure changes, such as: number of lanes increasing/decreasing, putting new traffic lights into operation, building new roads, entrances/exits, road junctions.

Mechanisms and Regulation of Mitotic Recombination in Saccharomyces cerevisiae

PubMed Central

Symington, Lorraine S.; Rothstein, Rodney; Lisby, Michael

2014-01-01

Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA repair, replication, and chromosome segregation during cell division as well as programmed cell developmental events. This chapter will focus on homologous mitotic recombination in budding yeast Saccharomyces cerevisiae. However, there is an important link between mitotic and meiotic recombination (covered in the forthcoming chapter by Hunter et al. 2015) and many of the functions are evolutionarily conserved. Here we will discuss several models that have been proposed to explain the mechanism of mitotic recombination, the genes and proteins involved in various pathways, the genetic and physical assays used to discover and study these genes, and the roles of many of these proteins inside the cell. PMID:25381364

Recovery from DNA damage-induced G2 arrest requires actin-binding protein filamin-A/actin-binding protein 280.

PubMed

Meng, Xiangbing; Yuan, Yuan; Maestas, Adrian; Shen, Zhiyuan

2004-02-13

Filamin-A (filamin-1) is an actin-binding protein involved in the organization of actin networks. Our previous study shows that filamin-A interacts with BRCA2, and lack of filamin-A expression results in increased cellular sensitivity to several DNA damaging agents in melanoma cells (Yuan, Y., and Shen, Z. (2001) J. Biol. Chem. 276, 48318-48324), suggesting a role of filamin-A in DNA damage response. In this report, we demonstrated that deficiency of filamin-A results in an 8-h delay in the recovery from G2 arrest in response to ionizing radiation. However, filamin-A deficiency does not affect the initial activation of the G2/M checkpoint. We also found that filamin-A deficiency results in sustained activation of Chk1 and Chk2 after irradiation. This in turn causes a delay in the dephosphorylation of phospho-Cdc2, which is inhibitory to the G2/M transition. In addition, filamin-A-deficient M2 cells undergo mitotic catastrophe-related nuclear fragmentation after they are released from the G2 arrest. Together, these data suggest a functional role of filamin-A in the recovery from G2 arrest and subsequent mitotic cell death after DNA damage.

The prolyl isomerase FKBP25 regulates microtubule polymerization impacting cell cycle progression and genomic stability

PubMed Central

Dilworth, David; Gudavicius, Geoff; Xu, Xiaoxue; Boyce, Andrew K J; O’Sullivan, Connor; Serpa, Jason J; Bilenky, Misha; Petrochenko, Evgeniy V; Borchers, Christoph H; Hirst, Martin; Swayne, Leigh Anne; Howard, Perry; Nelson, Christopher J

2018-01-01

Abstract FK506 binding proteins (FKBPs) catalyze the interconversion of cis-trans proline conformers in proteins. Importantly, FK506 drugs have anti-cancer and neuroprotective properties, but the effectors and mechanisms underpinning these properties are not well understood because the cellular function(s) of most FKBP proteins are unclear. FKBP25 is a nuclear prolyl isomerase that interacts directly with nucleic acids and is associated with several DNA/RNA binding proteins. Here, we show the catalytic FKBP domain binds microtubules (MTs) directly to promote their polymerization and stabilize the MT network. Furthermore, FKBP25 associates with the mitotic spindle and regulates entry into mitosis. This interaction is important for mitotic spindle dynamics, as we observe increased chromosome instability in FKBP25 knockdown cells. Finally, we provide evidence that FKBP25 association with chromatin is cell-cycle regulated by Protein Kinase C phosphorylation. This disrupts FKBP25–DNA contacts during mitosis while maintaining its interaction with the spindle apparatus. Collectively, these data support a model where FKBP25 association with chromatin and MTs is carefully choreographed to ensure faithful genome duplication. Additionally, they highlight that FKBP25 is a MT-associated FK506 receptor and potential therapeutic target in MT-associated diseases. PMID:29361176

Human Cdc14A regulates Wee1 stability by counteracting CDK-mediated phosphorylation

PubMed Central

Ovejero, Sara; Ayala, Patricia; Bueno, Avelino; Sacristán, María P.

2012-01-01

The activity of Cdk1–cyclin B1 mitotic complexes is regulated by the balance between the counteracting activities of Wee1/Myt1 kinases and Cdc25 phosphatases. These kinases and phosphatases must be strictly regulated to ensure proper mitotic timing. One masterpiece of this regulatory network is Cdk1, which promotes Cdc25 activity and suppresses inhibitory Wee1/Myt1 kinases through direct phosphorylation. The Cdk1-dependent phosphorylation of Wee1 primes phosphorylation by additional kinases such as Plk1, triggering Wee1 degradation at the onset of mitosis. Here we report that Cdc14A plays an important role in the regulation of Wee1 stability. Depletion of Cdc14A results in a significant reduction in Wee1 protein levels. Cdc14A binds to Wee1 at its amino-terminal domain and reverses CDK-mediated Wee1 phosphorylation. In particular, we found that Cdc14A inhibits Wee1 degradation through the dephosphorylation of Ser-123 and Ser-139 residues. Thus the lack of phosphorylation of these two residues prevents the interaction with Plk1 and the consequent efficient Wee1 degradation at the onset of mitosis. These data support the hypothesis that Cdc14A counteracts Cdk1–cyclin B1 activity through Wee1 dephosphorylation. PMID:23051732

Self organized spatio-temporal structure within the fractured Vadose Zone: The influence of dynamic overloading at fracture intersections

NASA Astrophysics Data System (ADS)

LaViolette, Randall A.; Glass, Robert J.

2004-09-01

Under low flow conditions (where gravity and capillary forces dominate) within an unsaturated fracture network, fracture intersections act as capillary barriers to integrate flow from above and then release it as a pulse below. Water exiting a fracture intersection is often thought to enter the single connected fracture with the lowest invasion pressure. When the accumulated volume varies between intersections, the smaller volume intersections can be overloaded to cause all of the available fractures exiting an intersection to flow. We included the dynamic overloading process at fracture intersections within our previously discussed model where intersections were modeled as tipping buckets connected within a two-dimensional diamond lattice. With dynamic overloading, the flow behavior transitioned smoothly from diverging to converging flow with increasing overload parameter, as a consequence of a heterogeneous field, and they impose a dynamic structure where additional pathways activate or deactivate in time.

An efficient collaborative approach for black hole attack discovery and mitigating its impact in manet

NASA Astrophysics Data System (ADS)

Devipriya, K.; Ivy, B. Persis Urbana; Prabha, D.

2018-04-01

A mobile ad hoc network (MANET) is an assemblage of nodes composed of mobile devices coupled in various ways wirelessly which do not have any central administration. Each node in MANET cooperates in forwarding packets in the network. This type of collaboration incurs high cost but there exits nodes that declines to cooperate leading to selfish conduct of nodes which effects overall network performance. To discover the attacks caused by such nodes, a renowned mechanism using watchdog can be deployed. In infrastructure less network attack detection and reaction and high false positives, false negatives initiating black hole attack becomes major issue in watchdog. This paper put forward a collaborative approach for identifying such attacks in MANET. Through abstract analysis and extensive simulation of this approach, the detection time of misbehaved nodes is reduced and substantial enhancement in overhead and throughput is witnessed.

A mathematical model for adaptive transport network in path finding by true slime mold.

PubMed

Tero, Atsushi; Kobayashi, Ryo; Nakagaki, Toshiyuki

2007-02-21

We describe here a mathematical model of the adaptive dynamics of a transport network of the true slime mold Physarum polycephalum, an amoeboid organism that exhibits path-finding behavior in a maze. This organism possesses a network of tubular elements, by means of which nutrients and signals circulate through the plasmodium. When the organism is put in a maze, the network changes its shape to connect two exits by the shortest path. This process of path-finding is attributed to an underlying physiological mechanism: a tube thickens as the flux through it increases. The experimental evidence for this is, however, only qualitative. We constructed a mathematical model of the general form of the tube dynamics. Our model contains a key parameter corresponding to the extent of the feedback regulation between the thickness of a tube and the flux through it. We demonstrate the dependence of the behavior of the model on this parameter.

Satellite fixed communications service: A forecast of potential domestic demand through the year 2000. Volume 3: Appendices

NASA Technical Reports Server (NTRS)

Kratochvil, D.; Bowyer, J.; Bhushan, C.; Steinnagel, K.; Kaushal, D.; Al-Kinani, G.

1983-01-01

Voice applications, data applications, video applications, impacted baseline forecasts, market distribution model, net long haul forecasts, trunking earth station definition and costs, trunking space segment cost, trunking entrance/exit links, trunking network costs and crossover distances with terrestrial tariffs, net addressable forecasts, capacity requirements, improving spectrum utilization, satellite system market development, and the 30/20 net accessible market are considered.

Satellite fixed communications service: A forecast of potential domestic demand through the year 2000. Volume 3: Appendices

NASA Astrophysics Data System (ADS)

Kratochvil, D.; Bowyer, J.; Bhushan, C.; Steinnagel, K.; Kaushal, D.; Al-Kinani, G.

1983-09-01

Voice applications, data applications, video applications, impacted baseline forecasts, market distribution model, net long haul forecasts, trunking earth station definition and costs, trunking space segment cost, trunking entrance/exit links, trunking network costs and crossover distances with terrestrial tariffs, net addressable forecasts, capacity requirements, improving spectrum utilization, satellite system market development, and the 30/20 net accessible market are considered.

Systems cell biology of the mitotic spindle.

PubMed

Saleem, Ramsey A; Aitchison, John D

2010-01-11

Cell division depends critically on the temporally controlled assembly of mitotic spindles, which are responsible for the distribution of duplicated chromosomes to each of the two daughter cells. To gain insight into the process, Vizeacoumar et al., in this issue (Vizeacoumar et al. 2010. J. Cell Biol. doi:10.1083/jcb.200909013), have combined systems genetics with high-throughput and high-content imaging to comprehensively identify and classify novel components that contribute to the morphology and function of the mitotic spindle.

Mitotic Recombination in the Heterochromatin of the Sex Chromosomes of DROSOPHILA MELANOGASTER

PubMed Central

Ripoll, P.; Garcia-Bellido, A.

1978-01-01

The frequency of spontaneous and X-ray-induced mitotic recombination involving the Y chromosome has been studied in individuals with a marked Y chromosome arm and different XY compound chromosomes. The genotypes used include X chromosomes with different amounts of X heterochromatin and either or both arms of the Y chromosome attached to either side of the centromere. Individuals with two Y chromosomes have also been studied. The results show that the bulk of mitotic recombination takes place between homologous regions. PMID:100372

Effects of polyamines and polyamine biosynthetic inhibitors on mitotic activity of Allium cepa root tips.

PubMed

Unal, Meral; Palavan-Unsal, Narcin; Tufekci, M A

2008-03-01

The genotoxic and cytotoxic effects of exogenous polyamines (PAs), putrescine (Put), spermidine (Spd), spermine (Spm) and PA biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO), cyclohexilamine (CHA), methylglioxal bis-(guanylhydrazone) (MGBG) were investigated in the root meristems of Allium cepa L. The reduction of mitotic index and the induction of chromosomal aberrations such as bridges, stickiness, c-mitotic anaphases, micronuclei, endoredupliction by PAs and PA biosynthetic inhibitors were observed and these were used as evidence of genotoxicity and cytotoxicity.

Mitotic and apoptotic activity in colorectal neoplasia.

PubMed

Kohoutova, Darina; Pejchal, Jaroslav; Bures, Jan

2018-05-18

Colorectal cancer (CRC) is third most commonly diagnosed cancer worldwide. The aim of the prospective study was to evaluate mitosis and apoptosis of epithelial cells at each stage of colorectal neoplasia. A total of 61 persons were enrolled into the study: 18 patients with non-advanced colorectal adenoma (non-a-A), 13 patients with advanced colorectal adenoma (a-A), 13 patients with CRC and 17 controls: individuals with normal findings on colonoscopy. Biopsy samples were taken from pathology (patients) and healthy mucosa (patients and healthy controls). Samples were formalin-fixed paraffin-embedded and stained with haematoxylin-eosin. Mitotic and apoptotic activity were evaluated in lower and upper part of the crypts and in the superficial compartment. Apoptotic activity was also assessed using detection of activated caspase-3. In controls, mitotic activity was present in lower part of crypts, accompanied with low apoptotic activity. Mitotic and apoptotic activity decreased (to almost zero) in upper part of crypts. In superficial compartment, increase in apoptotic activity was observed. Transformation of healthy mucosa into non-a-A was associated with significant increase of mitotic activity in lower and upper part of the crypts and with significant increase of apoptotic activity in all three compartments; p < 0.05. Transformation of non-a-A into a-A did not lead to any further significant increase in apoptotic activity, but was related to significant increase in mitotic activity in upper part of crypts and superficial compartment. A significant decrease in apoptotic activity was detected in all three comparments of CRC samples compared to a-A; p < 0.05. No differences in mitotic and apoptotic activity between biopsies in healthy controls and biopsy samples from healthy mucosa in patients with colorectal neoplasia were observed. Detection of activated caspase-3 confirmed the above findings in apoptotic activity. Significant dysregulation of mitosis and apoptosis during the progression of colorectal neoplasia, corresponding with histology, was confirmed. In patients with sporadic colorectal neoplasia, healthy mucosa does not display different mitotic and apoptotic activity compared to mucosa in healthy controls and therefore adequate endoscopic/surgical removal of colorectal neoplasia is sufficient.

Structure Shift of GaN Among Nanowall Network, Nanocolumn, and Compact Film Grown on Si (111) by MBE.

PubMed

Zhong, Aihua; Fan, Ping; Zhong, Yuanting; Zhang, Dongping; Li, Fu; Luo, Jingting; Xie, Yizhu; Hane, Kazuhiro

2018-02-13

Structure shift of GaN nanowall network, nanocolumn, and compact film were successfully obtained on Si (111) by plasma-assisted molecular beam epitaxy (MBE). As is expected, growth of the GaN nanocolumns was observed in N-rich condition on bare Si, and the growth shifted to compact film when the Ga flux was improved. Interestingly, if an aluminum (Al) pre-deposition for 40 s was carried out prior to the GaN growth, GaN grows in the form of the nanowall network. Results show that the pre-deposited Al exits in the form of droplets with typical diameter and height of ~ 80 and ~ 6.7 nm, respectively. A growth model for the nanowall network is proposed and the growth mechanism is discussed. GaN grows in the area without Al droplets while the growth above Al droplets is hindered, resulting in the formation of continuous GaN nanowall network that removes the obstacles of nano-device fabrication.

Structure Shift of GaN Among Nanowall Network, Nanocolumn, and Compact Film Grown on Si (111) by MBE

NASA Astrophysics Data System (ADS)

Zhong, Aihua; Fan, Ping; Zhong, Yuanting; Zhang, Dongping; Li, Fu; Luo, Jingting; Xie, Yizhu; Hane, Kazuhiro

2018-02-01

Structure shift of GaN nanowall network, nanocolumn, and compact film were successfully obtained on Si (111) by plasma-assisted molecular beam epitaxy (MBE). As is expected, growth of the GaN nanocolumns was observed in N-rich condition on bare Si, and the growth shifted to compact film when the Ga flux was improved. Interestingly, if an aluminum (Al) pre-deposition for 40 s was carried out prior to the GaN growth, GaN grows in the form of the nanowall network. Results show that the pre-deposited Al exits in the form of droplets with typical diameter and height of 80 and 6.7 nm, respectively. A growth model for the nanowall network is proposed and the growth mechanism is discussed. GaN grows in the area without Al droplets while the growth above Al droplets is hindered, resulting in the formation of continuous GaN nanowall network that removes the obstacles of nano-device fabrication.

ROADMs for reconfigurable metro networks

NASA Astrophysics Data System (ADS)

Homa, Jonathan; Bala, Krishna

2009-01-01

Reconfigurable Optical Add-Drop Multiplexers (ROADMs) are the key nodal sub-systems that are used to implement modern DWDM networks. They provide network flexibility by switching wavelengths among fibers under software control without expensive conversion to the electronic domain. They speed up provisioning time, reduce operational costs and eliminate human errors. Two general types of ROADMs are used in Metro optical networks, two-degree and multi-degree, where the degree refers to the numbers of DWDM fibers entering and exiting the ROADM node. A twodegree ROADM is like a location on a highway with off and on ramps to drop off and accept local traffic while a multidegree ROADM is like an interchange where highways meet and is used for interconnecting DWDM rings or for mesh networking. The paper describes two-degree and multi-degree ROADM architectures and how these relate to the technology alternatives used to implement the ROADMs themselves. Focus is provided on the role and expected evolution of the wavelength selective switch (WSS) which is the primary engine used to power ROADMs.

Induction of a Mitosis Delay and Cell Lysis by High-Level Secretion of Mouse α-Amylase from Saccharomyces cerevisiae

PubMed Central

Wang, Bi-Dar; Kuo, Tsong-Teh

2001-01-01

Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycle dependency is unknown. In this study, we found that Saccharomyces cerevisiae cells secreting high levels of mouse α-amylase have elongated buds and are delayed in cell cycle completion in mitosis. The delayed cell mitosis suggests that critical events during exit from mitosis might be disturbed. We found that the activities of PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in α-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, compared to the accumulation in wild-type cells. MPF inactivation is due to inhibitory phosphorylation on Cdc28, as a cdc28 mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and prevents the defective bud morphology induced by overproduction of α-amylase. Our data also suggest that high levels of α-amylase may downregulate PPH22, leading to cell lysis. In conclusion, overproduction of heterologous α-amylase in S. cerevisiae results in a negative regulation of PP2A, which causes mitotic delay and leads to cell lysis. PMID:11472949

Evaluation of allelopathic, decomposition and cytogenetic activities of Jasminum officinale L. f. var. grandiflorum (L.) Kob. on bioassay plants.

PubMed

Teerarak, Montinee; Laosinwattana, Chamroon; Charoenying, Patchanee

2010-07-01

Methanolic extracts prepared from dried leaves of Jasminum officinale f. var. grandiflorum (L.) Kob. (Spanish jasmine) inhibited seed germination and stunted both root and shoot length of the weeds Echinochloa crus-galli (L.) Beauv. and Phaseolus lathyroides L. The main active compound was isolated and determined by spectral data as a secoiridoid glucoside named oleuropein. In addition, a decrease in allelopathic efficacy appeared as the decomposition periods increased. The mitotic index in treated onion root tips decreased with increasing concentrations of the extracts and longer periods of treatment. Likewise, the mitotic phase index was altered in onion incubated with crude extract. Furthermore, crude extract produced mitotic abnormalities resulting from its action on chromatin organization and mitotic spindle. Copyright (c)2010 Elsevier Ltd. All rights reserved.

The magic of numbers: malignant melanoma between science and pseudoscience.

PubMed

Weyers, Wolfgang

2011-06-01

In 2009, a new system for staging and classification of malignant melanoma has been proposed by the American Joint Committee on Cancer (AJCC). The AJCC recommends that staging of primary melanoma be based on 3 criteria, namely, thickness, ulceration, and mitotic rate, the latter substituting Clark levels in the previous classification. In melanomas measuring ≤1 mm in thickness, ulceration or finding of single mitotic figure in the dermis defines stage T1b. According to the AJCC, sentinel lymph node dissection should be considered for those melanomas because of a significantly impaired prognosis. As with other prognostic parameters, however, assessment of mitotic rate, with one mitotic figure being the cutoff point, is highly unreliable, and statistics based on such data lack validity. Despite the large database being employed, they may be pseudoscience rather than science.

Effects of intracellular pH on the mitotic apparatus and mitotic stage in the sand dollar egg.

PubMed

Watanabe, K; Hamaguchi, M S; Hamaguchi, Y

1997-01-01

The effect of change in intracellular pH (pHi) on mitosis was investigated in the sand dollar egg. The pHi in the fertilized egg of Scaphechinus mirabilis and Clypeaster japonicus, which was 7.34 and 7.31, respectively, changed by means of treating the egg at nuclear envelope breakdown with sea water containing acetate and/or ammonia at various values of pH. The mitotic apparatus at pHi 6.70 became larger than that of normal fertilized eggs; that is, the mitotic spindle had the maximal size, especially in length at pHi 6.70. The spindle length linearly decreased when pHi increased from 6.70 to 7.84. By polarization microscopy, the increase in birefringence retardation was detected at slightly acidic pHi, suggesting that the increase in size of the spindle is caused by the increase in the amount of microtubules in the spindle. At pHi 6.30, the organization of the mitotic apparatus was inhibited. Furthermore, slightly acidic pHi caused cleavage retardation or inhibition. By counting the number of the eggs at various mitotic stages with time after treating them with the media, it is found that metaphase was persistent and most of the S. mirabilis eggs were arrested at metaphase under the condition of pHi 6.70. It is concluded that at slightly acidic pH, the microtubules in the spindle are stabilized and more microtubules assembled than those in the normal eggs.

Modulation of vinblastine cytotoxicity by dilantin (phenytoin) or the protein phosphatase inhibitor okadaic acid involves the potentiation of anti-mitotic effects and induction of apoptosis in human tumour cells.

PubMed Central

Kawamura, K. I.; Grabowski, D.; Weizer, K.; Bukowski, R.; Ganapathi, R.

1996-01-01

Cellular insensitivity to vinca alkaloids is suggested to be primarily due to drug efflux by P-glycoprotein (P-gp). The anti-epileptic phenytoin (DPH), which does not bind to P-gp, can selectively enhance vincristine (VCR) cytotoxicity in wild-type (WT) or multidrug-resistant (MDR) cells. We now demonstrate that the protein phosphatase inhibitor okadaic acid (OKA) can mimic the effect of DPH by selectively enhancing cytotoxicity of vinblastine (VBL), but not taxol and doxorubicin, in human leukaemia HL-60 cells. Both DPH and OKA potentiate the anti-mitotic effects of VBL by enhanced damage to the mitotic spindle, resulting in prolonged growth arrest. Also, unlike VBL alone, in human leukaemia or non-small-cell lung carcinoma cells treated with VBL plus DPH, recovery from damage to the mitotic spindle is compromised in drug-free medium and cell death by apoptosis in interphase ensues. Since protein phosphatases are involved with the regulation of metaphase to anaphase transit of cells during the mitotic cycle, enhanced VBL cytotoxicity in the presence of DPH or OKA may involve effects during metaphase on the mitotic spindle tubulin leading to growth arrest and apoptosis in interphase. These novel results suggest that DPH or OKA could be powerful tools to study cellular effects of vinca alkaloids and possibly for the development of novel therapeutic strategies. Images Figure 6 PMID:8546904

Genetic and evolutionary analysis of the Drosophila larval neuromuscular junction

NASA Astrophysics Data System (ADS)

Campbell, Megan

Although evolution of brains and behaviors is of fundamental biological importance, we lack comprehensive understanding of the general principles governing these processes or the specific mechanisms and molecules through which the evolutionary changes are effected. Because synapses are the basic structural and functional units of nervous systems, one way to address these problems is to dissect the genetic and molecular pathways responsible for morphological evolution of a defined synapse. I have undertaken such an analysis by examining morphology of the larval neuromuscular junction (NMJ) in wild caught D. melanogaster as well as in over 20 other species of Drosophila. Whereas variation in NMJ morphology within a species is limited, I discovered a surprisingly extensive variation among different species. Compared with evolution of other morphological traits, NMJ morphology appears to be evolving very rapidly. Moreover, my data indicate that natural selection rather than genetic drift is primarily responsible for evolution of NMJ morphology. To dissect underlying molecular mechanisms that may govern NMJ growth and evolutionary divergence, I focused on a naturally occurring variant in D. melanogaster that causes NMJ overgrowth. I discovered that the variant mapped to Mob2, a gene encoding a kinase adapter protein originally described in yeast as a member of the Mitotic Exit Network (MEN). I have subsequently examined mutations in the Drosophila orthologs of all the core components of the yeast MEN and found that all of them function as part of a common pathway that acts presynaptically to negatively regulate NMJ growth. As in the regulation of yeast cytokinesis, these components of the MEN appear to act ultimately by regulating actin dynamics during the process of bouton growth and division. These studies have thus led to the discovery of an entirely new role for the MEN---regulation of synaptic growth---that is separate from its function in cell division. This work has identified a rich source of material for discovery of novel genes and mechanisms that regulate synaptic growth and development, and has also provided new insights into the mechanisms that underlie morphological evolution of nervous systems.

Mechanical continuity and reversible chromosome disassembly within intact genomes removed from living cells

NASA Technical Reports Server (NTRS)

Maniotis, A. J.; Bojanowski, K.; Ingber, D. E.

1997-01-01

Chromatin is thought to be structurally discontinuous because it is packaged into morphologically distinct chromosomes that appear physically isolated from one another in metaphase preparations used for cytogenetic studies. However, analysis of chromosome positioning and movement suggest that different chromosomes often behave as if they were physically connected in interphase as well as mitosis. To address this paradox directly, we used a microsurgical technique to physically remove nucleoplasm or chromosomes from living cells under isotonic conditions. Using this approach, we found that pulling a single nucleolus or chromosome out from interphase or mitotic cells resulted in sequential removal of the remaining nucleoli and chromosomes, interconnected by a continuous elastic thread. Enzymatic treatments of interphase nucleoplasm and chromosome chains held under tension revealed that mechanical continuity within the chromatin was mediated by elements sensitive to DNase or micrococcal nuclease, but not RNases, formamide at high temperature, or proteases. In contrast, mechanical coupling between mitotic chromosomes and the surrounding cytoplasm appeared to be mediated by gelsolin-sensitive microfilaments. Furthermore, when ion concentrations were raised and lowered, both the chromosomes and the interconnecting strands underwent multiple rounds of decondensation and recondensation. As a result of these dynamic structural alterations, the mitotic chains also became sensitive to disruption by restriction enzymes. Ion-induced chromosome decondensation could be blocked by treatment with DNA binding dyes, agents that reduce protein disulfide linkages within nuclear matrix, or an antibody directed against histones. Fully decondensed chromatin strands also could be induced to recondense into chromosomes with pre-existing size, shape, number, and position by adding anti-histone antibodies. Conversely, removal of histones by proteolysis or heparin treatment produced chromosome decondensation which could be reversed by addition of histone H1, but not histones H2b or H3. These data suggest that DNA, its associated protein scaffolds, and surrounding cytoskeletal networks function as a structurally-unified system. Mechanical coupling within the nucleoplasm may coordinate dynamic alterations in chromatin structure, guide chromosome movement, and ensure fidelity of mitosis.

Impact of the 2009 AJCC staging guidelines for melanoma on the number of mitotic figures reported by dermatopathologists at one institution.

PubMed

Larson, Allison R; Rothschild, Brian; Walls, Andrew C; Granter, Scott R; Qureshi, Abrar A; Murphy, George F; Laga, Alvaro C

2015-08-01

In 2009 the revised seventh staging system for melanoma recommended the use of mitotic count to separate stage T1a from T1b. However, careful scrutiny of cases may lead to an inadvertent selection effect, with consequent increased reporting of mitotic counts. We investigated whether there is a significant increase in mitotic counts reported since 2009 for melanomas with a Breslow thickness of 1.0 mm or less. We conducted a retrospective, case-controlled study examining invasive melanoma cases at a large academic center. Mitotic counts were compared between pathology reports before 2009 (n = 61) and after 2009 (n = 125), with a subset of slides re-examined in a blinded fashion. Before the 2009 staging guidelines, 51% of cases had one or more mitosis reported compared to 38% after 2009 (p = 0.113). Blinded re-counting did not yield a significant difference when compared with the original pathology reports in either group. There was not a significant difference in the number of mitoses reported after the implementation of the new guidelines. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Clinicopathological relevance of tumour grading in canine osteosarcoma.

PubMed

Loukopoulos, P; Robinson, W F

2007-01-01

Tumour grading assesses biological aggressiveness and is of prognostic significance in many malignancies. The clinicopathological features of 140 primary canine osteosarcomas and their metastases were analysed, and the interrelations between them and an established grading system and its constituent parameters (mitotic index, necrosis, pleomorphism) were examined. Of these tumours, 35% were grade III (high-grade), 37% grade II and 28% grade I. Primary tumours that had metastasized were of significantly higher grade than non-metastatic osteosarcomas. Osteosarcomas belonging to the osteoblastic minimally productive subtype, but not chondroblastic or telangiectatic subtypes, differed from fibroblastic osteosarcomas in being associated with a significantly higher number of high-grade cases. Dogs younger than 4 years of age had osteosarcomas with higher grade, score and mitotic index than did older animals. Appendicular differed from axial tumours in having a higher mitotic index; distal differed from proximal tumours in being of higher grade; cranial tumours differed from tumours in most other sites in being of lower grade and lower mitotic index. Rib osteosarcomas showed a particularly high degree of necrosis. The mitotic index varied widely between tumour locations. Pleomorphism did not have prognostic merit when examined separately, as most osteosarcomas were highly pleomorphic.

Defects in chromosome congression and mitotic progression in KIF18A-deficient cells are partly mediated through impaired functions of CENP-E.

PubMed

Huang, Ying; Yao, Yixin; Xu, Han-Zhang; Wang, Zhu-Gang; Lu, Luo; Dai, Wei

2009-08-15

KIF18A, a molecular motor, is an essential component in the regulation of orderly chromosome congression by attenuation of the kinetochore oscillation amplitude at the midzone during mitosis in vertebrate cells. Here we report that KIF18A depletion resulted in mitotic arrest which was accompanied by the presence of unaligned chromosomes in HeLa cells. This resembles the phenotype induced by an impaired function of CENP-E, also a mitotic kinesin essential for the formation of the mitotic spindles. Our further analysis showed that KIF18A depletion caused specific downregulation of CENP-E. Downregulation of CENP-E as the result of KIF18A silencing was not due to reduced transcription but primarily due to the enhanced protein degradation. Co-immunoprecipitation revealed that KIF18A physically interacted with CENP-E and BubR1 during mitosis. Ectopic expression of the wild-type tail domain of CENP-E, but not a corresponding mutant, significantly suppressed chromosome congression defects in mitotic cells. Together, our studies strongly suggest that chromosome congression defects as the result of KIF18A depletion is at least in part mediated through destabilizing kinetochore CENP-E.

Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Feng; Camp, David G.; Gritsenko, Marina A.

2007-11-16

The chromosomal passenger complex (CPC) is a critical regulator of chromosome, cytoskeleton and membrane dynamics during mitosis. Here, we identified phosphopeptides and phosphoprotein complexes recognized by a phosphorylation specific antibody that labels the CPC using liquid chromatography coupled to mass spectrometry. A mitotic phosphorylation motif (PX{G/T/S}{L/M}[pS]P or WGL[pS]P) was identified in 11 proteins including Fzr/Cdh1 and RIC-8, two proteins with potential links to the CPC. Phosphoprotein complexes contained known CPC components INCENP, Aurora-B and TD-60, as well as SMAD2, 14-3-3 proteins, PP2A, and Cdk1, a likely kinase for this motif. Protein sequence analysis identified phosphorylation motifs in additional proteins includingmore » SMAD2, Plk3 and INCENP. Mitotic SMAD2 and Plk3 phosphorylation was confirmed using phosphorylation specific antibodies, and in the case of Plk3, phosphorylation correlates with its localization to the mitotic apparatus. A mutagenesis approach was used to show INCENP phosphorylation is required for midbody localization. These results provide evidence for a shared phosphorylation event that regulates localization of critical proteins during mitosis.« less

The Sin3p PAH Domains Provide Separate Functions Repressing Meiotic Gene Transcription in Saccharomyces cerevisiae ▿

PubMed Central

Mallory, Michael J.; Law, Michael J.; Buckingham, Lela E.; Strich, Randy

2010-01-01

Meiotic genes in budding yeast are repressed during vegetative growth but are transiently induced during specific stages of meiosis. Sin3p represses the early meiotic gene (EMG) by bridging the DNA binding protein Ume6p to the histone deacetylase Rpd3p. Sin3p contains four paired amphipathic helix (PAH) domains, one of which (PAH3) is required for repressing several genes expressed during mitotic cell division. This report examines the roles of the PAH domains in mediating EMG repression during mitotic cell division and following meiotic induction. PAH2 and PAH3 are required for mitotic EMG repression, while electrophoretic mobility shift assays indicate that only PAH2 is required for stable Ume6p-promoter interaction. Unlike mitotic repression, reestablishing EMG repression following transient meiotic induction requires PAH3 and PAH4. In addition, the role of Sin3p in reestablishing repression is expanded to include additional loci that it does not control during vegetative growth. These findings indicate that mitotic and postinduction EMG repressions are mediated by two separate systems that utilize different Sin3p domains. PMID:20971827

Real-time fluorescence imaging of the DNA damage repair response during mitosis.

PubMed

Miwa, Shinji; Yano, Shuya; Yamamoto, Mako; Matsumoto, Yasunori; Uehara, Fuminari; Hiroshima, Yukihiko; Toneri, Makoto; Murakami, Takashi; Kimura, Hiroaki; Hayashi, Katsuhiro; Yamamoto, Norio; Efimova, Elena V; Tsuchiya, Hiroyuki; Hoffman, Robert M

2015-04-01

The response to DNA damage during mitosis was visualized using real-time fluorescence imaging of focus formation by the DNA-damage repair (DDR) response protein 53BP1 linked to green fluorescent protein (GFP) (53BP1-GFP) in the MiaPaCa-2(Tet-On) pancreatic cancer cell line. To observe 53BP1-GFP foci during mitosis, MiaPaCa-2(Tet-On) 53BP1-GFP cells were imaged every 30 min by confocal microscopy. Time-lapse imaging demonstrated that 11.4 ± 2.1% of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells had increased focus formation over time. Non-mitotic cells did not have an increase in 53BP1-GFP focus formation over time. Some of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells with focus formation became apoptotic. The results of the present report suggest that DNA strand breaks occur during mitosis and undergo repair, which may cause some of the mitotic cells to enter apoptosis in a phenomenon possibly related to mitotic catastrophe. © 2014 Wiley Periodicals, Inc.

Mediator can regulate mitotic entry and direct periodic transcription in fission yeast.

PubMed

Banyai, Gabor; Lopez, Marcela Davila; Szilagyi, Zsolt; Gustafsson, Claes M

2014-11-01

Cdk8 is required for correct timing of mitotic progression in fission yeast. How the activity of Cdk8 is regulated is unclear, since the kinase is not activated by T-loop phosphorylation and its partner, CycC, does not oscillate. Cdk8 is, however, a component of the multiprotein Mediator complex, a conserved coregulator of eukaryotic transcription that is connected to a number of intracellular signaling pathways. We demonstrate here that other Mediator components regulate the activity of Cdk8 in vivo and thereby direct the timing of mitotic entry. Deletion of Mediator components Med12 and Med13 leads to higher cellular Cdk8 protein levels, premature phosphorylation of the Cdk8 target Fkh2, and earlier entry into mitosis. We also demonstrate that Mediator is recruited to clusters of mitotic genes in a periodic fashion and that the complex is required for the transcription of these genes. We suggest that Mediator functions as a hub for coordinated regulation of mitotic progression and cell cycle-dependent transcription. The many signaling pathways and activator proteins shown to function via Mediator may influence the timing of these cell cycle events. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Comparative effects of 60Co gamma-rays and neon and helium ions on cycle duration and division probability of EMT 6 cells. A time-lapse cinematography study.

PubMed

Collyn-d'Hooghe, M; Hemon, D; Gilet, R; Curtis, S B; Valleron, A J; Malaise, E P

1981-03-01

Exponentially growing cultures of EMT 6 cells were irradiated in vitro with neon ions, helium ions or 60Co gamma-rays. Time-lapse cinematography allowed the determination, for individual cells, of cycle duration, success of the mitotic division and the age of the cell at the moment of irradiation. Irradiation induced a significant mitotic delay increasing proportionally with the delivered dose. Using mitotic delay as an endpoint, the r.b.e. for neon ions with respect to 60Co gamma-rays was 3.3 +/- 0.2 while for helium ions it was 1.2 +/- 0.1. Mitotic delay was greatest in those cells that had progressed furthest in their cycle at the time of irradiation. No significant mitotic delay was observed in the post-irradiation generation. Division probability was significantly reduced by irradiation both in the irradiated and in the post-irradiated generation. The reduction in division probability obtained with 3 Gy of neon ions was similar to that obtained after irradiation with 6 Gy of helium ions or 60Co gamma-rays.

Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2

PubMed Central

Andersen, Joshua L; Johnson, Carrie E; Freel, Christopher D; Parrish, Amanda B; Day, Jennifer L; Buchakjian, Marisa R; Nutt, Leta K; Thompson, J Will; Moseley, M Arthur; Kornbluth, Sally

2009-01-01

The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1–cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1–cyclin B1 activity must be overcome for apoptosis to occur. PMID:19730412

Greatwall is essential to prevent mitotic collapse after nuclear envelope breakdown in mammals.

PubMed

Álvarez-Fernández, Mónica; Sánchez-Martínez, Ruth; Sanz-Castillo, Belén; Gan, Pei Pei; Sanz-Flores, María; Trakala, Marianna; Ruiz-Torres, Miguel; Lorca, Thierry; Castro, Anna; Malumbres, Marcos

2013-10-22

Greatwall is a protein kinase involved in the inhibition of protein phosphatase 2 (PP2A)-B55 complexes to maintain the mitotic state. Although its biochemical activity has been deeply characterized in Xenopus, its specific relevance during the progression of mitosis is not fully understood. By using a conditional knockout of the mouse ortholog, Mastl, we show here that mammalian Greatwall is essential for mouse embryonic development and cell cycle progression. Yet, Greatwall-null cells enter into mitosis with normal kinetics. However, these cells display mitotic collapse after nuclear envelope breakdown (NEB) characterized by defective chromosome condensation and prometaphase arrest. Intriguingly, Greatwall is exported from the nucleus to the cytoplasm in a CRM1-dependent manner before NEB. This export occurs after the nuclear import of cyclin B-Cdk1 complexes, requires the kinase activity of Greatwall, and is mediated by Cdk-, but not Polo-like kinase 1-dependent phosphorylation. The mitotic collapse observed in Greatwall-deficient cells is partially rescued after concomitant depletion of B55 regulatory subunits, which are mostly cytoplasmic before NEB. These data suggest that Greatwall is an essential protein in mammals required to prevent mitotic collapse after NEB.

Impaired mitotic progression and preimplantation lethality in mice lacking OMCG1, a new evolutionarily conserved nuclear protein.

PubMed

Artus, Jérôme; Vandormael-Pournin, Sandrine; Frödin, Morten; Nacerddine, Karim; Babinet, Charles; Cohen-Tannoudji, Michel

2005-07-01

While highly conserved through evolution, the cell cycle has been extensively modified to adapt to new developmental programs. Recently, analyses of mouse mutants revealed that several important cell cycle regulators are either dispensable for development or have a tissue- or cell-type-specific function, indicating that many aspects of cell cycle regulation during mammalian embryo development remain to be elucidated. Here, we report on the characterization of a new gene, Omcg1, which codes for a nuclear zinc finger protein. Embryos lacking Omcg1 die by the end of preimplantation development. In vitro cultured Omcg1-null blastocysts exhibit a dramatic reduction in the total cell number, a high mitotic index, and the presence of abnormal mitotic figures. Importantly, we found that Omcg1 disruption results in the lengthening of M phase rather than in a mitotic block. We show that the mitotic delay in Omcg1-/- embryos is associated with neither a dysfunction of the spindle checkpoint nor abnormal global histone modifications. Taken together, these results suggest that Omcg1 is an important regulator of the cell cycle in the preimplantation embryo.

Impaired Mitotic Progression and Preimplantation Lethality in Mice Lacking OMCG1, a New Evolutionarily Conserved Nuclear Protein†

PubMed Central

Artus, Jérôme; Vandormael-Pournin, Sandrine; Frödin, Morten; Nacerddine, Karim; Babinet, Charles; Cohen-Tannoudji, Michel

2005-01-01

While highly conserved through evolution, the cell cycle has been extensively modified to adapt to new developmental programs. Recently, analyses of mouse mutants revealed that several important cell cycle regulators are either dispensable for development or have a tissue- or cell-type-specific function, indicating that many aspects of cell cycle regulation during mammalian embryo development remain to be elucidated. Here, we report on the characterization of a new gene, Omcg1, which codes for a nuclear zinc finger protein. Embryos lacking Omcg1 die by the end of preimplantation development. In vitro cultured Omcg1-null blastocysts exhibit a dramatic reduction in the total cell number, a high mitotic index, and the presence of abnormal mitotic figures. Importantly, we found that Omcg1 disruption results in the lengthening of M phase rather than in a mitotic block. We show that the mitotic delay in Omcg1−/− embryos is associated with neither a dysfunction of the spindle checkpoint nor abnormal global histone modifications. Taken together, these results suggest that Omcg1 is an important regulator of the cell cycle in the preimplantation embryo. PMID:15988037

Value of counting positive PHH3 cells in the diagnosis of uterine smooth muscle tumors

PubMed Central

Pang, Shu-Jie; Li, Cheng-Cheng; Shen, Yan; Liu, Yian-Zhu; Shi, Yi-Quan; Liu, Yi-Xin

2015-01-01

The diagnosis of uterine smooth muscle tumors including leiomyosarcomas (LMS), smooth muscle tumors of uncertain malignant potential (STUMP), bizarre (atypical) leiomyoma (BLM), mitotically active leiomyoma (MAL) and leiomyoma (LM) depends on a combination of microscopic features, such as mitoses, cytologic atypia, and coagulative tumor cell necrosis. However, a small number of these tumors still pose difficult diagnostic challenges. The assessment of accurate mitotic figures (MF) is one of the major parameters in the proper classification of uterine smooth muscle tumors. This assessment can be hampered by the presence of increased number of apoptotic bodies or pyknotic nuclei, which frequently mimic mitoses. Phospho-histone H3 (PHH3) is a recently described immunomarker specific for cells undergoing mitoses. In our study, we collected 132 cases of uterine smooth muscle tumors, including 26 LMSs, 16 STUMPs, 30 BLMs, 30 MALs and 30 LMs. We used mitosis specific marker PHH3 to count mitotic indexes (MI) of uterine smooth muscle tumors and compared with the mitotic indexes of hematoxylin and eosin (H&E). There is a positive correlation with the number of mitotic figures in H&E-stained sections and PHH3-stained sections (r=0.944, P<0.05). The ratio of PHH3-MI to H&E-MI has no statistically significant difference in each group except for LMs (P>0.05). The counting value of PHH3 in LMSs have significantly higher than STUMPs, BLMs, MALs and LMs (P<0.001) and the counting value of PHH3 is 1.5±0.5 times of the number of mitotic indexes in H&E. To conclude, our results show that counting PHH3 is a useful index in the diagnosis of uterine smooth muscle tumors and it can provide a more accurate index instead of the time-honored mitotic figure counts at a certain ratio. PMID:26191133

Genetic effects of methyl benzimidazole-2-yl-carbamate on Saccharomyces cerevisiae.

PubMed Central

Wood, J S

1982-01-01

The genetic effects of the mitotic inhibitor methyl benzimidazole-2-yl-carbamate (MBC) have been studied in Saccharomyces cerevisiae. MBC had little or no effect on the frequency of mutation. In some experiments MBC caused an increase in the frequency of mitotic recombination; however, this effect was small and not reproducible. The primary genetic effect of MBC was to induce mitotic chromosome loss at a high frequency. Chromosome loss occurred at equal frequencies for all chromosomes tested (13 of 16). Cells which had lost multiple chromosomes were found more frequently than predicted if individual chromosome loss events were independent. The probability of loss for a particular chromosome increased with length of time cells were incubated with MBC. MBC treatment also increased the frequency at which polyploid cells were found. These results suggested that MBC acted to disrupt the structure or function of the mitotic spindle and cause chromosome nondisjunction. PMID:6757720

EGF Induced Centrosome Separation Promotes Mitotic Progression and Cell Survival

PubMed Central

Mardin, Balca R.; Isokane, Mayumi; Cosenza, Marco R.; Krämer, Alwin; Ellenberg, Jan; Fry, Andrew M.; Schiebel, Elmar

2014-01-01

Summary Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy since cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5. PMID:23643362

Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement

PubMed Central

Sorce, Barbara; Escobedo, Carlos; Toyoda, Yusuke; Stewart, Martin P.; Cattin, Cedric J.; Newton, Richard; Banerjee, Indranil; Stettler, Alexander; Roska, Botond; Eaton, Suzanne; Hyman, Anthony A.; Hierlemann, Andreas; Müller, Daniel J.

2015-01-01

Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells. PMID:26602832

A nontranscriptional role for Oct4 in the regulation of mitotic entry

PubMed Central

Zhao, Rui; Deibler, Richard W.; Lerou, Paul H.; Ballabeni, Andrea; Heffner, Garrett C.; Cahan, Patrick; Unternaehrer, Juli J.; Kirschner, Marc W.; Daley, George Q.

2014-01-01

Rapid progression through the cell cycle and a very short G1 phase are defining characteristics of embryonic stem cells. This distinct cell cycle is driven by a positive feedback loop involving Rb inactivation and reduced oscillations of cyclins and cyclin-dependent kinase (Cdk) activity. In this setting, we inquired how ES cells avoid the potentially deleterious consequences of premature mitotic entry. We found that the pluripotency transcription factor Oct4 (octamer-binding transcription factor 4) plays an unappreciated role in the ES cell cycle by forming a complex with cyclin–Cdk1 and inhibiting Cdk1 activation. Ectopic expression of Oct4 or a mutant lacking transcriptional activity recapitulated delayed mitotic entry in HeLa cells. Reduction of Oct4 levels in ES cells accelerated G2 progression, which led to increased chromosomal missegregation and apoptosis. Our data demonstrate an unexpected nontranscriptional function of Oct4 in the regulation of mitotic entry. PMID:25324523

Disappearance of nucleosome positioning in mitotic chromatin in vivo.

PubMed

Komura, Jun-ichiro; Ono, Tetsuya

2005-04-15

During mitosis, transcription is silenced and most transcription factors are displaced from their recognition sequences. By in vivo footprinting analysis, we have confirmed and extended previous studies showing loss of transcription factors from an RNA polymerase II promoter (c-FOS) and, for the first time, an RNA polymerase III promoter (U6) in HeLa cells. Because little was known about nucleosomal organization in mitotic chromosomes, we performed footprinting analysis for nucleosomes on these promoters in interphase and mitotic cells. During interphase, each of the promoters had a positioned nucleosome in the region intervening between proximal promoter elements and distal enhancer elements, but the strong nucleosome positioning disappeared during mitosis. Thus, the nucleosomal organization that appears to facilitate transcription in interphase cells may be lost in mitotic cells, and nucleosome positioning during mitosis does not seem to be a major component of the epigenetic mechanisms to mark genes for rapid reactivation after this phase.

Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement

NASA Astrophysics Data System (ADS)

Sorce, Barbara; Escobedo, Carlos; Toyoda, Yusuke; Stewart, Martin P.; Cattin, Cedric J.; Newton, Richard; Banerjee, Indranil; Stettler, Alexander; Roska, Botond; Eaton, Suzanne; Hyman, Anthony A.; Hierlemann, Andreas; Müller, Daniel J.

2015-11-01

Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

Joint effects of microwave and chromium trioxide on root tip cells of Vicia faba *

PubMed Central

Qian, Xiao-Wei; Luo, Wei-Hua; Zheng, Ou-Xiang

2006-01-01

The mutagenic effects of microwave and chromium trioxide (CrO3) on Vicia faba root tip were studied. Micronucleus assay and chromosomal aberration assay were used to determine the mitotic index, the micronucleus frequency and chromosomal aberration frequency of Vicia faba root tip cells induced by microwave and CrO3. The results showed that the micronucleus frequency decreased, and that the mitotic index and chromosomal aberration frequency showed linear dose responses to CrO3, in treatment of microwave for 5 s. In microwave of 25 s, the mitotic index decreased, the micronucleus frequency and chromosomal aberration frequency increased with increase of CrO3 concentration. We concluded that microwave and CrO3 had antagonistic effect on the mitotic index of Vicia faba root tip cells, but had synergetic effect on micronucleus frequency and chromosomal aberration frequency of Vicia faba root tip cells. PMID:16502510

Joint effects of microwave and chromium trioxide on root tip cells of Vicia faba.

PubMed

Qian, Xiao-wei; Luo, Wei-hua; Zheng, Ou-xiang

2006-03-01

The mutagenic effects of microwave and chromium trioxide (CrO(3)) on Vicia faba root tip were studied. Micronucleus assay and chromosomal aberration assay were used to determine the mitotic index, the micronucleus frequency and chromosomal aberration frequency of Vicia faba root tip cells induced by microwave and CrO(3). The results showed that the micronucleus frequency decreased, and that the mitotic index and chromosomal aberration frequency showed linear dose responses to CrO(3), in treatment of microwave for 5 s. In microwave of 25 s, the mitotic index decreased, the micronucleus frequency and chromosomal aberration frequency increased with increase of CrO(3) concentration. We concluded that microwave and CrO(3) had antagonistic effect on the mitotic index of Vicia faba root tip cells, but had synergetic effect on micronucleus frequency and chromosomal aberration frequency of Vicia faba root tip cells.

The pso4-1 mutation reduces spontaneous mitotic gene conversion and reciprocal recombination in Saccharomyces cerevisiae.

PubMed

Meira, L B; Fonseca, M B; Averbeck, D; Schenberg, A C; Henriques, J A

1992-11-01

Spontaneous mitotic recombination was examined in the haploid pso4-1 mutant of Saccharomyces cerevisiae and in the corresponding wild-type strain. Using a genetic system involving a duplication of the his4 gene it was shown that the pso4-1 mutation decreases at least fourfold the spontaneous rate of mitotic recombination. The frequency of spontaneous recombination was reduced tenfold in pso4-1 strains, as previously observed in the rad52-1 mutant. However, whereas the rad52-1 mutation specifically reduces gene conversion, the pso4-1 mutation reduces both gene conversion and reciprocal recombination. Induced mitotic recombination was also studied in pso4-1 mutant and wild-type strains after treatment with 8-methoxypsoralen plus UVA and 254 nm UV irradiation. Consistent with previous results, the pso4-1 mutation was found strongly to affect recombination induction.

Genetic variation in mitotic regulatory pathway genes is associated with breast tumor grade

PubMed Central

Purrington, Kristen S.; Slettedahl, Seth; Bolla, Manjeet K.; Michailidou, Kyriaki; Czene, Kamila; Nevanlinna, Heli; Bojesen, Stig E.; Andrulis, Irene L.; Cox, Angela; Hall, Per; Carpenter, Jane; Yannoukakos, Drakoulis; Haiman, Christopher A.; Fasching, Peter A.; Mannermaa, Arto; Winqvist, Robert; Brenner, Hermann; Lindblom, Annika; Chenevix-Trench, Georgia; Benitez, Javier; Swerdlow, Anthony; Kristensen, Vessela; Guénel, Pascal; Meindl, Alfons; Darabi, Hatef; Eriksson, Mikael; Fagerholm, Rainer; Aittomäki, Kristiina; Blomqvist, Carl; Nordestgaard, Børge G.; Nielsen, Sune F.; Flyger, Henrik; Wang, Xianshu; Olswold, Curtis; Olson, Janet E.; Mulligan, Anna Marie; Knight, Julia A.; Tchatchou, Sandrine; Reed, Malcolm W.R.; Cross, Simon S.; Liu, Jianjun; Li, Jingmei; Humphreys, Keith; Clarke, Christine; Scott, Rodney; Fostira, Florentia; Fountzilas, George; Konstantopoulou, Irene; Henderson, Brian E.; Schumacher, Fredrick; Le Marchand, Loic; Ekici, Arif B.; Hartmann, Arndt; Beckmann, Matthias W.; Hartikainen, Jaana M.; Kosma, Veli-Matti; Kataja, Vesa; Jukkola-Vuorinen, Arja; Pylkäs, Katri; Kauppila, Saila; Dieffenbach, Aida Karina; Stegmaier, Christa; Arndt, Volker; Margolin, Sara; Balleine, Rosemary; Arias Perez, Jose Ignacio; Pilar Zamora, M.; Menéndez, Primitiva; Ashworth, Alan; Jones, Michael; Orr, Nick; Arveux, Patrick; Kerbrat, Pierre; Truong, Thérèse; Bugert, Peter; Toland, Amanda E.; Ambrosone, Christine B.; Labrèche, France; Goldberg, Mark S.; Dumont, Martine; Ziogas, Argyrios; Lee, Eunjung; Dite, Gillian S.; Apicella, Carmel; Southey, Melissa C.; Long, Jirong; Shrubsole, Martha; Deming-Halverson, Sandra; Ficarazzi, Filomena; Barile, Monica; Peterlongo, Paolo; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Tollenaar, Robert A.E.M.; Seynaeve, Caroline; Brüning, Thomas; Ko, Yon-Dschun; Van Deurzen, Carolien H.M.; Martens, John W.M.; Kriege, Mieke; Figueroa, Jonine D.; Chanock, Stephen J.; Lissowska, Jolanta; Tomlinson, Ian; Kerin, Michael J.; Miller, Nicola; Schneeweiss, Andreas; Tapper, William J.; Gerty, Susan M.; Durcan, Lorraine; Mclean, Catriona; Milne, Roger L.; Baglietto, Laura; dos Santos Silva, Isabel; Fletcher, Olivia; Johnson, Nichola; Van'T Veer, Laura J.; Cornelissen, Sten; Försti, Asta; Torres, Diana; Rüdiger, Thomas; Rudolph, Anja; Flesch-Janys, Dieter; Nickels, Stefan; Weltens, Caroline; Floris, Giuseppe; Moisse, Matthieu; Dennis, Joe; Wang, Qin; Dunning, Alison M.; Shah, Mitul; Brown, Judith; Simard, Jacques; Anton-Culver, Hoda; Neuhausen, Susan L.; Hopper, John L.; Bogdanova, Natalia; Dörk, Thilo; Zheng, Wei; Radice, Paolo; Jakubowska, Anna; Lubinski, Jan; Devillee, Peter; Brauch, Hiltrud; Hooning, Maartje; García-Closas, Montserrat; Sawyer, Elinor; Burwinkel, Barbara; Marmee, Frederick; Eccles, Diana M.; Giles, Graham G.; Peto, Julian; Schmidt, Marjanka; Broeks, Annegien; Hamann, Ute; Chang-Claude, Jenny; Lambrechts, Diether; Pharoah, Paul D.P.; Easton, Douglas; Pankratz, V. Shane; Slager, Susan; Vachon, Celine M.; Couch, Fergus J.

2014-01-01

Mitotic index is an important component of histologic grade and has an etiologic role in breast tumorigenesis. Several small candidate gene studies have reported associations between variation in mitotic genes and breast cancer risk. We measured associations between 2156 single nucleotide polymorphisms (SNPs) from 194 mitotic genes and breast cancer risk, overall and by histologic grade, in the Breast Cancer Association Consortium (BCAC) iCOGS study (n = 39 067 cases; n = 42 106 controls). SNPs in TACC2 [rs17550038: odds ratio (OR) = 1.24, 95% confidence interval (CI) 1.16–1.33, P = 4.2 × 10−10) and EIF3H (rs799890: OR = 1.07, 95% CI 1.04–1.11, P = 8.7 × 10−6) were significantly associated with risk of low-grade breast cancer. The TACC2 signal was retained (rs17550038: OR = 1.15, 95% CI 1.07–1.23, P = 7.9 × 10−5) after adjustment for breast cancer risk SNPs in the nearby FGFR2 gene, suggesting that TACC2 is a novel, independent genome-wide significant genetic risk locus for low-grade breast cancer. While no SNPs were individually associated with high-grade disease, a pathway-level gene set analysis showed that variation across the 194 mitotic genes was associated with high-grade breast cancer risk (P = 2.1 × 10−3). These observations will provide insight into the contribution of mitotic defects to histological grade and the etiology of breast cancer. PMID:24927736

Mitotic control of human papillomavirus genome-containing cells is regulated by the function of the PDZ-binding motif of the E6 oncoprotein.

PubMed

Marsh, Elizabeth K; Delury, Craig P; Davies, Nicholas J; Weston, Christopher J; Miah, Mohammed A L; Banks, Lawrence; Parish, Joanna L; Higgs, Martin R; Roberts, Sally

2017-03-21

The function of a conserved PDS95/DLG1/ZO1 (PDZ) binding motif (E6 PBM) at the C-termini of E6 oncoproteins of high-risk human papillomavirus (HPV) types contributes to the development of HPV-associated malignancies. Here, using a primary human keratinocyte-based model of the high-risk HPV18 life cycle, we identify a novel link between the E6 PBM and mitotic stability. In cultures containing a mutant genome in which the E6 PBM was deleted there was an increase in the frequency of abnormal mitoses, including multinucleation, compared to cells harboring the wild type HPV18 genome. The loss of the E6 PBM was associated with a significant increase in the frequency of mitotic spindle defects associated with anaphase and telophase. Furthermore, cells carrying this mutant genome had increased chromosome segregation defects and they also exhibited greater levels of genomic instability, as shown by an elevated level of centromere-positive micronuclei. In wild type HPV18 genome-containing organotypic cultures, the majority of mitotic cells reside in the suprabasal layers, in keeping with the hyperplastic morphology of the structures. However, in mutant genome-containing structures a greater proportion of mitotic cells were retained in the basal layer, which were often of undefined polarity, thus correlating with their reduced thickness. We conclude that the ability of E6 to target cellular PDZ proteins plays a critical role in maintaining mitotic stability of HPV infected cells, ensuring stable episome persistence and vegetative amplification.

The relative effect of citral on mitotic microtubules in wheat roots and BY2 cells.

PubMed

Chaimovitsh, D; Rogovoy Stelmakh, O; Altshuler, O; Belausov, E; Abu-Abied, M; Rubin, B; Sadot, E; Dudai, N

2012-03-01

The plant volatile monoterpene citral is a highly active compound with suggested allelopathic traits. Seed germination and seedling development are inhibited in the presence of citral, and it disrupts microtubules in both plant and animal cells in interphase. We addressed the following additional questions: can citral interfere with cell division; what is the relative effect of citral on mitotic microtubules compared to interphase cortical microtubules; what is its effect on newly formed cell plates; and how does it affect the association of microtubules with γ-tubulin? In wheat seedlings, citral led to inhibition of root elongation, curvature of newly formed cell walls and deformation of microtubule arrays. Citral's effect on microtubules was both dose- and time-dependent, with mitotic microtubules appearing to be more sensitive to citral than cortical microtubules. Association of γ-tubulin with microtubules was more sensitive to citral than were the microtubules themselves. To reveal the role of disrupted mitotic microtubules in dictating aberrations in cell plates in the presence of citral, we used tobacco BY2 cells expressing GFP-Tua6. Citral disrupted mitotic microtubules, inhibited the cell cycle and increased the frequency of asymmetric cell plates in these cells. The time scale of citral's effect in BY2 cells suggested a direct influence on cell plates during their formation. Taken together, we suggest that at lower concentrations, citral interferes with cell division by disrupting mitotic microtubules and cell plates, and at higher concentrations it inhibits cell elongation by disrupting cortical microtubules. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

Relationship between plant growth and cytological effect in root apical meristem after exposure of wheat dry seeds to carbon ion beams

NASA Astrophysics Data System (ADS)

Liu, Qingfang; Wang, Zhuanzi; Zhou, Libin; Qu, Ying; Lu, Dong; Yu, Lixia; Du, Yan; Jin, Wenjie; Li, Wenjian

2013-06-01

In order to analyze the relationship between plant growth and cytological effects, wheat dry seeds were exposed to various doses of 12C6+ beams and the biological endpoints reflecting plant growth and root apical meristem (RAM) activities were investigated. The results showed that most of the seeds were able to germinate normally within all dose range, while the plant survival rate descended at higher doses. The seedling growth including root length and seedling height also decreased significantly at higher doses. Mitotic index (MI) in RAM had no changes at 10 and 20 Gy and decreased obviously at higher doses and the proportion of prophase cells had the same trend with MI. These data suggested that RAM cells experienced cell cycle arrest, which should be responsible for the inhibition of root growth after exposure to higher doses irradiation. Moreover, various types of chromosome aberrations (CAs) were observed in the mitotic cells. The frequencies of mitotic cells with lagging chromosomes and these with anaphase bridges peaked around 60 Gy, while the frequencies of these with fragments increased as the irradiation doses increased up to 200 Gy. The total frequencies of mitotic cells with CAs induced by irradiation increased significantly with the increasing doses. The serious damage of mitotic chromosomes maybe caused cell cycle arrest or cell death. These findings suggested that the influences of 12C6+ beams irradiation on plant growth were related to the alternation of mitotic activities and the chromosomal damages in RAM.

TC Mps1 12, a novel Mps1 inhibitor, suppresses the growth of hepatocellular carcinoma cells via the accumulation of chromosomal instability.

PubMed

Choi, Minji; Min, Yoo Hong; Pyo, Jaehyuk; Lee, Chang-Woo; Jang, Chang-Young; Kim, Ja-Eun

2017-06-01

Chromosomal instability is not only a hallmark of cancer but also an attractive therapeutic target. A diverse set of mitotic kinases maintains chromosomal stability. One of these is monopolar spindle 1 (Mps1, also known as TTK), which is essential for chromosome alignment and for the spindle assembly checkpoint (SAC). Pharmacological inhibition of Mps1 has been suggested as a cancer therapeutic; however, despite the existence of a novel Mps1 inhibitor, TC Mps1 12, no such studies have been performed. The effects of TC Mps1 12 on cell viability, chromosome alignment, centrosome number, mitotic duration, apoptosis and SAC were determined in hepatocellular carcinoma (HCC) cells. In addition, the association of Mps1 expression with the overall survival of HCC patients was analysed. Treatment of human HCC cells with TC Mps1 12 led to chromosome misalignment and missegregation, and disorganization of centrosomes. Even in the presence of these errors, TC Mps1 12-treated cells overrode the SAC, resulting in a shortened mitotic duration and mitotic slippage. This mitotic catastrophe triggered apoptosis and, finally, inhibited the growth of HCC cells. In addition, the expression of the Mps1-encoding TTK gene was associated with poor overall survival of HCC patients. TC Mps1 12 results in the accumulation of chromosomal instabilities and mitotic catastrophe in HCC cells. Overall, these data demonstrate that the inhibition of Mps1 kinase using TC Mps1 12 is a promising therapeutic approach for liver cancer. © 2017 The British Pharmacological Society.

High throughput screening of natural products for anti-mitotic effects in MDA-MB-231 human breast carcinoma cells

PubMed Central

Mazzio, E; Badisa, R; Mack, N; Deiab, S; Soliman, KFA

2013-01-01

Some of the most effective anti-mitotic microtubule-binding agents, such as paclitaxel (Taxus brevifolia) were originally discovered through robust NCI botanical screenings. In this study, a high-through microarray format was utilized to screen 897 aqueous extracts of commonly used natural products (0.00015–0.5 mg/ml) relative to paclitaxel for anti-mitotic effects (independent of toxicity) on proliferation of MDA-MB-231 cells. The data obtained showed that less than 1.34 % tested showed inhibitory growth (IG50) properties <0.0183 mg/ml. The most potent anti-mitotics (independent of toxicity) were Mandrake root (Podophyllum peltatum), Truja Twigs (Thuja occidentalis), Colorado desert mistletoe (Phoradendron flavescens), Tou Gu Cao Speranskia Herb (Speranskia tuberculata), Bentonite Clay, Bunge Root (Pulsatilla chinensis), Brucea Fruit (Brucea javanica), Madder Root (Rubia tinctorum), Gallnut of Chinese Sumac (Melaphis chinensis), Elecampane Root (Inula Helenium), Yuan Zhi Root (Polygala tenuifolia), Pagoda Tree Fruit (Melia Toosendan), Stone Root (Collinsonia Canadensis) and others such as American Witchhazel, Arjun and Bladderwrack. The strongest tumoricidal herbs identified from amongst the subset evaluated for anti-mitotic properties were wild yam (Dioscorea villosa), beth-root (Trillium Pendulum) and alkanet-root (Lithospermum canescens). Additional data was obtained on a lesser-recognized herb: (Speranskia tuberculata) which showed growth inhibition on BT-474 (human ductal breast carcinoma) and Ishikawa (human endometrial adenocarcinoma) cells with ability to block replicative DNA synthesis leading to G2 arrest in MDA-MB-231 cells. In conclusion, these findings present relative potency of natural anti-mitotic resources effective against human breast carcinoma MDA-MB-231 cell division. PMID:24105850

Genome accessibility is widely preserved and locally modulated during mitosis

PubMed Central

Hsiung, Chris C.-S.; Morrissey, Christapher S.; Udugama, Maheshi; Frank, Christopher L.; Keller, Cheryl A.; Baek, Songjoon; Giardine, Belinda; Crawford, Gregory E.; Sung, Myong-Hee; Hardison, Ross C.

2015-01-01

Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that such mitotically retained molecular signatures could provide transcriptional memory through mitosis. To understand the role of chromatin structure in mitotic memory, we performed the first genome-wide comparison of DNase I sensitivity of chromatin in mitosis and interphase, using a murine erythroblast model. Despite chromosome condensation during mitosis visible by microscopy, the landscape of chromatin accessibility at the macromolecular level is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly DNase hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility more strongly, whereas distal regulatory elements tend to lose accessibility. Large domains of DNA hypomethylation mark a subset of promoters that retain accessibility during mitosis and across many cell types in interphase. Erythroid transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but has little influence on mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis, but are modulated at the level of individual genes and regulatory elements. PMID:25373146

Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity.

PubMed

Dewey, Evan B; Johnston, Christopher A

2017-09-15

Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial-mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation. © 2017 Dewey and Johnston. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Two Bistable Switches Govern M Phase Entry.

PubMed

Mochida, Satoru; Rata, Scott; Hino, Hirotsugu; Nagai, Takeharu; Novák, Béla

2016-12-19

The abrupt and irreversible transition from interphase to M phase is essential to separate DNA replication from chromosome segregation. This transition requires the switch-like phosphorylation of hundreds of proteins by the cyclin-dependent kinase 1 (Cdk1):cyclin B (CycB) complex. Previous studies have ascribed these switch-like phosphorylations to the auto-activation of Cdk1:CycB through the removal of inhibitory phosphorylations on Cdk1-Tyr15 [1, 2]. The positive feedback in Cdk1 activation creates a bistable switch that makes mitotic commitment irreversible [2-4]. Here, we surprisingly find that Cdk1 auto-activation is dispensable for irreversible, switch-like mitotic entry due to a second mechanism, whereby Cdk1:CycB inhibits its counteracting phosphatase (PP2A:B55). We show that the PP2A:B55-inhibiting Greatwall (Gwl)-endosulfine (ENSA) pathway is both necessary and sufficient for switch-like phosphorylations of mitotic substrates. Using purified components of the Gwl-ENSA pathway in a reconstituted system, we found a sharp Cdk1 threshold for phosphorylation of a luminescent mitotic substrate. The Cdk1 threshold to induce mitotic phosphorylation is distinctly higher than the Cdk1 threshold required to maintain these phosphorylations-evidence for bistability. A combination of mathematical modeling and biochemical reconstitution show that the bistable behavior of the Gwl-ENSA pathway emerges from its mutual antagonism with PP2A:B55. Our results demonstrate that two interlinked bistable mechanisms provide a robust solution for irreversible and switch-like mitotic entry. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of Phosphohistone H3 Cutoff Values Corresponding to Original WHO Grades but Distinguishable in Well-Differentiated Gastrointestinal Neuroendocrine Tumors.

PubMed

Kim, Min Jeong; Kwon, Mi Jung; Kang, Ho Suk; Choi, Kyung Chan; Nam, Eun Sook; Cho, Seong Jin; Park, Hye-Rim; Min, Soo Kee; Seo, Jinwon; Choe, Ji-Young; Park, Hyoung-Chul

2018-01-01

Mitotic counts in the World Health Organization (WHO) grading system have narrow cutoff values. True mitotic figures, however, are not always distinguishable from apoptotic bodies and darkly stained nuclei, complicating the ability of the WHO grading system to diagnose well-differentiated neuroendocrine tumors (NETs). The mitosis-specific marker phosphohistone H3 (PHH3) can identify true mitoses and grade tumors reliably. The aim of this study was to investigate the correspondence of tumor grades, as determined by PHH3 mitotic index (MI) and mitotic counts according to WHO criteria, and to determine the clinically relevant cutoffs of PHH3 MI in rectal and nonrectal gastrointestinal NETs. Mitotic counts correlated with both the Ki-67 labeling index and PHH3 MI, but the correlation with PHH3 MI was slightly higher. The PHH3 MI cutoff ≥4 correlated most closely with original WHO grades for both rectal NETs. A PHH3 MI cutoff ≥4, which could distinguish between G1 and G2 tumors, was associated with disease-free survival in patients with rectal NETs, whereas that cutoff value showed marginal significance for overall survival in patient with rectal NETs. In conclusion, the use of PHH3 ≥4 correlated most closely with original WHO grades.

Identification of Phosphohistone H3 Cutoff Values Corresponding to Original WHO Grades but Distinguishable in Well-Differentiated Gastrointestinal Neuroendocrine Tumors

PubMed Central

Kim, Min Jeong; Kang, Ho Suk; Choi, Kyung Chan; Nam, Eun Sook; Cho, Seong Jin; Park, Hye-Rim; Seo, Jinwon; Choe, Ji-Young

2018-01-01

Mitotic counts in the World Health Organization (WHO) grading system have narrow cutoff values. True mitotic figures, however, are not always distinguishable from apoptotic bodies and darkly stained nuclei, complicating the ability of the WHO grading system to diagnose well-differentiated neuroendocrine tumors (NETs). The mitosis-specific marker phosphohistone H3 (PHH3) can identify true mitoses and grade tumors reliably. The aim of this study was to investigate the correspondence of tumor grades, as determined by PHH3 mitotic index (MI) and mitotic counts according to WHO criteria, and to determine the clinically relevant cutoffs of PHH3 MI in rectal and nonrectal gastrointestinal NETs. Mitotic counts correlated with both the Ki-67 labeling index and PHH3 MI, but the correlation with PHH3 MI was slightly higher. The PHH3 MI cutoff ≥4 correlated most closely with original WHO grades for both rectal NETs. A PHH3 MI cutoff ≥4, which could distinguish between G1 and G2 tumors, was associated with disease-free survival in patients with rectal NETs, whereas that cutoff value showed marginal significance for overall survival in patient with rectal NETs. In conclusion, the use of PHH3 ≥4 correlated most closely with original WHO grades. PMID:29780816

Regulating positioning and orientation of mitotic spindles via cell size and shape

NASA Astrophysics Data System (ADS)

Li, Jingchen; Jiang, Hongyuan

2018-01-01

Proper location of the mitotic spindle is critical for chromosome segregation and the selection of the cell division plane. However, how mitotic spindles sense cell size and shape to regulate their own position and orientation is still largely unclear. To investigate this question systematically, we used a general model by considering chromosomes, microtubule dynamics, and forces of various molecular motors. Our results show that in cells of various sizes and shapes, spindles can always be centered and oriented along the long axis robustly in the absence of other specified mechanisms. We found that the characteristic time of positioning and orientation processes increases with cell size. Spindles sense the cell size mainly by the cortical force in small cells and by the cytoplasmic force in large cells. In addition to the cell size, the cell shape mainly influences the orientation process. We found that more slender cells have a faster orientation process, and the final orientation is not necessarily along the longest axis but is determined by the radial profile and the symmetry of the cell shape. Finally, our model also reproduces the separation and repositioning of the spindle poles during the anaphase. Therefore, our work provides a general tool for studying the mitotic spindle across the whole mitotic phase.

Picropodophyllin causes mitotic arrest and catastrophe by depolymerizing microtubules via Insulin-like growth factor-1 receptor-independent mechanism

PubMed Central

Waraky, Ahmed; Akopyan, Karen; Parrow, Vendela; Strömberg, Thomas; Axelson, Magnus; Abrahmsén, Lars; Lindqvist, Arne; Larsson, Olle; Aleem, Eiman

2014-01-01

Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has been shown to suppress IGF-1R signaling and to induce a G2/M cell cycle phase arrest but the exact mechanisms remain to be elucidated. The present study identified an IGF-1-independent mechanism of PPP leading to pro-metaphase arrest. The mitotic block was induced in human cancer cell lines and in an A549 xenograft mouse but did not occur in normal hepatocytes/mouse tissues. Cell cycle arrest by PPP occurred in vitro and in vivo accompanied by prominent CDK1 activation, and was IGF-1R-independent since it occurred also in IGF-1R-depleted and null cells. The tumor cells were not arrested in G2/M but in mitosis. Centrosome separation was prevented during mitotic entry, resulting in a monopolar mitotic spindle with subsequent prometaphase-arrest, independent of Plk1/Aurora A or Eg5, and leading to cell features of mitotic catastrophe. PPP also increased soluble tubulin and decreased spindle-associated tubulin within minutes, indicating that it interfered with microtubule dynamics. These results provide a novel IGF-1R-independent mechanism of antitumor effects of PPP. PMID:25268741

Cyclin K dependent regulation of Aurora B affects apoptosis and proliferation by induction of mitotic catastrophe in prostate cancer.

PubMed

Schecher, Sabrina; Walter, Britta; Falkenstein, Michael; Macher-Goeppinger, Stephan; Stenzel, Philipp; Krümpelmann, Kristina; Hadaschik, Boris; Perner, Sven; Kristiansen, Glen; Duensing, Stefan; Roth, Wilfried; Tagscherer, Katrin E

2017-10-15

Cyclin K plays a critical role in transcriptional regulation as well as cell development. However, the role of Cyclin K in prostate cancer is unknown. Here, we describe the impact of Cyclin K on prostate cancer cells and examine the clinical relevance of Cyclin K as a biomarker for patients with prostate cancer. We show that Cyclin K depletion in prostate cancer cells induces apoptosis and inhibits proliferation accompanied by an accumulation of cells in the G2/M phase. Moreover, knockdown of Cyclin K causes mitotic catastrophe displayed by multinucleation and spindle multipolarity. Furthermore, we demonstrate a Cyclin K dependent regulation of the mitotic kinase Aurora B and provide evidence for an Aurora B dependent induction of mitotic catastrophe. In addition, we show that Cyclin K expression is associated with poor biochemical recurrence-free survival in patients with prostate cancer treated with an adjuvant therapy. In conclusion, targeting Cyclin K represents a novel, promising anti-cancer strategy to induce cell cycle arrest and apoptotic cell death through induction of mitotic catastrophe in prostate cancer cells. Moreover, our results indicate that Cyclin K is a putative predictive biomarker for clinical outcome and therapy response for patients with prostate cancer. © 2017 UICC.

Cell Cycle Synchronization of HeLa Cells to Assay EGFR Pathway Activation.

PubMed

Wee, Ping; Wang, Zhixiang

2017-01-01

Progression through the cell cycle causes changes in the cell's signaling pathways that can alter EGFR signal transduction. Here, we describe drug-derived protocols to synchronize HeLa cells in various phases of the cell cycle, including G1 phase, S phase, G2 phase, and mitosis, specifically in the mitotic stages of prometaphase, metaphase, and anaphase/telophase. The synchronization procedures are designed to allow synchronized cells to be treated for EGF and collected for the purpose of Western blotting for EGFR signal transduction components.S phase synchronization is performed by thymidine block, G2 phase with roscovitine, prometaphase with nocodazole, metaphase with MG132, and anaphase/telophase with blebbistatin. G1 phase synchronization is performed by culturing synchronized mitotic cells obtained by mitotic shake-off. We also provide methods to validate the synchronization methods. For validation by Western blotting, we provide the temporal expression of various cell cycle markers that are used to check the quality of the synchronization. For validation of mitotic synchronization by microscopy, we provide a guide that describes the physical properties of each mitotic stage, using their cellular morphology and DNA appearance. For validation by flow cytometry, we describe the use of imaging flow cytometry to distinguish between the phases of the cell cycle, including between each stage of mitosis.

The Stil protein regulates centrosome integrity and mitosis through suppression of Chfr

PubMed Central

Castiel, Asher; Danieli, Michal Mark; David, Ahuvit; Moshkovitz, Sharon; Aplan, Peter D.; Kirsch, Ilan R.; Brandeis, Michael; Krämer, Alwin; Izraeli, Shai

2011-01-01

Stil (Sil, SCL/TAL1 interrupting locus) is a cytosolic and centrosomal protein expressed in proliferating cells that is required for mouse and zebrafish neural development and is mutated in familial microcephaly. Recently the Drosophila melanogaster ortholog of Stil was found to be important for centriole duplication. Consistent with this finding, we report here that mouse embryonic fibroblasts lacking Stil are characterized by slow growth, low mitotic index and absence of clear centrosomes. We hypothesized that Stil regulates mitosis through the tumor suppressor Chfr, an E3 ligase that blocks mitotic entry in response to mitotic stress. Mouse fibroblasts lacking Stil by genomic or RNA interference approaches, as well as E9.5 Stil−/− embryos, express high levels of the Chfr protein and reduced levels of the Chfr substrate Plk1. Exogenous expression of Stil, knockdown of Chfr or overexpression of Plk1 reverse the abnormal mitotic phenotypes of fibroblasts lacking Stil. We further demonstrate that Stil increases Chfr auto-ubiquitination and reduces its protein stability. Thus, Stil is required for centrosome organization, entry into mitosis and cell proliferation, and these functions are at least partially mediated by Chfr and its targets. This is the first identification of a negative regulator of the Chfr mitotic checkpoint. PMID:21245198

The Bio-Logic and machinery of plant morphogenesis.

PubMed

Niklas, Karl J

2003-04-01

Morphogenesis (the development of organic form) requires signal-trafficking and cross-talking across all levels of organization to coordinate the operation of metabolic and genomic networked systems. Many biologists are currently converging on the pictorial conventions of computer scientists to render biological signaling as logic circuits supervising the operation of one or more signal-activated metabolic or gene networks. This approach can redact and simplify complex morphogenetic phenomena and allows for their aggregation into diagrams of larger, more "global" networked systems. This conceptualization is discussed in terms of how logic circuits and signal-activated subsystems work, and it is illustrated for examples of increasingly more complex morphogenetic phenomena, e.g., auxin-mediated cell expansion, entry into the mitotic cell cycle phases, and polar/lateral intercellular auxin transport. For each of these phenomena, a posited circuit/subsystem diagram draws rapid attention to missing components, either in the logic circuit or in the subsystem it supervises. These components must be identified experimentally if each of these basic phenomena is to be fully understood. Importantly, the power of the circuit/subsystem approach to modeling developmental phenomena resides not in its pictorial appeal but in the mathematical tools that are sufficiently strong to reveal and quantify the synergistics of networked systems and thus foster a better understanding of morphogenesis.

The endophytic symbiont Epichloë festucae establishes an epiphyllous net on the surface of Lolium perenne leaves by development of an expressorium, an appressorium-like leaf exit structure.

PubMed

Becker, Matthias; Becker, Yvonne; Green, Kimberly; Scott, Barry

2016-07-01

Epichloë festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network. E. festucae also grows as an epiphyte, but the mechanism for leaf surface colonization is not known. Here we identify an appressorium-like structure, which we call an expressorium that allows endophytic hyphae to penetrate the cuticle from the inside of the leaf to establish an epiphytic hyphal net on the surface of the leaf. We used a combination of scanning electron, transmission electron and confocal laser scanning microscopy to characterize this novel fungal structure and determine the composition of the hyphal cell wall using aniline blue and wheat germ agglutinin labelled with Alexafluor-488. Expressoria differentiate immediately below the cuticle in the leaf blade and leaf sheath intercalary cell division zones where the hyphae grow by tip growth. Differentiation of this structure requires components of both the NoxA and NoxB NADPH oxidase complexes. Major remodelling of the hyphal cell wall occurs following exit from the leaf. These results establish that the symbiotic association of E. festucae with L. perenne involves an interconnected hyphal network of both endophytic and epiphytic hyphae. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Micromechanical study of mitotic chromosome structure

NASA Astrophysics Data System (ADS)

Marko, John

2011-03-01

Our group has developed micromanipulation techniques for study of the highly compacted mitotic form of chromosome found in eukaryote cells during cell division. Each metaphase chromosome contains two duplicate centimeter-long DNA molecules, folded up by proteins into cylindrical structures several microns in length. Native chromosomes display linear and reversible stretching behavior over a wide range of extensions (up to 5x native length for amphibian chromosomes), described by a Young modulus of about 300 Pa. Studies using DNA-cutting and protein-cutting enzymes have revealed that metaphase chromosomes behave as a network of chromatin fibers held together by protein-based isolated crosslinks. Our results are not consistent with the more classical model of loops of chromatin attached to a protein-based structural organizer or ``scaffold". In short, our experiments indicate that metaphase chromosomes can be considered to be ``gels" of chromatin; the stretching modulus of a whole chromosome is consistent with stretching of the chromatin fibers contained within it. Experiments using topoisomerases suggest that topological constraints may play an appreciable role in confining chromatin in the metaphase chromosome. Finally, recent experiments on human chromosomes will be reviewed, including results of experiments where chromosome-folding proteins are specifically depleted using siRNA methods. Supported by NSF-MCB-1022117, DMR-0715099, PHY-0852130, DMR-0520513, NCI 1U54CA143869-01 (NU-PS-OC), and the American Heart Association.

Arp2 depletion inhibits sheet-like protrusions but not linear protrusions of fibroblasts and lymphocytes

PubMed Central

Nicholson-Dykstra, Susan M.; Higgs, Henry N.

2009-01-01

The Arp2/3 complex-mediated assembly and protrusion of a branched actin network at the leading edge occurs during cell migration, although some studies suggest it is not essential. In order to test the role of Arp2/3 complex in leading edge protrusion, Swiss 3T3 fibroblasts and Jurkat T cells were depleted of Arp2 and evaluated for defects in cell morphology and spreading efficiency. Arp2-depleted fibroblasts exhibit severe defects in formation of sheet-like protrusions at early time points of cell spreading, with sheet-like protrusions limited to regions along the length of linear protrusions. However, Arp2-depleted cells are able to spread fully after extended times. Similarly, Arp2-depleted Jurkat T lymphocytes exhibit defects in spreading on anti-CD3. Interphase Jurkats in suspension are covered with large ruffle structures, whereas mitotic Jurkats are covered by finger-like linear protrusions. Arp2-depleted Jurkats exhibit defects in ruffle assembly but not in assembly of mitotic linear protrusions. Similarly, Arp2-depletion has no effect on the highly dynamic linear protrusion of another suspended lymphocyte line. We conclude that Arp2/3 complex plays a significant role in assembly of sheet-like protrusions, especially during early stages of cell spreading, but is not required for assembly of a variety of linear actin-based protrusions. PMID:18720401

The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

PubMed

Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

2014-09-01

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis. Copyright © 2014 the American Physiological Society.

Inversin modulates the cortical actin network during mitosis

PubMed Central

Werner, Michael E.; Ward, Heather H.; Phillips, Carrie L.; Miller, Caroline; Gattone, Vincent H.

2013-01-01

Mutations in inversin cause nephronophthisis type II, an autosomal recessive form of polycystic kidney disease associated with situs inversus, dilatation, and kidney cyst formation. Since cyst formation may represent a planar polarity defect, we investigated whether inversin plays a role in cell division. In developing nephrons from inv−/− mouse embryos we observed heterogeneity of nuclear size, increased cell membrane perimeters, cells with double cilia, and increased frequency of binuclear cells. Depletion of inversin by siRNA in cultured mammalian cells leads to an increase in bi- or multinucleated cells. While spindle assembly, contractile ring formation, or furrow ingression appears normal in the absence of inversin, mitotic cell rounding and the underlying rearrangement of the cortical actin cytoskeleton are perturbed. We find that inversin loss causes extensive filopodia formation in both interphase and mitotic cells. These cells also fail to round up in metaphase. The resultant spindle positioning defects lead to asymmetric division plane formation and cell division. In a cell motility assay, fibroblasts isolated from inv−/− mouse embryos migrate at half the speed of wild-type fibroblasts. Together these data suggest that inversin is a regulator of cortical actin required for cell rounding and spindle positioning during mitosis. Furthermore, cell division defects resulting from improper spindle position and perturbed actin organization contribute to altered nephron morphogenesis in the absence of inversin. PMID:23515530

Perspectives of Youth in Foster Care on Essential Ingredients for Promoting Self-determination and Successful Transition to Adult Life: My Life Model.

PubMed

Powers, Laurie E; Fullerton, Ann; Schmidt, Jessica; Geenen, Sarah; Oberweiser-Kennedy, Molly; Dohn, JoAnn; Nelson, May; Iavanditti, Rosemary; Blakeslee, Jennifer

2018-02-01

Research clearly documents the serious challenges and poor outcomes experienced by many young people exiting foster care, as well as compounded disparities for the high percentage of youth in care who are identified with disabilities and/or mental health challenges. However, very little research has been conducted to specify or validate effective models for improving the transition trajectories of youth exiting care. Evidence suggests the My Life self-determination enhancement model offers a promising approach for supporting youths' self-determined and positive transition to adulthood. The model includes youth-directed, experientially oriented coaching in the application of self-determination skills to achieve youth-identified transition goals, coupled with peer mentoring workshops that provide opportunities for learning, networking and fun. This in depth qualitative study of 10 youth who completed the My Life intervention focused on investigating coaching and mentoring elements and processes that youth participants identify as most important to their success, with the intention of informing the further development of youth-directed approaches to supporting young people who are transitioning to adulthood. Themes emerged around the centrality of youth self-direction, important processes in the coaching relationship, the essential value of experiential activities and self-determination skill development, and peer mentoring experiences that youth identified as fostering their success. Implications are discussed for research and practice in supporting youth exiting foster care.

Driving simulator evaluation of drivers' response to intersections with dynamic use of exit-lanes for left-turn.

PubMed

Zhao, Jing; Yun, Meiping; Zhang, H Michael; Yang, Xiaoguang

2015-08-01

With the worsening of urban traffic congestion in large cities around the world, researchers have been looking for unconventional designs and/or controls to squeeze more capacity out of intersections, the most common bottlenecks of the road network. One of these innovative intersection designs, known as the exit-lanes for left-turn (EFL), opens up exit-lanes to be used by left-turn traffic with the help of an additional traffic light installed at the median opening (the pre-signal). This paper studies how drivers respond to EFL intersections with a series of driving simulator experiments. In our experiments, 64 drivers were recruited and divided into two groups. One group is trained to use the EFL while the other group is not. In addition, four scenarios were considered with different sign and marking designs and traffic conditions in the experiments. Results indicate that drivers show certain amount of confusion and hesitation when encountering an EFL intersection for the first time. They can be overcome, however, by increasing exposure through driver education or by cue provided from other vehicles. Moreover, drivers unfamiliar with EFL operation can make a left turn using the conventional left-turn lanes as usual. The EFL operation is not likely to pose any serious safety risk of the intersection in real life operations. Copyright © 2015 Elsevier Ltd. All rights reserved.

Emissions Prediction and Measurement for Liquid-Fueled TVC Combustor with and without Water Injection

NASA Technical Reports Server (NTRS)

Brankovic, A.; Ryder, R. C., Jr.; Hendricks, R. C.; Liu, N.-S.; Shouse, D. T.; Roquemore, W. M.

2005-01-01

An investigation is performed to evaluate the performance of a computational fluid dynamics (CFD) tool for the prediction of the reacting flow in a liquid-fueled combustor that uses water injection for control of pollutant emissions. The experiment consists of a multisector, liquid-fueled combustor rig operated at different inlet pressures and temperatures, and over a range of fuel/air and water/fuel ratios. Fuel can be injected directly into the main combustion airstream and into the cavities. Test rig performance is characterized by combustor exit quantities such as temperature and emissions measurements using rakes and overall pressure drop from upstream plenum to combustor exit. Visualization of the flame is performed using gray scale and color still photographs and high-frame-rate videos. CFD simulations are performed utilizing a methodology that includes computer-aided design (CAD) solid modeling of the geometry, parallel processing over networked computers, and graphical and quantitative post-processing. Physical models include liquid fuel droplet dynamics and evaporation, with combustion modeled using a hybrid finite-rate chemistry model developed for Jet-A fuel. CFD and experimental results are compared for cases with cavity-only fueling, while numerical studies of cavity and main fueling was also performed. Predicted and measured trends in combustor exit temperature, CO and NOx are in general agreement at the different water/fuel loading rates, although quantitative differences exist between the predictions and measurements.

Colonization by Legionella spp. of water networks in residential buildings of the Province of Pisa, Italy.

PubMed

Baggiani, A; Casini, B; Totaro, M; Aquino, F; Valentini, P; Bruni, B; Porretta, A; Casalini, F; Miccoli, M; Privitera, G

2015-01-01

Despite the increase of community acquired cases of legionellosis in Italy over the last years, the Italian guidelines do not give indications for prevention and control of Legionella in the hot water networks (or centralized conditioning systems) of residential buildings. We performed a survey on eight medium sized apartment buildings in the Pisa district to assess the prevalence of Legionella spp. in the water network and the respondance to drinking water requisites at the point of use, according to the Italian norms. For each building two hot water and three cold water samples (located at water entrance from the aqueduct network into the building pipework, at the exit from pressure autoclave, and at a remote tap) were collected. Legionella was detected in 20% of residential buildings, mostly in those with a central hot water production system. The study highlights a condition of potential risk for susceptible population subgroups and supports the need for measures of risk assessment and control.

A Clonal Genetic Screen for Mutants Causing Defects in Larval Tracheal Morphogenesis in Drosophila

PubMed Central

Baer, Magdalena M.; Bilstein, Andreas; Leptin, Maria

2007-01-01

The initial establishment of the tracheal network in the Drosophila embryo is beginning to be understood in great detail, both in its genetic control cascades and in its cell biological events. By contrast, the vast expansion of the system during larval growth, with its extensive ramification of preexisting tracheal branches, has been analyzed less well. The mutant phenotypes of many genes involved in this process are probably not easy to reveal, as these genes may be required for other functions at earlier developmental stages. We therefore conducted a screen for defects in individual clonal homozygous mutant cells in the tracheal network of heterozygous larvae using the mosaic analysis with a repressible cell marker (MARCM) system to generate marked, recombinant mitotic clones. We describe the identification of a set of mutants with distinct phenotypic effects. In particular we found a range of defects in terminal cells, including failure in lumen formation and reduced or extensive branching. Other mutations affect cell growth, cell shape, and cell migration. PMID:17603107

Mitotic rate in primary melanoma: interobserver and intraobserver reliability, analyzed using H&E sections and immunohistochemistry.

PubMed

Garbe, Claus; Eigentler, Thomas K; Bauer, Jürgen; Blödorn-Schlicht, Norbert; Cerroni, Lorenzo; Fend, Falko; Hantschke, Markus; Kurschat, Peter; Kutzner, Heinz; Metze, Dieter; Mielke, Volker; Preßler, Harald; Reusch, Michael; Reusch, Ursula; Stadler, Rudolf; Tronnier, Michael; Yazdi, Amir; Metzler, Gisela

2016-09-01

In 2009, the AJCC issued a revised melanoma staging system. In addition to tumor thickness and ulceration, the mitotic rate was introduced as the third major prognostic parameter for the classification of primary cutaneous melanoma. Given that, according to the 2009 AJCC classification, the detection of one or more dermal tumor mitoses leads to an upstaging - from stage Ia to Ib - of melanomas with a tumor thickness of ≤ 1.0 mm, we set out to investigate the reproducibility of this new parameter. In order to assess interobserver reliability, 17 dermatopathologists und pathologists - all well versed in the diagnosis of cutaneous melanoma - analyzed the mitotic rate in 15 thin primary cutaneous melanomas (mean tumor thickness 0.91 mm) using identical slides. Mitotic rates were determined on H&E and phosphohistone H3 (Ser10)-stained samples. Without knowledge of their previous assessment, five of the aforementioned examiners reevaluated the samples after more than one year in order to ascertain intraobserver reliability. Interobserver reliability of the mitotic rate in thin primary melanomas is disappointing and independent of whether H&E or immunohistochemically stained samples are used (kappa value: 0.088 [H&E], 0.154 [IH], respectively). Kappa values improved to 0.345 (H&E) and 0.403 (IH) when using a cutoff of 0/1 vs. 2+ mitoses. Similarly unsatisfactory, kappa values for intraobserver reliability ranged from 0.18 and 0.348, depending on the individual examiner. Given the unsatisfactory reproducibility and large variations in assessing the mitotic rate, it remains a matter of debate whether this diagnostic parameter should play a role in therapeutic decisions. © 2016 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd.

Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage.

PubMed

Hamaguchi, Y; Toriyama, M; Sakai, H; Hiramoto, Y

1985-04-01

Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC-labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin.

Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage

PubMed Central

1985-01-01

Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC- labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin. PMID:3920225

Genotoxicity of multi-walled carbon nanotubes at occupationally relevant doses

PubMed Central

2014-01-01

Carbon nanotubes are commercially-important products of nanotechnology; however, their low density and small size makes carbon nanotube respiratory exposures likely during their production or processing. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to single-walled carbon nanotubes (SWCNT). In this study, we examined whether multi-walled carbon nanotubes (MWCNT) cause mitotic spindle damage in cultured cells at doses equivalent to 34 years of exposure at the NIOSH Recommended Exposure Limit (REL). MWCNT induced a dose responsive increase in disrupted centrosomes, abnormal mitotic spindles and aneuploid chromosome number 24 hours after exposure to 0.024, 0.24, 2.4 and 24 μg/cm2 MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 μm optical sections showed carbon nanotubes integrated with microtubules, DNA and within the centrosome structure. Cell cycle analysis demonstrated a greater number of cells in S-phase and fewer cells in the G2 phase in MWCNT-treated compared to diluent control, indicating a G1/S block in the cell cycle. The monopolar phenotype of the disrupted mitotic spindles and the G1/S block in the cell cycle is in sharp contrast to the multi-polar spindle and G2 block in the cell cycle previously observed following exposure to SWCNT. One month following exposure to MWCNT there was a dramatic increase in both size and number of colonies compared to diluent control cultures, indicating a potential to pass the genetic damage to daughter cells. Our results demonstrate significant disruption of the mitotic spindle by MWCNT at occupationally relevant exposure levels. PMID:24479647

TC Mps1 12, a novel Mps1 inhibitor, suppresses the growth of hepatocellular carcinoma cells via the accumulation of chromosomal instability

PubMed Central

Choi, Minji; Min, Yoo Hong; Pyo, Jaehyuk; Lee, Chang‐Woo; Jang, Chang‐Young

2017-01-01

Background and Purpose Chromosomal instability is not only a hallmark of cancer but also an attractive therapeutic target. A diverse set of mitotic kinases maintains chromosomal stability. One of these is monopolar spindle 1 (Mps1, also known as TTK), which is essential for chromosome alignment and for the spindle assembly checkpoint (SAC). Pharmacological inhibition of Mps1 has been suggested as a cancer therapeutic; however, despite the existence of a novel Mps1 inhibitor, TC Mps1 12, no such studies have been performed. Experimental Approach The effects of TC Mps1 12 on cell viability, chromosome alignment, centrosome number, mitotic duration, apoptosis and SAC were determined in hepatocellular carcinoma (HCC) cells. In addition, the association of Mps1 expression with the overall survival of HCC patients was analysed. Key Results Treatment of human HCC cells with TC Mps1 12 led to chromosome misalignment and missegregation, and disorganization of centrosomes. Even in the presence of these errors, TC Mps1 12‐treated cells overrode the SAC, resulting in a shortened mitotic duration and mitotic slippage. This mitotic catastrophe triggered apoptosis and, finally, inhibited the growth of HCC cells. In addition, the expression of the Mps1‐encoding TTK gene was associated with poor overall survival of HCC patients. Conclusion and Implications TC Mps1 12 results in the accumulation of chromosomal instabilities and mitotic catastrophe in HCC cells. Overall, these data demonstrate that the inhibition of Mps1 kinase using TC Mps1 12 is a promising therapeutic approach for liver cancer. PMID:28299790

Effects of heat stress in the leaf mitotic cell cycle and chromosomes of four wine-producing grapevine varieties.

PubMed

Carvalho, Ana; Leal, Fernanda; Matos, Manuela; Lima-Brito, José

2018-05-22

Grapevine varieties respond differentially to heat stress (HS). HS ultimately reduces the photosynthesis and respiratory performance. However, the HS effects in the leaf nuclei and mitotic cells of grapevine are barely known. This work intends to evaluate the HS effects in the leaf mitotic cell cycle and chromosomes of four wine-producing varieties: Touriga Franca (TF), Touriga Nacional (TN), Rabigato, and Viosinho. In vitro plants with 11 months were used in a stepwise acclimation and recovery (SAR) experimental setup comprising different phases: heat acclimation period (3 h-32 °C), extreme HS (1 h-42 °C), and two recovery periods (3 h-32 °C and 24 h-25 °C), and compared to control plants (maintained in vitro at 25 °C). At the end of each SAR phase, leaves were collected, fixed, and used for cell suspensions and chromosome preparations. Normal and abnormal interphase and mitotic cells were observed, scored, and statistically analyzed in all varieties and treatments (control and SAR phases). Different types of chromosomal anomalies in all mitotic phases, treatments, and varieties were found. In all varieties, the percentage of dividing cells with anomalies (%DCA) after extreme HS increased relative to control. TF and Viosinho were considered the most tolerant to HS. TF showed a gradual MI reduction from heat acclimation to HS and the lowest %DCA after HS and 24 h of recovery. Only Viosinho reached the control values after the long recovery period. Extrapolating these data to the field, we hypothesize that during consecutive hot summer days, the grapevine plants will not have time or capacity to recover from the mitotic anomalies caused by high temperatures.

Genome accessibility is widely preserved and locally modulated during mitosis.

PubMed

Hsiung, Chris C-S; Morrissey, Christapher S; Udugama, Maheshi; Frank, Christopher L; Keller, Cheryl A; Baek, Songjoon; Giardine, Belinda; Crawford, Gregory E; Sung, Myong-Hee; Hardison, Ross C; Blobel, Gerd A

2015-02-01

Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that such mitotically retained molecular signatures could provide transcriptional memory through mitosis. To understand the role of chromatin structure in mitotic memory, we performed the first genome-wide comparison of DNase I sensitivity of chromatin in mitosis and interphase, using a murine erythroblast model. Despite chromosome condensation during mitosis visible by microscopy, the landscape of chromatin accessibility at the macromolecular level is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly DNase hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility more strongly, whereas distal regulatory elements tend to lose accessibility. Large domains of DNA hypomethylation mark a subset of promoters that retain accessibility during mitosis and across many cell types in interphase. Erythroid transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but has little influence on mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis, but are modulated at the level of individual genes and regulatory elements. © 2015 Hsiung et al.; Published by Cold Spring Harbor Laboratory Press.

The phosphorylation-dependent regulation of nuclear SREBP1 during mitosis links lipid metabolism and cell growth

PubMed Central

Bengoechea-Alonso, Maria Teresa; Ericsson, Johan

2016-01-01

ABSTRACT The SREBP transcription factors are major regulators of lipid metabolism. Disturbances in lipid metabolism are at the core of several health issues facing modern society, including cardiovascular disease, obesity and diabetes. In addition, the role of lipid metabolism in cancer cell growth is receiving increased attention. Transcriptionally active SREBP molecules are unstable and rapidly degraded in a phosphorylation-dependent manner by Fbw7, a ubiquitin ligase that targets several cell cycle regulatory proteins for degradation. We have previously demonstrated that active SREBP1 is stabilized during mitosis. We have now delineated the mechanisms involved in the stabilization of SREBP1 in mitotic cells. This process is initiated by the phosphorylation of a specific serine residue in nuclear SREBP1 by the mitotic kinase Cdk1. The phosphorylation of this residue creates a docking site for a separate mitotic kinase, Plk1. Plk1 interacts with nuclear SREBP1 in mitotic cells and phosphorylates a number of residues in the C-terminal domain of the protein, including a threonine residue in close proximity of the Fbw7 docking site in SREBP1. The phosphorylation of these residues by Plk1 blocks the interaction between SREBP1 and Fbw7 and attenuates the Fbw7-dependent degradation of nuclear SREBP1 during cell division. Inactivation of SREBP1 results in a mitotic defect, suggesting that SREBP1 could regulate cell division. We propose that the mitotic phosphorylation and stabilization of nuclear SREBP1 during cell division provides a link between lipid metabolism and cell proliferation. Thus, the current study provides additional support for the emerging hypothesis that SREBP-dependent lipid metabolism may be important for cell growth. PMID:27579997

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Masatoshi, E-mail: msuzuki@nagasaki-u.ac.jp; Yamauchi, Motohiro; Oka, Yasuyoshi

Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% andmore » 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO{sub 2}-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ss-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.« less

Changes in basal cell mitosis and transepidermal water loss in skin cultures treated with vitamins C and E.

PubMed

Parish, W E; Read, J; Paterson, S E

2005-09-01

Three variants of the living skin equivalent cultures were compared in order to determine the most suitable to grow human differentiated epidermis to test beneficial properties of nutrients. Criteria of culture quality were mitotic index and transepidermal water loss (TEWL) assayed by means of a ServoMed Evaporimeter EP-2TM (ServoMed, Kinna, Sweden). Standards were donor skin mean mitotic index 11.1% and TEWL of living subjects mean 6.4 g/m(2)/h. Cultures (i) in 5% serum, 10 ng/ml of epidermal growth factor (EGF) at 37 degrees C and 95% relative humidity (RH); mitotic index on day 14, 19.2%, but on day 21, 1.8% and TEWL 9.5 g/m(2)/h on day 18. (ii) In 1% serum, no EGF, 33 degrees C and 95% RH, mitotic index on day 21, 9.1% and TEWL, 9.5% on day 18. (iii) Culture in same medium, 33 degrees C and 60% RH, mitotic index on day 28, 9.5% and TEWL 6.1 g/m(2)/h on day 18 as in vivo. Incubation in 60% RH was achieved using a novel chamber and dishes exposing only the corneum, sealing the medium. Vitamins C and E were used as model test nutrients. Culture conditions were 1% serum, no EGF at 33 degrees C and 95% RH. Vitamin C at 142 and 284 microM increased the mitotic index after 10- and 15-day treatment, but at 586 microM it was weakly toxic. Vitamin E at 20 and 40 microM did not. Both vitamins reduced TEWL providing functional data in support of previous reports on barrier properties. These are functional biomarkers of skin benefit relevant to skin in vivo.

Presence of Legionella spp. in Hot Water Networks of Different Italian Residential Buildings: A Three-Year Survey.

PubMed

Totaro, Michele; Valentini, Paola; Costa, Anna Laura; Frendo, Lorenzo; Cappello, Alessia; Casini, Beatrice; Miccoli, Mario; Privitera, Gaetano; Baggiani, Angelo

2017-10-26

Although the European reports highlight an increase in community-acquired Legionnaires' disease cases, the risk of Legionella spp. in private houses is underestimated. In Pisa (Italy) we performed a three-year survey on Legionella presence in 121 buildings with an independent hot water production (IB); 64 buildings with a central hot water production (CB); and 35 buildings with a solar thermal system for hot water production (TB). From all the 220 buildings Legionella spp. was researched in two hot water samples collected either at the recirculation point or on the first floor and on the last floor, while the potable water quality was analysed in three cold water samples collected at the inlet from the aqueduct network, at the exit from the autoclave, and at the most remote tap. Legionella pneumophila sg1, Legionella pneumophila sg2-16, and non- pneumophila Legionella species were detected in 26% of the hot water networks, mostly in CB and TB. In these buildings we detected correlations between the presence of Legionella and the total chlorine concentration decrease and/or the increase of the temperature. Cold water resulted free from microbiological hazards, with the exception of Serratia liquefaciens and Enterobacter cloacae isolated at the exit from two different autoclaves. We observed an increase in total microbial counts at 22 °C and 37 °C between the samples collected at the most remote taps compared to the ones collected at the inlet from the aqueduct. The study highlights a condition of potential risk for susceptible categories of population and supports the need for measures of risk assessment and control.

The SmartOR: a distributed sensor network to improve operating room efficiency.

PubMed

Huang, Albert Y; Joerger, Guillaume; Fikfak, Vid; Salmon, Remi; Dunkin, Brian J; Bass, Barbara L; Garbey, Marc

2017-09-01

Despite the significant expense of OR time, best practice achieves only 70% efficiency. Compounding this problem is a lack of real-time data. Most current OR utilization programs require manual data entry. Automated systems require installation and maintenance of expensive tracking hardware throughout the institution. This study developed an inexpensive, automated OR utilization system and analyzed data from multiple operating rooms. OR activity was deconstructed into four room states. A sensor network was then developed to automatically capture these states using only three sensors, a local wireless network, and a data capture computer. Two systems were then installed into two ORs, recordings captured 24/7. The SmartOR recorded the following events: any room activity, patient entry/exit time, anesthesia time, laparoscopy time, room turnover time, and time of preoperative patient identification by the surgeon. From November 2014 to December 2015, data on 1003 cases were collected. The mean turnover time was 36 min, and 38% of cases met the institutional goal of ≤30 min. Data analysis also identified outlier cases (>1 SD from mean) in the domains of time from patient entry into the OR to intubation (11% of cases) and time from extubation to patient exiting the OR (11% of cases). Time from surgeon identification of patient to scheduled procedure start time was 11 min (institution bylaws require 20 min before scheduled start time), yet OR teams required 22 min on average to bring a patient into the room after surgeon identification. The SmartOR automatically and reliably captures data on OR room state and, in real time, identifies outlier cases that may be examined closer to improve efficiency. As no manual entry is required, the data are indisputable and allow OR teams to maintain a patient-centric focus.

Presence of Legionella spp. in Hot Water Networks of Different Italian Residential Buildings: A Three-Year Survey

PubMed Central

Totaro, Michele; Valentini, Paola; Costa, Anna Laura; Frendo, Lorenzo; Cappello, Alessia; Casini, Beatrice; Miccoli, Mario; Privitera, Gaetano; Baggiani, Angelo

2017-01-01

Although the European reports highlight an increase in community-acquired Legionnaires’ disease cases, the risk of Legionella spp. in private houses is underestimated. In Pisa (Italy) we performed a three-year survey on Legionella presence in 121 buildings with an independent hot water production (IB); 64 buildings with a central hot water production (CB); and 35 buildings with a solar thermal system for hot water production (TB). From all the 220 buildings Legionella spp. was researched in two hot water samples collected either at the recirculation point or on the first floor and on the last floor, while the potable water quality was analysed in three cold water samples collected at the inlet from the aqueduct network, at the exit from the autoclave, and at the most remote tap. Legionella pneumophila sg1, Legionella pneumophila sg2–16, and non-pneumophila Legionella species were detected in 26% of the hot water networks, mostly in CB and TB. In these buildings we detected correlations between the presence of Legionella and the total chlorine concentration decrease and/or the increase of the temperature. Cold water resulted free from microbiological hazards, with the exception of Serratia liquefaciens and Enterobacter cloacae isolated at the exit from two different autoclaves. We observed an increase in total microbial counts at 22 °C and 37 °C between the samples collected at the most remote taps compared to the ones collected at the inlet from the aqueduct. The study highlights a condition of potential risk for susceptible categories of population and supports the need for measures of risk assessment and control. PMID:29072607

Hydrogen-Bonded Network and Water Dynamics in the D-channel of Cytochrome c Oxidase.

PubMed

Ghane, Tahereh; Gorriz, Rene F; Wrzalek, Sandro; Volkenandt, Senta; Dalatieh, Ferand; Reidelbach, Marco; Imhof, Petra

2018-02-12

Proton transfer in cytochrome c oxidase (CcO) from the cellular inside to the binuclear redox centre as well as proton pumping through the membrane takes place through proton entrance via two distinct pathways, the D- and K-channel. Both channels show a dependence of their hydration level on the protonation states of their key residues, K362 for the K-channel, and E286 or D132 for the D-channel. In the oxidative half of CcO's catalytic cycle the D-channel is the proton-conducting path. For this channel, an interplay of protonation state of the D-channel residues with the water and hydrogen-bond dynamics has been observed in molecular dynamics simulations of the CcO protein, embedded in a lipid bi-layer, modelled in different protonation states. Protonation of residue E286 at the end of the D-channel results in a hydrogen-bonded network pointing from E286 to N139, that is against proton transport, and favouring N139 conformations which correspond to a closed asparagine gate (formed by residues N121 and N139). Consequently, the hydration level is lower than with unprotonated E286. In those models, the Asn gate is predominantly open, allowing water molecules to pass and thus increase the hydration level. The hydrogen-bonded network in these states exhibits longer life times of the Asn residues with water than other models and shows the D-channel to be traversable from the entrance, D132, to exit, E286. The D-channel can thus be regarded as auto-regulated with respect to proton transport, allowing proton passage only when required, that is the proton is located at the lower part of the D-channel (D132 to Asn gate) and not at the exit (E286).

Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis

PubMed Central

Pillai, Smitha; Nguyen, Jonathan; Johnson, Joseph; Haura, Eric; Coppola, Domenico; Chellappan, Srikumar

2015-01-01

TANK Binding Kinase 1 (TBK1) is a non-canonical IκB kinase that contributes to KRAS-driven lung cancer. Here we report that TBK1 plays essential roles in mammalian cell division. Specifically, levels of active phospho-TBK1 increase during mitosis and localize to centrosomes, mitotic spindles and midbody, and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and to the mitotic apparatus protein NuMA, and both CEP170 and NuMA are TBK1 substrates. Further, TBK1 is necessary for CEP170 centrosomal localization and binding to the microtubule depolymerase Kif2b, and for NuMA binding to dynein. Finally, selective disruption of the TBK1–CEP170 complex augments microtubule stability and triggers defects in mitosis, suggesting that TBK1 functions as a mitotic kinase necessary for microtubule dynamics and mitosis. PMID:26656453

GAK, a regulator of clathrin-mediated membrane traffic, also controls centrosome integrity and chromosome congression.

PubMed

Shimizu, Hiroyuki; Nagamori, Ippei; Yabuta, Norikazu; Nojima, Hiroshi

2009-09-01

Cyclin G-associated kinase (GAK) is an association partner of clathrin heavy chain (CHC) and is essential for clathrin-mediated membrane trafficking. Here, we report two novel functions of GAK: maintenance of proper centrosome maturation and of mitotic chromosome congression. Indeed, GAK knockdown by siRNA caused cell-cycle arrest at metaphase, which indicates that GAK is required for proper mitotic progression. We found that this impaired mitotic progression was due to activation of the spindle-assembly checkpoint, which senses protruded, misaligned or abnormally condensed chromosomes in GAK-siRNA-treated cells. GAK knockdown also caused multi-aster formation, which was due to abnormal fragmentation of pericentriolar material, but not of the centrioles. Moreover, GAK and CHC cooperated in the same pathway and interacted in mitosis to regulate the formation of a functional spindle. Taken together, we conclude that GAK and clathrin function cooperatively not only in endocytosis, but also in mitotic progression.

Release of Mps1 from kinetochores is crucial for timely anaphase onset.

PubMed

Jelluma, Nannette; Dansen, Tobias B; Sliedrecht, Tale; Kwiatkowski, Nicholas P; Kops, Geert J P L

2010-10-18

Mps1 kinase activity is required for proper chromosome segregation during mitosis through its involvements in microtubule-chromosome attachment error correction and the mitotic checkpoint. Mps1 dynamically exchanges on unattached kinetochores but is largely removed from kinetochores in metaphase. Here we show that Mps1 promotes its own turnover at kinetochores and that removal of Mps1 upon chromosome biorientation is a prerequisite for mitotic checkpoint silencing. Inhibition of Mps1 activity increases its half-time of recovery at unattached kinetochores and causes accumulation of Mps1 protein at these sites. Strikingly, preventing dissociation of active Mps1 from kinetochores delays anaphase onset despite normal chromosome attachment and alignment, and high interkinetochore tension. This delay is marked by continued recruitment of Mad1 and Mad2 to bioriented chromosomes and is attenuated by Mad2 depletion, indicating chronic engagement of the mitotic checkpoint in metaphase. We propose that release of Mps1 from kinetochores is essential for mitotic checkpoint silencing and a fast metaphase-to-anaphase transition.

SIRT6 deacetylates H3K18Ac at pericentric chromatin to prevent mitotic errors and cell senescence

PubMed Central

Tasselli, Luisa; Xi, Yuanxin; Zheng, Wei; Tennen, Ruth I.; Odrowaz, Zaneta; Simeoni, Federica; Li, Wei; Chua, Katrin F.

2018-01-01

Pericentric heterochromatin silencing at mammalian centromeres is essential for mitotic fidelity and genomic stability. Defective pericentric silencing is observed in senescent cells, aging tissues, and mammalian tumors, but the underlying mechanisms and functional consequences of these defects are unclear. Here, we uncover a pivotal role of the human SIRT6 enzyme in pericentric transcriptional silencing, and show that this function protects against mitotic defects, genomic instability, and cellular senescence. At pericentric heterochromatin, SIRT6 promotes deacetylation of a new substrate, histone H3 lysine K18 (H3K18), and inactivation of SIRT6 in cells leads to H3K18 hyperacetylation and aberrant accumulation of pericentric transcripts. Strikingly, RNAi-depletion of these transcripts rescues the mitotic and senescence phenotypes of SIRT6-deficient cells. Together, our findings reveal a new function for SIRT6 and H3K18Ac regulation at heterochromatin, and demonstrate the pathogenic role of de-regulated pericentric transcription in aging- and cancer- related cellular dysfunction. PMID:27043296

γH2AX foci formation in the absence of DNA damage: mitotic H2AX phosphorylation is mediated by the DNA-PKcs/CHK2 pathway.

PubMed

Tu, Wen-Zhi; Li, Bing; Huang, Bo; Wang, Yu; Liu, Xiao-Dan; Guan, Hua; Zhang, Shi-Meng; Tang, Yan; Rang, Wei-Qing; Zhou, Ping-Kun

2013-11-01

Phosphorylated H2AX is considered to be a biomarker for DNA double-strand breaks (DSB), but recent evidence suggests that γH2AX does not always indicate the presence of DSB. Here we demonstrate the bimodal dynamic of H2AX phosphorylation induced by ionizing radiation, with the second peak appearing when G2/M arrest is induced. An increased level of γH2AX occurred in mitotic cells, and this increase was attenuated by DNA-PKcs inactivation or Chk2 depletion, but not by ATM inhibition. The phosphorylation-mimic CHK2-T68D abrogated the attenuation of mitotic γH2AX induced by DNA-PKcs inactivation. Thus, the DNA-PKcs/CHK2 pathway mediates the mitotic phosphorylation of H2AX in the absence of DNA damage. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Prolonged Mitosis of Neural Progenitors Alters Cell Fate in the Developing Brain.

PubMed

Pilaz, Louis-Jan; McMahon, John J; Miller, Emily E; Lennox, Ashley L; Suzuki, Aussie; Salmon, Edward; Silver, Debra L

2016-01-06

Embryonic neocortical development depends on balanced production of progenitors and neurons. Genetic mutations disrupting progenitor mitosis frequently impair neurogenesis; however, the link between altered mitosis and cell fate remains poorly understood. Here we demonstrate that prolonged mitosis of radial glial progenitors directly alters neuronal fate specification and progeny viability. Live imaging of progenitors from a neurogenesis mutant, Magoh(+/-), reveals that mitotic delay significantly correlates with preferential production of neurons instead of progenitors, as well as apoptotic progeny. Independently, two pharmacological approaches reveal a causal relationship between mitotic delay and progeny fate. As mitotic duration increases, progenitors produce substantially more apoptotic progeny or neurons. We show that apoptosis, but not differentiation, is p53 dependent, demonstrating that these are distinct outcomes of mitotic delay. Together our findings reveal that prolonged mitosis is sufficient to alter fates of radial glia progeny and define a new paradigm to understand how mitosis perturbations underlie brain size disorders such as microcephaly. Copyright © 2016 Elsevier Inc. All rights reserved.

Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

PubMed

Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

2015-11-05

Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division. Copyright © 2015 Elsevier Inc. All rights reserved.

Hunting the mechanisms of self-renewal of immortal cell populations by means of real-time imaging of living cells.

PubMed

Kvitko, O V; Koneva, I I; Sheiko, Y I; Anisovich, M V

2005-12-01

The causes of the indefinite propagation of immortalized cell populations remain insufficiently understood, that hinders the research of such fundamental processes as ageing and cancer. In this study the interrelations between clonal proliferation and abnormalities of mitotic divisions in the immortalized cell line established from the mouse embryo were investigated with the aid of computerized microscopy of living cells. 3 mitoses with three daughter cells and 7 asymmetric mitoses which generated two daughter cells of conspicuously different sizes were registered among 71 mitotic divisions in the individual cell genealogy. Abnormal mitotic divisions either did not slow the proliferation in cell clones compared with progenies of cells that divided by means of normal mitoses or were followed by the acceleration of divisions in consecutive cell generations. These data suggest that abnormal mitotic divisions may contribute to the maintenance of the immortalized state of cell populations by means of generating chromosomal instability.

BRCA1 interaction of centrosomal protein Nlp is required for successful mitotic progression.

PubMed

Jin, Shunqian; Gao, Hua; Mazzacurati, Lucia; Wang, Yang; Fan, Wenhong; Chen, Qiang; Yu, Wei; Wang, Mingrong; Zhu, Xueliang; Zhang, Chuanmao; Zhan, Qimin

2009-08-21

Breast cancer susceptibility gene BRCA1 is implicated in the control of mitotic progression, although the underlying mechanism(s) remains to be further defined. Deficiency of BRCA1 function leads to disrupted mitotic machinery and genomic instability. Here, we show that BRCA1 physically interacts and colocalizes with Nlp, an important molecule involved in centrosome maturation and spindle formation. Interestingly, Nlp centrosomal localization and its protein stability are regulated by normal cellular BRCA1 function because cells containing BRCA1 mutations or silenced for endogenous BRCA1 exhibit disrupted Nlp colocalization to centrosomes and enhanced Nlp degradation. Its is likely that the BRCA1 regulation of Nlp stability involves Plk1 suppression. Inhibition of endogenous Nlp via the small interfering RNA approach results in aberrant spindle formation, aborted chromosomal segregation, and aneuploidy, which mimic the phenotypes of disrupted BRCA1. Thus, BRCA1 interaction of Nlp might be required for the successful mitotic progression, and abnormalities of Nlp lead to genomic instability.

BRCA1 Interaction of Centrosomal Protein Nlp Is Required for Successful Mitotic Progression*♦

PubMed Central

Jin, Shunqian; Gao, Hua; Mazzacurati, Lucia; Wang, Yang; Fan, Wenhong; Chen, Qiang; Yu, Wei; Wang, Mingrong; Zhu, Xueliang; Zhang, Chuanmao; Zhan, Qimin

2009-01-01

Breast cancer susceptibility gene BRCA1 is implicated in the control of mitotic progression, although the underlying mechanism(s) remains to be further defined. Deficiency of BRCA1 function leads to disrupted mitotic machinery and genomic instability. Here, we show that BRCA1 physically interacts and colocalizes with Nlp, an important molecule involved in centrosome maturation and spindle formation. Interestingly, Nlp centrosomal localization and its protein stability are regulated by normal cellular BRCA1 function because cells containing BRCA1 mutations or silenced for endogenous BRCA1 exhibit disrupted Nlp colocalization to centrosomes and enhanced Nlp degradation. Its is likely that the BRCA1 regulation of Nlp stability involves Plk1 suppression. Inhibition of endogenous Nlp via the small interfering RNA approach results in aberrant spindle formation, aborted chromosomal segregation, and aneuploidy, which mimic the phenotypes of disrupted BRCA1. Thus, BRCA1 interaction of Nlp might be required for the successful mitotic progression, and abnormalities of Nlp lead to genomic instability. PMID:19509300

The timing of synthesis of proteins required for mitotic spindle and phragmoplast in partially synchronized root meristems of Vicia faba L.

PubMed

Olszewska, M J; Marciniak, K; Kuran, H

1990-10-01

After cycloheximide treatment (1 h, 2.5 micrograms/ml) protein synthesis was decreased by 70% and was partially restored after 7 h of postincubation (still 20% decrease). In partially synchronized root meristems of Vicia faba L. treated with cycloheximide at middle G2, a strong decrease of the mitotic index was observed. Exposure to the drug at late G2 did not modify the mitotic index; the changes in the phase indices suggested that the course of mitosis was blocked at prophase-metaphase/anaphase-telophase transitions. The use of indirect immunocytochemical staining of tubulin (second antibody labeled with peroxidase) made it possible to show a decreased number of cells with preprophase bands in cycloheximide-treated meristems and the mitotic spindles and phragmoplasts containing a reduced number of shortened bands of microtubules. As a result of these structural and functional disturbances, binucleate cells and polyploid nuclei were observed.

Automatic digital image analysis for identification of mitotic cells in synchronous mammalian cell cultures.

PubMed

Eccles, B A; Klevecz, R R

1986-06-01

Mitotic frequency in a synchronous culture of mammalian cells was determined fully automatically and in real time using low-intensity phase-contrast microscopy and a newvicon video camera connected to an EyeCom III image processor. Image samples, at a frequency of one per minute for 50 hours, were analyzed by first extracting the high-frequency picture components, then thresholding and probing for annular objects indicative of putative mitotic cells. Both the extraction of high-frequency components and the recognition of rings of varying radii and discontinuities employed novel algorithms. Spatial and temporal relationships between annuli were examined to discern the occurrences of mitoses, and such events were recorded in a computer data file. At present, the automatic analysis is suited for random cell proliferation rate measurements or cell cycle studies. The automatic identification of mitotic cells as described here provides a measure of the average proliferative activity of the cell population as a whole and eliminates more than eight hours of manual review per time-lapse video recording.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hitomi, Toshiaki; Habu, Toshiyuki; Kobayashi, Hatasu

Highlights: •Overexpression of RNF213 R4810K inhibited cell proliferation. •Overexpression of RNF213 R4810K had the time of mitosis 4-fold and mitotic failure. •R4810K formed a complex with MAD2 more readily than wild-type. •iPSECs from the MMD patients had elevated mitotic failure compared from the control. •RNF213 R4810K induced mitotic abnormality and increased risk of aneuploidy. -- Abstract: Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the Circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. In the present study, we characterized phenotypes caused by overexpression of RNF213 wild type and R4810K variant in the cellmore » cycle to investigate the mechanism of proliferation inhibition. Overexpression of RNF213 R4810K in HeLa cells inhibited cell proliferation and extended the time of mitosis 4-fold. Ablation of spindle checkpoint by depletion of mitotic arrest deficiency 2 (MAD2) did not shorten the time of mitosis. Mitotic morphology in HeLa cells revealed that MAD2 colocalized with RNF213 R4810K. Immunoprecipitation revealed an RNF213/MAD2 complex: R4810K formed a complex with MAD2 more readily than RNF213 wild-type. Desynchronized localization of MAD2 was observed more frequently during mitosis in fibroblasts from patients (n = 3, 61.0 ± 8.2%) compared with wild-type subjects (n = 6, 13.1 ± 7.7%; p < 0.01). Aneuploidy was observed more frequently in fibroblasts (p < 0.01) and induced pluripotent stem cells (iPSCs) (p < 0.03) from patients than from wild-type subjects. Vascular endothelial cells differentiated from iPSCs (iPSECs) of patients and an unaffected carrier had a longer time from prometaphase to metaphase than those from controls (p < 0.05). iPSECs from the patients and unaffected carrier had significantly increased mitotic failure rates compared with controls (p < 0.05). Thus, RNF213 R4810K induced mitotic abnormalities and increased risk of genomic instability.« less

[Epidemiology of catheter-related infections in intensive care unit].

PubMed

Merrer, J

2005-03-01

Catheter-related infections remain an important cause of nosocomial infection in the ICU. They include colonization of the device, exit-site infection and catheter-related bloodstream infection with or without bacteraemia. Data from clinical studies and surveillance networks should be compared cautiously due to important methodological differences and wide variations of device-utilization ratio between units or countries. In France, two regional networks (C-CLIN Paris-Nord and C-CLIN Sud-Est) produced comparable and reproducible results. Colonization represents five-six cases per 1000 catheter-days and bacteraemia represents one case per 1000 catheter-days. Incidence rates from North American studies are usually four to five times higher. Numerous risk factors have been identified. Some of them could be used to stratify patients according to risk of catheter-related infection and to allow more valid comparison between ICU's performances. Participation of French ICUs to the recent national surveillance networks (REA RAISIN and REACAT RAISIN) should be encouraged.

Comparison of cytotoxic and genotoxic effects of plutonium-239 alpha particles and mobile phone GSM 900 radiation in the Allium cepa test.

PubMed

Pesnya, Dmitry S; Romanovsky, Anton V

2013-01-20

The goal of this study was to compare the cytotoxic and genotoxic effects of plutonium-239 alpha particles and GSM 900 modulated mobile phone (model Sony Ericsson K550i) radiation in the Allium cepa test. Three groups of bulbs were exposed to mobile phone radiation during 0 (sham), 3 and 9h. A positive control group was treated during 20min with plutonium-239 alpha-radiation. Mitotic abnormalities, chromosome aberrations, micronuclei and mitotic index were analyzed. Exposure to alpha-radiation from plutonium-239 and exposure to modulated radiation from mobile phone during 3 and 9h significantly increased the mitotic index. GSM 900 mobile phone radiation as well as alpha-radiation from plutonium-239 induced both clastogenic and aneugenic effects. However, the aneugenic activity of mobile phone radiation was more pronounced. After 9h of exposure to mobile phone radiation, polyploid cells, three-groups metaphases, amitoses and some unspecified abnormalities were detected, which were not registered in the other experimental groups. Importantly, GSM 900 mobile phone radiation increased the mitotic index, the frequency of mitotic and chromosome abnormalities, and the micronucleus frequency in a time-dependent manner. Due to its sensitivity, the A. cepa test can be recommended as a useful cytogenetic assay to assess cytotoxic and genotoxic effects of radiofrequency electromagnetic fields. Copyright © 2012 Elsevier B.V. All rights reserved.

CDK-dependent potentiation of MPS1 kinase activity is essential to the mitotic checkpoint.

PubMed

Morin, Violeta; Prieto, Susana; Melines, Sabrina; Hem, Sonia; Rossignol, Michel; Lorca, Thierry; Espeut, Julien; Morin, Nathalie; Abrieu, Ariane

2012-02-21

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached. Copyright © 2012 Elsevier Ltd. All rights reserved.

900 MHz radiation does not induce micronucleus formation in different cell types.

PubMed

Hintzsche, Henning; Jastrow, Christian; Kleine-Ostmann, Thomas; Schrader, Thorsten; Stopper, Helga

2012-07-01

The exposure of the population to non-ionising electromagnetic radiation is still increasing, mainly due to mobile communication. Whether low-intensity electromagnetic fields can cause other effects apart from heating has been a subject of debate. One of the effects, which were proposed to be caused by mobile phone radiation, is the occurrence of mitotic disturbances. The aim of this study was to investigate possible consequences of these mitotic disturbances as manifest genomic damage, i.e. micronucleus induction. Cells were irradiated at a frequency of 900 MHz, which is located in one of the main frequency bands applied for mobile communication. Two cell types were used, HaCaT cells as human cells and A(L) cells (human-hamster hybrid cells), in which mitotic disturbances had been reported to occur. After different post-exposure incubation periods, cells were fixed and micronucleus frequencies were evaluated. Both cell types did not show any genomic damage after exposure. To adapt the protocol for the micronucleus test into the direction of the protocol for mitotic disturbances, the post-exposure incubation period was reduced and exposure time was extended to one cell cycle length. This did not result in any increase of the genomic damage. In conclusion, micronucleus induction was not observed as a consequence of exposure to non-ionising radiation, even though this agent was reported to cause mitotic disturbances under similar experimental conditions.

Deficiency of RITA results in multiple mitotic defects by affecting microtubule dynamics.

PubMed

Steinhäuser, K; Klöble, P; Kreis, N-N; Ritter, A; Friemel, A; Roth, S; Reichel, J M; Michaelis, J; Rieger, M A; Louwen, F; Oswald, F; Yuan, J

2017-04-01

Deregulation of mitotic microtubule (MT) dynamics results in defective spindle assembly and chromosome missegregation, leading further to chromosome instability, a hallmark of tumor cells. RBP-J interacting and tubulin-associated protein (RITA) has been identified as a negative regulator of the Notch signaling pathway. Intriguingly, deregulated RITA is involved in primary hepatocellular carcinoma and other malignant entities. We were interested in the potential molecular mechanisms behind its involvement. We show here that RITA binds to tubulin and localizes to various mitotic MT structures. RITA coats MTs and affects their structures in vitro as well as in vivo. Tumor cell lines deficient of RITA display increased acetylated α-tubulin, enhanced MT stability and reduced MT dynamics, accompanied by multiple mitotic defects, including chromosome misalignment and segregation errors. Re-expression of wild-type RITA, but not RITA Δtub ineffectively binding to tubulin, restores the phenotypes, suggesting that the role of RITA in MT modulation is mediated via its interaction with tubulin. Mechanistically, RITA interacts with tubulin/histone deacetylase 6 (HDAC6) and its suppression decreases the binding of the deacetylase HDAC6 to tubulin/MTs. Furthermore, the mitotic defects and increased MT stability are also observed in RITA -/- mouse embryonic fibroblasts. RITA has thus a novel role in modulating MT dynamics and its deregulation results in erroneous chromosome segregation, one of the major reasons for chromosome instability in tumor cells.

A study of knowledge supernetworks and network robustness in different business incubators

NASA Astrophysics Data System (ADS)

Zhang, Haihong; Wu, Wenqing; Zhao, Liming

2016-04-01

As the most important intangible resource of the new generation of business incubators, knowledge has been studied extensively, particularly with respect to how it spreads among incubating firms through knowledge networks. However, these homogeneous networks do not adequately describe the heterogeneity of incubating firms in different types of business incubators. To solve the problem of heterogeneity, the notion of a knowledge supernetwork has been used both to construct a knowledge interaction model among incubating firms and to distinguish social network relationships from knowledge network relationships. The process of knowledge interaction and network evolution can then be simulated with a few rules for incubating firms regarding knowledge innovation/absorption, social network connection, and entry and exit, among other aspects. Knowledge and networks have been used as performance indicators to evaluate the evolution of knowledge supernetworks. Moreover, we study the robustness of incubating firms' social networks by employing four types of attack strategies. Based on our simulation results, we conclude that there have been significant knowledge interaction and network evolution among incubating firms on a periodic basis and that both specialized and diversified business incubators have every advantage necessary in terms of both knowledge and networks to cultivate start-up companies. As far as network robustness is concerned, there is no obvious difference between the two types of business incubators with respect to the stability of their network structures, but specialized business incubators have stronger network communication abilities than diversified business incubators.

Battlefield Acoustic Sensing, Multimodal Sensing, and Networked Sensing for Intelligence, Surveillance, and Reconnaissance (ISR) Applications

DTIC Science & Technology

2015-09-01

seen in Fig. 9, were placed at the rear of the vehicle to minimize the noise and vibration from the engine and its intake and exhaust from the...Specific/unique algorithm approaches attempted and results 4) Observations related to wind noise rejection and/or effects 5) Limitations of technology 6...After a certain time, the muzzle blast is detected, which results from the exit of the munition at the muzzle. Figure 6 shows an example of a single

Automated analysis of Physarum network structure and dynamics

NASA Astrophysics Data System (ADS)

Fricker, Mark D.; Akita, Dai; Heaton, Luke LM; Jones, Nick; Obara, Boguslaw; Nakagaki, Toshiyuki

2017-06-01

We evaluate different ridge-enhancement and segmentation methods to automatically extract the network architecture from time-series of Physarum plasmodia withdrawing from an arena via a single exit. Whilst all methods gave reasonable results, judged by precision-recall analysis against a ground-truth skeleton, the mean phase angle (Feature Type) from intensity-independent, phase-congruency edge enhancement and watershed segmentation was the most robust to variation in threshold parameters. The resultant single pixel-wide segmented skeleton was converted to a graph representation as a set of weighted adjacency matrices containing the physical dimensions of each vein, and the inter-vein regions. We encapsulate the complete image processing and network analysis pipeline in a downloadable software package, and provide an extensive set of metrics that characterise the network structure, including hierarchical loop decomposition to analyse the nested structure of the developing network. In addition, the change in volume for each vein and intervening plasmodial sheet was used to predict the net flow across the network. The scaling relationships between predicted current, speed and shear force with vein radius were consistent with predictions from Murray’s law. This work was presented at PhysNet 2015.

The peroxisomal multifunctional protein interacts with cortical microtubules in plant cells

PubMed Central

2005-01-01

Background The plant peroxisomal multifunctional protein (MFP) possesses up to four enzymatic activities that are involved in catalyzing different reactions of fatty acid β-oxidation in the peroxisome matrix. In addition to these peroxisomal activities, in vitro assays revealed that rice MFP possesses microtubule- and RNA-binding activities suggesting that this protein also has important functions in the cytosol. Results We demonstrate that MFP is an authentic microtubule-binding protein, as it localized to the cortical microtubule array in vivo, in addition to its expected targeting to the peroxisome matrix. MFP does not, however, interact with the three mitotic microtubule arrays. Microtubule co-sedimentation assays of truncated versions of MFP revealed that multiple microtubule-binding domains are present on the MFP polypeptide. This indicates that these regions function together to achieve high-affinity binding of the full-length protein. Real-time imaging of a transiently expressed green fluorescent protein-MFP chimera in living plant cells illustrated that a dynamic, spatial interaction exits between peroxisomes and cortical microtubules as peroxisomes move along actin filaments or oscillate at fixed locations. Conclusion Plant MFP is associated with the cortical microtubule array, in addition to its expected localization in the peroxisome. This observation, coupled with apparent interactions that frequently occur between microtubules and peroxisomes in the cell cortex, supports the hypothesis that MFP is concentrated on microtubules in order to facilitate the regulated import of MFP into peroxisomes. PMID:16313672

Sporophytic and gametophytic functions of the cell cycle-associated Mob1 gene in Arabidopsis thaliana L.

PubMed

Galla, Giulio; Zenoni, Sara; Marconi, Gianpiero; Marino, Giada; Botton, Alessandro; Pinosa, Francesco; Citterio, Sandra; Ruperti, Benedetto; Palme, Klaus; Albertini, Emidio; Pezzotti, Mario; Mau, Martin; Sharbel, Timothy F; De Storme, Nico; Geelen, Danny; Barcaccia, Gianni

2011-09-15

Mob1 genes are primarily involved in the cell cycle progression and mitosis exit in yeasts and animals. The function of a Mob1-like gene (At5g45550) from Arabidopsis thaliana was investigated using RNAi and immunological staining. AtMob1-like RNAi silenced lines showed a reduced radial expansion of the inflorescence stem and a reduced elongation zone of the primary root. Morphological features of plant organs were accompanied by a reduction in cell size. The fertility of AtMob1-like RNAi silenced lines was very low as seed production was strongly reduced. About 2% of the progeny of AtMob1-like RNAi silenced plants were tetraploid. The female and male sporogenesis was affected differentially. The ovules developed irregularly and one third of the megaspores and embryo sacs degenerated prematurely. Up to 20% of the ovules produced binucleated megaspores that failed to develop further, being their degeneration likely accompanied with a delayed programmed cell death. The anthers produced about 30% of aborted pollen grains, showing also a strong variation in their size. Together, the results show that Arabidopsis MOB1-like is required to regulate cell expansion and cell division, presumably by affecting the mitotic as well as the meiotic cell cycle. Copyright © 2011 Elsevier B.V. All rights reserved.

NON-TOXIC MELANOMA THERAPY BY A NOVEL TUBULIN-BINDING AGENT

PubMed Central

Aneja, Ritu; Asress, Seneshaw; Dhiman, Neerupma; Awasthi, Anshumali; Rida, Padmashree C.G.; Arora, Sudarshan K.; Zhou, Jun; Glass, Jonathan D.; Joshi, Harish C.

2009-01-01

(S)-3-((R)-9-bromo-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquino-lin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (EM011) is a tubulin-binding agent with significant anticancer activity. Here we show that EM011 modulates microtubule dynamics at concentrations that do not alter the total polymer mass of tubulin. In particular, EM011 decreases the transition frequencies between growth and shortening phases and increases the duration microtubules spend in an idle ‘pause’ state. Using B16LS9 murine melanoma cells, we show that EM011 briefly arrests cell-cycle progression at the G2/M phase by formation of multiple aster spindles. An aberrant mitotic exit without cytokinesis then occurs, leading to the accumulation of abnormal multinucleated cells prior to apoptosis. Our pharmacokinetic studies conformed to a linear dose-response relationship upto 150 mg/kg. However, non-linearity was observed at 300 mg/kg. In a syngeneic murine model of subcutaneous melanoma, better antitumor responses were seen at 150 mg/kg compared to 300 mg/kg of EM011. Unlike currently available chemotherapeutics, EM011 is non-toxic to normal tissues and most importantly, does not cause any immunosuppression and neurotoxicity. Our data thus warrant a clinical evaluation of EM011 for melanoma therapy. PMID:19626589

Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function

PubMed Central

Zhang, Lixia; Kendrick, Christina; Jülich, Dörthe; Holley, Scott A.

2010-01-01

Summary Cell division, differentiation and morphogenesis are coordinated during embryonic development and frequently in disarray in pathologies such as cancer. Here, we present a zebrafish mutant that ceases mitosis at the beginning of gastrulation, but undergoes axis elongation and develops blood, muscle and a beating heart. We identify the mutation as being in early mitotic inhibitor 1 (emi1), a negative regulator of the Anaphase Promoting Complex, and utilize the mutant to examine the role of the cell cycle in somitogenesis. The mutant phenotype indicates that axis elongation during the segmentation period is substantially driven by cell migration. We find that the segmentation clock, which regulates somitogenesis, functions normally in the absence of cell cycle progression and observe that mitosis is a modest source of noise for the clock. Somite morphogenesis involves the epithelialization of the somite border cells around a core of mesenchyme. As in wild-type embryos, somite boundary cells are polarized along a Fibronectin matrix in emi1−/−. The mutants also display evidence of segment polarity. However, in the absence of a normal cell cycle, somites appear to hyper-epithelialize as the internal mesenchymal cells exit the core of the somite after initial boundary formation. Thus, cell cycle progression is not required during the segmentation period for segmentation clock function but is necessary for normal segmental arrangement of epithelial borders and internal mesenchymal cells. PMID:18480162

Loss of the Mammalian DREAM Complex Deregulates Chondrocyte Proliferation

PubMed Central

Forristal, Chantal; Henley, Shauna A.; MacDonald, James I.; Bush, Jason R.; Ort, Carley; Passos, Daniel T.; Talluri, Srikanth; Ishak, Charles A.; Thwaites, Michael J.; Norley, Chris J.; Litovchick, Larisa; DeCaprio, James A.; DiMattia, Gabriel; Holdsworth, David W.; Beier, Frank

2014-01-01

Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes. PMID:24710275

Estimation of mutagenic effect and modifications of mitosis by silver nanoparticles.

PubMed

Prokhorova, I M; Kibrik, B S; Pavlov, A V; Pesnya, D S

2013-12-01

We analyzed mutagenic and mitosis-modifying effects of silver nanoparticles (Allium test). Chromosome aberrations and laggings and micronuclei were simultaneously registered in the same sample. Mitotic and phase indexes were calculated. No mutagenic effects were detected after treatment with silver nanoparticles in doses of 1.0, 2.5, 5.0, and 50 mg/liter. Silver nanoparticles in a concentration of 50 mg/liter significantly increased the mitotic index. Nanoparticles in a dose of 5 mg/liter induced slight, but significant increase in mitotic index, but did not affect the ratio of phase indexes. Exposure to silver nanoparticles in concentrations of 1.0 and 2.5 mg/liter was not followed by modification of mitosis.

29 CFR 1917.122 - Employee exits.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 29 Labor 7 2010-07-01 2010-07-01 false Employee exits. 1917.122 Section 1917.122 Labor Regulations...) MARINE TERMINALS Terminal Facilities § 1917.122 Employee exits. (a) Employee exits shall be clearly marked. (b) If an employee exit is not visible from employees' work stations, directional signs...

Advanced Diesel Oil Fuel Processor Development

DTIC Science & Technology

1986-06-01

water exit 29 sample quencher: gas sample line inlet 30 sample quencher: gas sample line exit 31 sample quencher: cooling water inlet 32 desulfuriser ...exit line 33, 34 desulfurimer 35 heat exchanger: process gas exit (to desulfuriser ) 38 shift reactor inlet (top) 37 shift reactor: cooling air exit

Antagonistic effects of pemoline to colchicine and caffeine.

PubMed

Röper, W

1975-10-15

Pemoline, the constituent of Tradon, is able to slow down the decrease of the mitotic index caused by 0.1% caffeine in roots of Vicia faba, and mitotic aberrations are reduced. With 0.005% colchicine and 3 x 10(-4) g/ml pemoline, no metaphase-accumulation can be observed, and anaphase-disorder is delayed.

The Aurora kinase A inhibitor TC-A2317 disrupts mitotic progression and inhibits cancer cell proliferation

PubMed Central

Min, Yoo Hong; Kim, Wootae; Kim, Ja-Eun

2016-01-01

Mitotic progression is crucial for the maintenance of chromosomal stability. A proper progression is ensured by the activities of multiple kinases. One of these enzymes, the serine/threonine kinase Aurora A, is required for proper mitosis through the regulation of centrosome and spindle assembly. In this study, we functionally characterized a newly developed Aurora kinase A inhibitor, TC-A2317. In human lung cancer cells, TC-A2317 slowed proliferation by causing aberrant formation of centrosome and microtubule spindles and prolonging the duration of mitosis. Abnormal mitotic progression led to accumulation of cells containing micronuclei or multinuclei. Furthermore, TC-A2317–treated cells underwent apoptosis, autophagy or senescence depending on cell type. In addition, TC-A2317 inactivated the spindle assembly checkpoint triggered by paclitaxel, thereby exacerbating mitotic catastrophe. Consistent with this, the expression level of Aurora A in tumors was inversely correlated with survival in lung cancer patients. Collectively, these data suggest that inhibition of Aurora kinase A using TC-A2317 is a promising target for anti-cancer therapeutics. PMID:27713168

Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

PubMed Central

Gergely, Zachary R.; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Betterton, Meredith D.

2016-01-01

Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen. PMID:27146110

Tripolar mitosis in human cells and embryos: occurrence, pathophysiology and medical implications.

PubMed

Kalatova, Beata; Jesenska, Renata; Hlinka, Daniel; Dudas, Marek

2015-01-01

Tripolar mitosis is a specific case of cell division driven by typical molecular mechanisms of mitosis, but resulting in three daughter cells instead of the usual count of two. Other variants of multipolar mitosis show even more mitotic poles and are relatively rare. In nature, this phenomenon was frequently observed or suspected in multiple common cancers, infected cells, the placenta, and in early human embryos with impaired pregnancy-yielding potential. Artificial causes include radiation and various toxins. Here we combine several pieces of the most recent evidence for the existence of different types of multipolar mitosis in preimplantation embryos together with a detailed review of the literature. The related molecular and cellular mechanisms are discussed, including the regulation of centriole duplication, mitotic spindle biology, centromere functions, cell cycle checkpoints, mitotic autocorrection mechanisms, and the related complicating factors in healthy and affected cells, including post-mitotic cell-cell fusion often associated with multipolar cell division. Clinical relevance for oncology and embryo selection in assisted reproduction is also briefly discussed in this context. Copyright © 2014 Elsevier GmbH. All rights reserved.

Tumor Environmental Factors Glucose Deprivation and Lactic Acidosis Induce Mitotic Chromosomal Instability – An Implication in Aneuploid Human Tumors

PubMed Central

Zhu, Chunpeng; Hu, Xun

2013-01-01

Mitotic chromosomal instability (CIN) plays important roles in tumor progression, but what causes CIN is incompletely understood. In general, tumor CIN arises from abnormal mitosis, which is caused by either intrinsic or extrinsic factors. While intrinsic factors such as mitotic checkpoint genes have been intensively studied, the impact of tumor microenvironmental factors on tumor CIN is largely unknown. We investigate if glucose deprivation and lactic acidosis – two tumor microenvironmental factors – could induce cancer cell CIN. We show that glucose deprivation with lactic acidosis significantly increases CIN in 4T1, MCF-7 and HCT116 scored by micronuclei, or aneuploidy, or abnormal mitosis, potentially via damaging DNA, up-regulating mitotic checkpoint genes, and/or amplifying centrosome. Of note, the feature of CIN induced by glucose deprivation with lactic acidosis is similar to that of aneuploid human tumors. We conclude that tumor environmental factors glucose deprivation and lactic acidosis can induce tumor CIN and propose that they are potentially responsible for human tumor aneuploidy. PMID:23675453

A TPR domain–containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B

PubMed Central

Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H.J.; Snel, Berend

2013-01-01

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B–dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization. PMID:23569217

A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B.

PubMed

Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H J; Snel, Berend; Perrakis, Anastassis; Kops, Geert J P L

2013-04-15

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

Axin localizes to mitotic spindles and centrosomes in mitotic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Shi-Mun; Choi, Eun-Jin; Song, Ki-Joon

2009-04-01

Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3{beta}) without producing any notable changes inmore » cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3{beta} in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.« less

Mitosis, double strand break repair, and telomeres: a view from the end: how telomeres and the DNA damage response cooperate during mitosis to maintain genome stability.

PubMed

Cesare, Anthony J

2014-11-01

Double strand break (DSB) repair is suppressed during mitosis because RNF8 and downstream DNA damage response (DDR) factors, including 53BP1, do not localize to mitotic chromatin. Discovery of the mitotic kinase-dependent mechanism that inhibits DSB repair during cell division was recently reported. It was shown that restoring mitotic DSB repair was detrimental, resulting in repair dependent genome instability and covalent telomere fusions. The telomere DDR that occurs naturally during cellular aging and in cancer is known to be refractory to G2/M checkpoint activation. Such DDR-positive telomeres, and those that occur as part of the telomere-dependent prolonged mitotic arrest checkpoint, normally pass through mitosis without covalent ligation, but result in cell growth arrest in G1 phase. The discovery that suppressing DSB repair during mitosis may function primarily to protect DDR-positive telomeres from fusing during cell division reinforces the unique cooperation between telomeres and the DDR to mediate tumor suppression. © 2014 The Author. Bioessays published by WILEY Periodicals, Inc.

Phospho-H1 Decorates the Inter-chromatid Axis and Is Evicted along with Shugoshin by SET during Mitosis.

PubMed

Krishnan, Swathi; Smits, Arne H; Vermeulen, Michiel; Reinberg, Danny

2017-08-17

Precise control of sister chromatid separation during mitosis is pivotal to maintaining genomic integrity. Yet, the regulatory mechanisms involved are not well understood. Remarkably, we discovered that linker histone H1 phosphorylated at S/T18 decorated the inter-chromatid axial DNA on mitotic chromosomes. Sister chromatid resolution during mitosis required the eviction of such H1S/T18ph by the chaperone SET, with this process being independent of and most likely downstream of arm-cohesin dissociation. SET also directed the disassembly of Shugoshins in a polo-like kinase 1-augmented manner, aiding centromere resolution. SET ablation compromised mitotic fidelity as evidenced by unresolved sister chromatids with marked accumulation of H1S/T18ph and centromeric Shugoshin. Thus, chaperone-assisted eviction of linker histones and Shugoshins is a fundamental step in mammalian mitotic progression. Our findings also elucidate the functional implications of the decades-old observation of mitotic linker histone phosphorylation, serving as a paradigm to explore the role of linker histones in bio-signaling processes. Copyright © 2017 Elsevier Inc. All rights reserved.

PLK1 Activation in Late G2 Sets Up Commitment to Mitosis.

PubMed

Gheghiani, Lilia; Loew, Damarys; Lombard, Bérangère; Mansfeld, Jörg; Gavet, Olivier

2017-06-06

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.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

PubMed

Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

2015-10-13

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. Copyright © 2015, American Association for the Advancement of Science.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells

PubMed Central

Rusin, Scott F.; Schlosser, Kate A.; Adamo, Mark E.; Kettenbach, Arminja N.

2017-01-01

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry–based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c–dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2–dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

Functional organization of mitotic microtubules. Physical chemistry of the in vivo equilibrium system.

PubMed Central

Inoué, S; Fuseler, J; Salmon, E D; Ellis, G W

1975-01-01

Equilibrium between mitotic microtubules and tubulin is analyzed, using birefringence of mitotic spindle to measure microtubule concentration in vivo. A newly designed temperature-controlled slide and miniature, thermostated hydrostatic pressure chamber permit rapid alteration of temperature and of pressure. Stress birefringence of the windows is minimized, and a system for rapid recording of compensation is incorporated, so that birefringence can be measured to 0.1 nm retardation every few seconds. Both temperature and pressure data yield thermodynamic values (delta H similar to 35 kcal/mol, delta S similar to 120 entropy units [eu], delta V similar to 400 ml/mol of subunit polymerized) consistent with the explanation that polymerization of tubulin is entropy driven and mediated by hydrophobic interactions. Kinetic data suggest pseudo-zero-order polymerization and depolymerization following rapid temperature shifts, and a pseudo-first-order depolymerization during anaphase at constant temperature. The equilibrium properties of the in vivo mitotic microtubules are compared with properties of isolated brain tubules. Images FIGURE 1 FIGURE 2 FIGURE 5 FIGURE 12 FIGURE 13 FIGURE 14 FIGURE 19 PMID:1139037

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Linlin; Sun, Xiaodong; Xie, Songbo

Highlights: • DIMEN displays higher anti-proliferative activity than enastron. • DIMEN induced mitotic arrest and apoptosis more significantly than enastron. • DIMEN blocked the conformational change of ADP-binding pocket more effectively. • DIMEN hindered ADP release more potently than enastron. - Abstract: Eg5 is a mitotic kinesin that plays a crucial role in the formation of bipolar mitotic spindles, by hydrolyzing ATP to push apart anti-parallel microtubules. Dimethylenastron is potent specific small molecule inhibitor of Eg5. The mechanism by which dimethylenastron inhibits Eg5 function remains unclear. By comparing with enastron, here we report that dimethylenastron prevents the growth of pancreaticmore » and lung cancer cells more effectively, by halting mitotic progression and triggering apoptosis. We analyze their interactions with ADP-bound Eg5 crystal structure, and find that dimethylenastron binds Eg5 motor domain with higher affinity. In addition, dimethylenastron allosterically blocks the conformational change of the “sandwich”-like ADP-binding pocket more effectively. We subsequently use biochemical approach to reveal that dimethylenastron slows ADP release more significantly than enastron. These data thus provide biological, structural and mechanistic insights into the potent inhibitory activity of dimethylenastron.« less

Nuclear Chk1 prevents premature mitotic entry.

PubMed

Matsuyama, Makoto; Goto, Hidemasa; Kasahara, Kousuke; Kawakami, Yoshitaka; Nakanishi, Makoto; Kiyono, Tohru; Goshima, Naoki; Inagaki, Masaki

2011-07-01

Chk1 inhibits the premature activation of the cyclin-B1-Cdk1. However, it remains controversial whether Chk1 inhibits Cdk1 in the centrosome or in the nucleus before the G2-M transition. In this study, we examined the specificity of the mouse monoclonal anti-Chk1 antibody DCS-310, with which the centrosome was stained. Conditional Chk1 knockout in mouse embryonic fibroblasts reduced nuclear but not centrosomal staining with DCS-310. In Chk1(+/myc) human colon adenocarcinoma (DLD-1) cells, Chk1 was detected in the nucleus but not in the centrosome using an anti-Myc antibody. Through the combination of protein array and RNAi technologies, we identified Ccdc-151 as a protein that crossreacted with DCS-310 on the centrosome. Mitotic entry was delayed by expression of the Chk1 mutant that localized in the nucleus, although forced immobilization of Chk1 to the centrosome had little impact on the timing of mitotic entry. These results suggest that nuclear but not centrosomal Chk1 contributes to correct timing of mitotic entry.

Curcumin-treated cancer cells show mitotic disturbances leading to growth arrest and induction of senescence phenotype.

PubMed

Mosieniak, Grażyna; Sliwinska, Małgorzata A; Przybylska, Dorota; Grabowska, Wioleta; Sunderland, Piotr; Bielak-Zmijewska, Anna; Sikora, Ewa

2016-05-01

Cellular senescence is recognized as a potent anticancer mechanism that inhibits carcinogenesis. Cancer cells can also undergo senescence upon chemo- or radiotherapy. Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, shows anticancer properties both in vitro and in vivo. Previously, we have shown that treatment with curcumin leads to senescence of human cancer cells. Now we identified the molecular mechanism underlying this phenomenon. We observed a time-dependent accumulation of mitotic cells upon curcumin treatment. The time-lapse analysis proved that those cells progressed through mitosis for a significantly longer period of time. A fraction of cells managed to divide or undergo mitotic slippage and then enter the next phase of the cell cycle. Cells arrested in mitosis had an improperly formed mitotic spindle and were positive for γH2AX, which shows that they acquired DNA damage during prolonged mitosis. Moreover, the DNA damage response pathway was activated upon curcumin treatment and the components of this pathway remained upregulated while cells were undergoing senescence. Inhibition of the DNA damage response decreased the number of senescent cells. Thus, our studies revealed that the induction of cell senescence upon curcumin treatment resulted from aberrant progression through the cell cycle. Moreover, the DNA damage acquired by cancer cells, due to mitotic disturbances, activates an important molecular mechanism that determines the potential anticancer activity of curcumin. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Yeast Polo Kinase Cdc5 Regulates the Shape of the Mitotic Nucleus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walters, Alison D.; May, Christopher K.; Dauster, Emma S.

Abnormal nuclear size and shape are hallmarks of aging and cancer. However, the mechanisms regulating nuclear morphology and nuclear envelope (NE) expansion are poorly understood. In metazoans, the NE disassembles prior to chromosome segregation and reassembles at the end of mitosis. In budding yeast, the NE remains intact. The nucleus elongates as chromosomes segregate and then divides at the end of mitosis to form two daughter nuclei without NE disassembly. The budding yeast nucleus also undergoes remodeling during a mitotic arrest; the NE continues to expand despite the pause in chromosome segregation, forming a nuclear extension, or "flare," that encompassesmore » the nucleolus. The distinct nucleolar localization of the mitotic flare indicates that the NE is compartmentalized and that there is a mechanism by which NE expansion is confined to the region adjacent to the nucleolus. Here we show that mitotic flare formation is dependent on the yeast polo kinase Cdc5. This function of Cdc5 is independent of its known mitotic roles, including rDNA condensation. High-resolution imaging revealed that following Cdc5 inactivation, nuclei expand isometrically rather than forming a flare, indicating that Cdc5 is needed for NE compartmentalization. Lastly, even in an uninterrupted cell cycle, a small NE expansion occurs adjacent to the nucleolus prior to anaphase in a Cdc5-dependent manner. Our data provide the first evidence that polo kinase, a key regulator of mitosis, plays a role in regulating nuclear morphology and NE expansion.« less

The Yeast Polo Kinase Cdc5 Regulates the Shape of the Mitotic Nucleus

DOE PAGES

Walters, Alison D.; May, Christopher K.; Dauster, Emma S.; ...

2014-11-20

Abnormal nuclear size and shape are hallmarks of aging and cancer. However, the mechanisms regulating nuclear morphology and nuclear envelope (NE) expansion are poorly understood. In metazoans, the NE disassembles prior to chromosome segregation and reassembles at the end of mitosis. In budding yeast, the NE remains intact. The nucleus elongates as chromosomes segregate and then divides at the end of mitosis to form two daughter nuclei without NE disassembly. The budding yeast nucleus also undergoes remodeling during a mitotic arrest; the NE continues to expand despite the pause in chromosome segregation, forming a nuclear extension, or "flare," that encompassesmore » the nucleolus. The distinct nucleolar localization of the mitotic flare indicates that the NE is compartmentalized and that there is a mechanism by which NE expansion is confined to the region adjacent to the nucleolus. Here we show that mitotic flare formation is dependent on the yeast polo kinase Cdc5. This function of Cdc5 is independent of its known mitotic roles, including rDNA condensation. High-resolution imaging revealed that following Cdc5 inactivation, nuclei expand isometrically rather than forming a flare, indicating that Cdc5 is needed for NE compartmentalization. Lastly, even in an uninterrupted cell cycle, a small NE expansion occurs adjacent to the nucleolus prior to anaphase in a Cdc5-dependent manner. Our data provide the first evidence that polo kinase, a key regulator of mitosis, plays a role in regulating nuclear morphology and NE expansion.« less

The GPER agonist G-1 induces mitotic arrest and apoptosis in human vascular smooth muscle cells independent of GPER.

PubMed

Gui, Yu; Shi, Zhan; Wang, ZengYong; Li, Jing-Jing; Xu, Can; Tian, RuiJuan; Song, XinXing; Walsh, Michael P; Li, Dong; Gao, Jie; Zheng, Xi-Long

2015-04-01

The G protein-coupled estrogen receptor (GPER) has been implicated in the regulation of smooth muscle cell (SMC) proliferation. The GPER selective agonist G-1 has been a useful tool for exploring the biological roles of GPER in a variety of experimental settings, including SMC proliferation. The present study, originally designed to investigate cellular and signaling mechanisms underlying the regulatory role of GPER in vascular SMC proliferation using G-1, unexpectedly revealed off-target effects of G-1. G-1(1-10 μM) inhibited bromodeoxyuridine (BrdU) incorporation of human SMCs and caused G2/M cell accumulation. G-1 treatment also increased mitotic index concurrent with a decrease in phosphorylation of Cdk1 (Tyr 15) and an increase in phosphorylation of the mitotic checkpoint protein BuBR1. Furthermore, G-1 caused microtubule disruption, mitotic spindle damage, and tubulin depolymerization. G-1 induced cell apoptosis as indicated by the appearance of TUNEL-positive and annexin V-positive cells with enhanced cleavage of caspases 3 and 9. However, neither the GPER antagonist G-15 nor the MAPK kinase inhibitor PD98059 prevented these G-1 effects. Down-regulation of GPER or p44/42 MAPK with siRNA transfection also did not affect the G-1-induced apoptosis. We conclude that G-1 inhibits proliferation of SMCs through mechanisms involving mitotic arrest and apoptosis, independent of GPER and the MAPK pathway. © 2014 Wiley Periodicals, Inc.

Functional Characterization of G12, a Gene Required for Mitotic Progression during Gastrulation in Zebrafish

NASA Technical Reports Server (NTRS)

Reinsch, Sigrid; Conway, Gregory; Dalton, Bonnie P. (Technical Monitor)

2002-01-01

In a differential RNA display screen we have isolated a zebrafish gene, G12, for which homologs can only be found in DNA databases for vertebrates, but not invertebrates. This suggests that this is a gene required specifically in vertebrates. G12 expression is upregulated at mid-blastula transition (MBT). Morpholino inactivation of this gene by injection into 1-cell embryos results in mitotic defects and apoptosis shortly after MBT. Nuclei in morpholino treated embryos also display segregation defects. We have characterized the localization of this gene as a GFP fusion in live and fixed embryos. Overexpression of G12-GFP is non-toxic. Animals retain GFP expression for at least 7 days with no developmental defects, Interestingly in these animals G12-GFP is never detectable in blood cells though blood is present. In the deep cells of early embryos, G 12GFP is localized to nuclei and cytoskeletal elements in interphase and to the centrosome and spindle apparatus during mitosis. In the EVL, G12-GFP shows additional localization to the cell periphery, especially in mitosis. In the yolk syncytium, G12-GFP again localizes to nuclei and strongly to cytoplasmic microtubules of migrating nuclei at the YSL margin. Morpholinc, injection specifically into the YSL after cellularization blocks epiboly and nuclei of the YSL show mitotic defects while deep cells show no mitotic defects and continue to divide. Rescue experiments in which morpholino and G12-GFP RNA are co-injected indicate partial rescue by the G12-GFP. The rescue is cell autonomous; that is, regions of the embryo with higher G12-GFP expression show fewer mitotic defects. Spot 14, the human bomolog of G12, has been shown to be amplified in aggressive breast tumors. This finding, along with our functional and morphological data suggest that G12 and spot 14 are vertebrate-specific and may function either as mitotic checkpoints or as structural components of the spindle apparatus.

Effects of nozzle exit geometry and pressure ratio on plume shape for nozzles exhausting into quiescent air

NASA Technical Reports Server (NTRS)

Scallion, William I.

1991-01-01

The effects of varying the exit geometry on the plume shapes of supersonic nozzles exhausting into quiescent air at several exit-to-ambient pressure ratios are given. Four nozzles having circular throat sections and circular, elliptical and oval exit cross sections were tested and the exit plume shapes are compared at the same exit-to-ambient pressure ratios. The resulting mass flows were calculated and are also presented.

Primary mesenchymal (nonangiomatous/nonlymphomatous) neoplasms occurring in the canine spleen: anatomic classification, immunohistochemistry, and mitotic activity correlated with patient survival.

PubMed

Spangler, W L; Culbertson, M R; Kass, P H

1994-01-01

Surgical submissions from canine splenectomy cases spanning a 3-year period (1988-1990) were evaluated. Eighty seven neoplasms of the spleen considered to be of nonangiomatous and nonlymphomatous origin were selected for morphologic classification, mitotic index determination, immunohistochemical analysis, and patient survival determination. In 76/87 cases, patient survival information was available, and the mitotic index was determined in 83/87 cases. Immunohistochemistry for selected antigens (vimentin, desmin, smooth muscle actin, myosin, and factor VIII-related antigen) was performed in 58/87 of the cases. Morphologic classification of these lesions in standard HE preparations yielded the following neoplastic groups: fibrosarcoma (19/87), undifferentiated sarcoma (19/87), leiomyosarcoma (14/87), osteosarcoma (8/87), mesenchymoma (7/87), myxosarcoma (6/87), histiocytic sarcoma (6/87), leiomyoma (3/87), lipoma-myelolipoma (2/87), liposarcoma (2/87), and malignant fibrous histiocytoma (1/87). A lack of distinct morphologic characteristics among many of the neoplasms that were classified as either fibrosarcoma, leiomyosarcoma, or undifferentiated sarcoma contrasted these groups with the relatively unambiguous features that distinguished the other sarcoma groups. Using immunohistochemical staining for muscle-specific antigens (desmin, smooth muscle actin, and myosin), specific staining often overlapped extensively within the neoplastic groups of fibrosarcomas, leiomyosarcomas, and undifferentiated sarcomas, suggesting either ambiguous morphologic findings or the possibility of a common histogenesis from smooth muscle trabeculae or a distinct population of splenic myofibroblasts. The biological behavior of all tumors examined could be placed into three categories of patient survival: (1) benign, noninvasive tumors (leiomyoma, lipoma) with prolonged survival intervals; (2) malignant tumors (fibrosarcoma, undifferentiated sarcoma, leiomyosarcoma, osteosarcoma, myxosarcoma, histiocytic sarcoma, and liposarcoma), showing severely truncated survival (median 4 months with 80-100% mortality after 12 months; and (3) intermediate survival periods (median 12 months with 50% 1 year survival) attributed to a single group of neoplasm, the mesenchymomas. The biological behavior of primary splenic nonangiomatous, nonlymphomatous sarcomas was most closely correlated with observed mitotic index. Splenic neoplasms of this type with a mitotic index < 9 showed significantly (P < 0.0001) longer survival intervals than those with an index > 9. With the exception of osteosarcoma, all anatomically defined tumor groups contained one or more specimens with a mitotic index < 9. The clinical prognosis given for splenic sarcomas should be modified according to the mitotic index as a predictive value for patient survival.

Role of apoptosis and mitosis during human eye development.

PubMed

Bozanić, Darka; Tafra, Robert; Saraga-Babić, Mirna

2003-08-01

The spatial and temporal distribution as well as ultrastructural and biochemical characteristics of apoptotic and mitotic cells during human eye development were investigated in 14 human conceptuses of 4-9 postovulatory weeks, using electron and light microscopy. In the 5th developmental week, apoptotic and mitotic cells were found in the neuroepithelium of the optic cup and stalk, being the most numerous at the borderline between the two layers of the optic cup, and at the place of transition of the optic cup into stalk. They were also found at the region of detachment of the lens pit from the surface ectoderm. In the later developmental stages (the 6th-the 9th week), apoptotic and mitotic cells were observed in the neural retina and the anterior lens epithelium. Throughout all stages examined, mitotic cells were found exclusively adjacent to the lumen either of the intraretinal space or the optic stalk ventricle, or were restricted to the superficial epithelial layer of the lens primordium. Unlike mitotic cells, apoptotic cells occurred throughout the whole width both of the neuroepithelium and the surface epithelium. Ultrastructurally, apoptotic cells were characterised by round- or crescent-shaped condensations of chromatin near the nuclear membrane, while in the more advanced stages of apoptosis by apoptotic bodies. The distribution of caspase-3-positive cells coincided with the location of apoptotic cells described by morphological techniques indicating that the caspase-3-dependent apoptotic pathway operates during the all stages of human eye development. The location of cells positive for anti-apoptotic bcl-2 protein was in accordance with the regions of eye with high mitotic activity, confirming the role of bcl-2 in protecting cells from apoptosis. In the earliest stage of eye development, apoptosis and mitosis might be associated with the sculpturing of the walls of optic cup and stalk, while high mitotic activity along the intraretinal space and optic stalk ventricle indicates its role in the gradual luminal closure. These processes also participate in the detachment of the lens pit epithelium from the surface ectoderm as well as in further closure of the lens vesicle. Later on, both processes seem to be involved in the neural retina differentiation, lens morphogenesis and secondary lens fibre differentiation.

14 CFR 27.805 - Flight crew emergency exits.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Flight crew emergency exits. 27.805 Section... § 27.805 Flight crew emergency exits. (a) For rotorcraft with passenger emergency exits that are not convenient to the flight crew, there must be flight crew emergency exits, on both sides of the rotorcraft or...

14 CFR 29.805 - Flight crew emergency exits.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Flight crew emergency exits. 29.805 Section... Accommodations § 29.805 Flight crew emergency exits. (a) For rotorcraft with passenger emergency exits that are not convenient to the flight crew, there must be flight crew emergency exits, on both sides of the...

14 CFR 29.805 - Flight crew emergency exits.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Flight crew emergency exits. 29.805 Section... Accommodations § 29.805 Flight crew emergency exits. (a) For rotorcraft with passenger emergency exits that are not convenient to the flight crew, there must be flight crew emergency exits, on both sides of the...

14 CFR 27.805 - Flight crew emergency exits.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Flight crew emergency exits. 27.805 Section... § 27.805 Flight crew emergency exits. (a) For rotorcraft with passenger emergency exits that are not convenient to the flight crew, there must be flight crew emergency exits, on both sides of the rotorcraft or...

The transcription factor Nerfin-1 prevents reversion of neurons into neural stem cells.

PubMed

Froldi, Francesca; Szuperak, Milan; Weng, Chen-Fang; Shi, Wei; Papenfuss, Anthony T; Cheng, Louise Y

2015-01-15

Cellular dedifferentiation is the regression of a cell from a specialized state to a more multipotent state and is implicated in cancer. However, the transcriptional network that prevents differentiated cells from reacquiring stem cell fate is so far unclear. Neuroblasts (NBs), the Drosophila neural stem cells, are a model for the regulation of stem cell self-renewal and differentiation. Here we show that the Drosophila zinc finger transcription factor Nervous fingers 1 (Nerfin-1) locks neurons into differentiation, preventing their reversion into NBs. Following Prospero-dependent neuronal specification in the ganglion mother cell (GMC), a Nerfin-1-specific transcriptional program maintains differentiation in the post-mitotic neurons. The loss of Nerfin-1 causes reversion to multipotency and results in tumors in several neural lineages. Both the onset and rate of neuronal dedifferentiation in nerfin-1 mutant lineages are dependent on Myc- and target of rapamycin (Tor)-mediated cellular growth. In addition, Nerfin-1 is required for NB differentiation at the end of neurogenesis. RNA sequencing (RNA-seq) and chromatin immunoprecipitation (ChIP) analysis show that Nerfin-1 administers its function by repression of self-renewing-specific and activation of differentiation-specific genes. Our findings support the model of bidirectional interconvertibility between neural stem cells and their post-mitotic progeny and highlight the importance of the Nerfin-1-regulated transcriptional program in neuronal maintenance. © 2015 Froldi et al.; Published by Cold Spring Harbor Laboratory Press.

"Getting out of downtown": a longitudinal study of how street-entrenched youth attempt to exit an inner city drug scene.

PubMed

Knight, Rod; Fast, Danya; DeBeck, Kora; Shoveller, Jean; Small, Will

2017-05-02

Urban drug "scenes" have been identified as important risk environments that shape the health of street-entrenched youth. New knowledge is needed to inform policy and programing interventions to help reduce youths' drug scene involvement and related health risks. The aim of this study was to identify how young people envisioned exiting a local, inner-city drug scene in Vancouver, Canada, as well as the individual, social and structural factors that shaped their experiences. Between 2008 and 2016, we draw on 150 semi-structured interviews with 75 street-entrenched youth. We also draw on data generated through ethnographic fieldwork conducted with a subgroup of 25 of these youth between. Youth described that, in order to successfully exit Vancouver's inner city drug scene, they would need to: (a) secure legitimate employment and/or obtain education or occupational training; (b) distance themselves - both physically and socially - from the urban drug scene; and (c) reduce their drug consumption. As youth attempted to leave the scene, most experienced substantial social and structural barriers (e.g., cycling in and out of jail, the need to access services that are centralized within a place that they are trying to avoid), in addition to managing complex individual health issues (e.g., substance dependence). Factors that increased youth's capacity to successfully exit the drug scene included access to various forms of social and cultural capital operating outside of the scene, including supportive networks of friends and/or family, as well as engagement with addiction treatment services (e.g., low-threshold access to methadone) to support cessation or reduction of harmful forms of drug consumption. Policies and programming interventions that can facilitate young people's efforts to reduce engagement with Vancouver's inner-city drug scene are critically needed, including meaningful educational and/or occupational training opportunities, 'low threshold' addiction treatment services, as well as access to supportive housing outside of the scene.

Do spouses coordinate their work exits? A combined survey and register analysis from Norway.

PubMed

Syse, Astri; Solem, Per Erik; Ugreninov, Elisabeth; Mykletun, Reidar; Furunes, Trude

2014-09-01

Research on spouses' joint work exits is scarce, although household factors such as spouses' work status, marital quality, and caregiving burdens are likely to affect seniors' work engagement. We therefore examine whether the work exit probability of one spouse affects that of the other. Discrete-time hazard regression analyses of survey data linked to later registry information including all gainfully employed married respondents aged 50-74 with a working spouse (N = 1,764) were used to assess subsequent work exits. A spouse's work exit is a strong predictor of a respondent's work exit (hazard ratio 3.1, 95% confidence interval [2.5, 4.0]). Educational attainment, poor marital quality, and spouses' health and care needs do not predict work exits. Surprisingly, no gender differences are observed. Research on larger survey samples to distinguish different work exit routes and reasons for spouses' joint work exits appears warranted. To account for cultural and welfare state characteristics, cross-national studies ought to be undertaken. © The Author(s) 2013.

Noise Tolerance of Attractor and Feedforward Memory Models

PubMed Central

Lim, Sukbin; Goldman, Mark S.

2017-01-01

In short-term memory networks, transient stimuli are represented by patterns of neural activity that persist long after stimulus offset. Here, we compare the performance of two prominent classes of memory networks, feedback-based attractor networks and feedforward networks, in conveying information about the amplitude of a briefly presented stimulus in the presence of gaussian noise. Using Fisher information as a metric of memory performance, we find that the optimal form of network architecture depends strongly on assumptions about the forms of nonlinearities in the network. For purely linear networks, we find that feedforward networks outperform attractor networks because noise is continually removed from feedforward networks when signals exit the network; as a result, feedforward networks can amplify signals they receive faster than noise accumulates over time. By contrast, attractor networks must operate in a signal-attenuating regime to avoid the buildup of noise. However, if the amplification of signals is limited by a finite dynamic range of neuronal responses or if noise is reset at the time of signal arrival, as suggested by recent experiments, we find that attractor networks can out-perform feedforward ones. Under a simple model in which neurons have a finite dynamic range, we find that the optimal attractor networks are forgetful if there is no mechanism for noise reduction with signal arrival but nonforgetful (perfect integrators) in the presence of a strong reset mechanism. Furthermore, we find that the maximal Fisher information for the feedforward and attractor networks exhibits power law decay as a function of time and scales linearly with the number of neurons. These results highlight prominent factors that lead to trade-offs in the memory performance of networks with different architectures and constraints, and suggest conditions under which attractor or feedforward networks may be best suited to storing information about previous stimuli. PMID:22091664

Performance Characteristics of Flush and Shielded Auxiliary Exits at Mach Numbers of 1.5 to 2.0

NASA Technical Reports Server (NTRS)

Abdalla, Kaleel L.

1959-01-01

The performance characteristics of several flush and shielded auxiliary exits were investigated at Mach numbers of 1.5 to 2.0, and jet pressure ratios from jet off to 10. The results indicate that the shielded configurations produced better overall performance than the corresponding flush exits over the Mach-number and pressure-ratio ranges investigated. Furthermore, the full-length shielded exit was highest in performance of all the configurations. The flat-exit nozzle block provided considerably improved performance compared with the curved-exit nozzle block.

The quality of service in passenger transport terminals

NASA Astrophysics Data System (ADS)

Oprea, C.; Roşca, E.; Popa, M.; Ilie, A.; Dinu, O.; Roşca, M.

2016-11-01

The quality of service in transport terminals is differently perceived by engineers, economists, transport operators and sociologists. The traveler's perception is nevertheless decisive. The quality of service is well connected with the inside design of terminals, with the facilities in terminals and with the provided service standards. In order to provide a high level of service, the activities taking place in the public transport terminal and the maximum travelers flow size must be carefully analyzed and dimensioned. The purpose of modelling is to find the best route for each traveler from origin (entrance) to destination (exit) through all the intermediate service points, taking into consideration the instant network conditions. In developing the model we consider the walking, the waiting and the serving time. Using a simulation program written in ARENA we determine the waiting time. For validation, the model is used to evaluate the performance level in Bucharest Basarab station. By comparing the total walking distance for the possible routes and the utility function that describes the utility of all activities from entrance to exit we can find the optimal route.

Can favourable psychosocial working conditions in midlife moderate the risk of work exit for chronically ill workers? A 20-year follow-up of the Whitehall II study.

PubMed

Fleischmann, Maria; Carr, Ewan; Stansfeld, Stephen A; Xue, Baowen; Head, Jenny

2018-03-01

To investigate if favourable psychosocial working conditions can reduce the risk of work exit and specifically for workers with chronic disease. Men and women (32%) aged 35-55, working and having no chronic disease at baseline of the Whitehall II study of London-based civil servants were selected (n=9040). We observed participants' exit from work through retirement, health-related exit and unemployment, new diagnosis of chronic disease (ie, coronary heart disease, diabetes, stroke and cancer) and their psychosocial working conditions in midlife. Using cause-specific Cox models, we examined the association of chronic disease and favourable psychosocial working conditions and their interaction, with the three types of work exit. We adjusted for gender, occupational grade, educational level, remaining in civil service, spouse's employment status and mental health. Chronic disease significantly increased the risk of any type of work exit (HR 1.27) and specifically the risk of health-related exit (HR 2.42). High skill discretion in midlife reduced the risk of any type of work exit (HR 0.90), retirement (HR 0.91) and health-related exit (HR 0.68). High work social support in midlife decreased the risk of health-related exit (HR 0.79) and unemployment (HR 0.71). Favourable psychosocial working conditions in midlife did not attenuate the association between chronic disease and work exit significantly. The chronically ill have increased risks of work exit, especially through health-related exit routes. Chronic disease is an obstacle to extended working lives. Favourable working conditions directly relate to reduced risks of work exit. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Detection of mitotic figures and G2+ tumor nuclei with histone markers correlates with worse overall survival in patients with Merkel cell carcinoma.

PubMed

Henderson, Samuel A; Tetzlaff, Michael T; Pattanaprichakul, Penvadee; Fox, Patricia; Torres-Cabala, Carlos A; Bassett, Roland L; Prieto, Victor G; Richards, Hunter W; Curry, Jonathan L

2014-11-01

High mitotic figure count (MFC) correlates with low survival rate in Merkel cell carcinoma (MCC). However, the prognostic impact of histone biomarkers as surrogates of MFC in MCC is unknown. We evaluated the prognostic significance of the immunodetection of mitotic figures and of G2+ tumor nuclei with histone-associated mitotic markers H3K79me3T80ph (H3KT) and phosphohistone H3 (PHH3) in MCC. Immunohistochemical analyses of H3KT and PHH3 and proliferative marker Ki-67 were performed in a series of 21 cases of MCC. The significance of the pathologic data and immunoreactivity with these markers was evaluated with Pearson correlation and paired Student t-test. Univariate Cox proportional hazards regression models were performed to assess the relationships between these markers and survival. H3KT detected a higher number of mitotic figure (p<0.0001) and G2+ tumor nuclei (p<0.0052) than did PHH3. Furthermore, the MFC combined with G2+ tumor nuclei detected with H3KT compared to PHH3 and manual MFC was a significant predictor of impaired survival in patients with MCC (p=0.035; HR=1.0172), corresponding to a 1.72% increased risk of death for each unit increase in H3KT. Biomarker analysis of proliferative rates with histone markers may have relevance in stratifying risk in patients with MCC. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The Echinoid Mitotic Gradient: Effect of Cell Size on the Micromere Cleavage Cycle

PubMed Central

Langelan Duncan, Rosalie E.; Whiteley, Arthur H.

2012-01-01

SUMMARY Like other euechinoids, the fertilized eggs of the sand dollar Dendraster excentricus proceed through cleavages that produce a pattern of macromeres, mesomeres, and micromeres at the 4th division. The 8 cells of the macro-mesomere lineage proceed through 6 additional cleavages before hatching. At the fifth overall division, the 4 micromeres produce a lineage of large micromeres that will divide 3 additional times, and a lineage of small micromeres that will divide once more before hatching. Irrespective of lineage, the length of the cell cycles is closely related to the size of the blastomere; cells of the same size have the same cell cycle time. A consequence is that at the fourth cleavage, there is a gradient of mitotic activity from the fastest dividers at the animal pole and the slowest cleacing micromeres at the vegetal pole. By the time of hatching, which is the 10th division of meso-macromeres, all cells are the same small size, the metachronic pattern of division gives way to asynchrony, and the mitotic gradient along the polar axis is lost. Experimental pre-exposure to sodium dodecyl sulfate (SDS), however, blocks the appearance of the gradients in cell size, the mitotic gradient, and the differential in cell cycle times. It is proposed that the mitotic gradients, cell cycle times, and attainment of a state of asynchrony are functions of cell size. Developmental consequences of the transition are large, and include coordinated activation of transcriptions, synthesis of new patterns of proteins, alterations of metabolism, and onset of morphogenesis. PMID:22006441

Cleavage in conical sand dollar eggs.

PubMed

Rappaport, R; Rappaport, B N

1994-07-01

Previous experiments have shown that the mitotic apparatus and the surface can interact and produce functional furrows in various unusual geometrical circumstances. The consistent development of the furrow in the plane equidistant from the aster centers has led to conjecture about the need for a special structural configuration of the subsurface in the future cleavage plane. In most experiments involving altered cell geometry, the relation between each aster and nearby surface was symmetrical, and the effect of that symmetry upon the position and orientation of the cleavage mechanism in the cortex has not been systematically analyzed. The normal symmetry of sand dollar eggs can be changed by reshaping them into cones. When the cone and mitotic axes are parallel, the aster center closer to the vertex is also closer to the nearby surface, and the cleavage plane develops on the vertex side of the midpoint between the asters. A mitotic apparatus oriented perpendicular to the cone axis produces in the base of the cone a normal unilateral furrow that advances toward the vertex, and a second contractile band that isolates the vertex region. This event only occurs when the surface is conical and the mitotic apparatus is perpendicular to the cone axis. Furrow formation is not restricted to the plane of the metaphase plate or the midpoint between the aster centers. The orientation of mitotic apparatus-produced contractile bands is not limited to the circumstances in normal cytokinesis, but may vary according to surface contour. These results confirm predictions of the Harris and Gewalt model of contractile ring induction.

NUP98 fusion oncoproteins interact with the APC/C(Cdc20) as a pseudosubstrate and prevent mitotic checkpoint complex binding.

PubMed

Salsi, Valentina; Fantini, Sebastian; Zappavigna, Vincenzo

2016-09-01

NUP98 is a recurrent partner gene in translocations causing acute myeloid leukemias and myelodisplastic syndrome. The expression of NUP98 fusion oncoproteins has been shown to induce mitotic spindle defects and chromosome missegregation, which correlate with the capability of NUP98 fusions to cause mitotic checkpoint attenuation. We show that NUP98 oncoproteins physically interact with the APC/C(Cdc20) in the absence of the NUP98 partner protein RAE1, and prevent the binding of the mitotic checkpoint complex to the APC/C(Cdc20). NUP98 oncoproteins require the GLEBS-like domain present in their NUP98 moiety to bind the APC/C(Cdc20). We found that NUP98 wild-type is a substrate of APC/C(Cdc20) prior to mitotic entry, and that its binding to APC/C(Cdc20) is controlled via phosphorylation of a PEST sequence located within its C-terminal portion. We identify S606, within the PEST sequence, as a key target site, whose phosphorylation modulates the capability of NUP98 to interact with APC/C(Cdc20). We finally provide evidence for an involvement of the peptidyl-prolyl isomerase PIN1 in modulating the possible conformational changes within NUP98 that lead to its dissociation from the APC/C(Cdc20) during mitosis. Our results provide novel insight into the mechanisms underlying the aberrant capability of NUP98 oncoproteins to interact with APC/C(Cdc20) and to interfere with its function.

Determining local and contextual features describing appearance of difficult to identify mitotic figures

NASA Astrophysics Data System (ADS)

Gandomkar, Ziba; Brennan, Patrick C.; Mello-Thoms, Claudia

2017-03-01

Mitotic count is helpful in determining the aggressiveness of breast cancer. In previous studies, it was shown that the agreement among pathologists for grading mitotic index is fairly modest, as mitoses have a large variety of appearances and they could be mistaken for other similar objects. In this study, we determined local and contextual features that differ significantly between easily identifiable mitoses and challenging ones. The images were obtained from the Mitosis-Atypia 2014 challenge. In total, the dataset contained 453 mitotic figures. Two pathologists annotated each mitotic figure. In case of disagreement, an opinion from a third pathologist was requested. The mitoses were grouped into three categories, those recognized as "a true mitosis" by both pathologists ,those labelled as "a true mitosis" by only one of the first two readers and also the third pathologist, and those annotated as "probably a mitosis" by all readers or the majority of them. After color unmixing, the mitoses were segmented from H channel. Shape-based features along with intensity-based and textural features were extracted from H-channel, blue ratio channel and five different color spaces. Holistic features describing each image were also considered. The Kruskal-Wallis H test was used to identify significantly different features. Multiple comparisons were done using the rank-based version of Tukey-Kramer test. The results indicated that there are local and global features which differ significantly among different groups. In addition, variations between mitoses in different groups were captured in the features from HSL and LCH color space more than other ones.

Kinome-wide transcriptional profiling of uveal melanoma reveals new vulnerabilities to targeted therapeutics.

PubMed

Bailey, Fiona P; Clarke, Kim; Kalirai, Helen; Kenyani, Jenna; Shahidipour, Haleh; Falciani, Francesco; Coulson, Judy M; Sacco, Joseph J; Coupland, Sarah E; Eyers, Patrick A

2018-03-01

Metastatic uveal melanoma (UM) is invariably fatal, usually within a year of diagnosis. There are currently no effective therapies, and clinical studies employing kinase inhibitors have so far demonstrated limited success. This is despite common activating mutations in GNAQ/11 genes, which trigger signalling pathways that might predispose tumours to a variety of targeted drugs. In this study, we have profiled kinome expression network dynamics in various human ocular melanomas. We uncovered a shared transcriptional profile in human primary UM samples and across a variety of experimental cell-based models. The poor overall response of UM cells to FDA-approved kinase inhibitors contrasted with much higher sensitivity to the bromodomain inhibitor JQ1, a broad transcriptional repressor. Mechanistically, we identified a repressed FOXM1-dependent kinase subnetwork in JQ1-exposed cells that contained multiple cell cycle-regulated protein kinases. Consistently, we demonstrated vulnerability of UM cells to inhibitors of mitotic protein kinases within this network, including the investigational PLK1 inhibitor BI6727. We conclude that analysis of kinome-wide signalling network dynamics has the potential to reveal actionable drug targets and inhibitors of potential therapeutic benefit for UM patients. © 2017 The Authors. Pigment Cell & Melanoma Research Published by John Wiley & Sons.

Conserved molecular interactions in centriole-to-centrosome conversion.

PubMed

Fu, Jingyan; Lipinszki, Zoltan; Rangone, Hélène; Min, Mingwei; Mykura, Charlotte; Chao-Chu, Jennifer; Schneider, Sandra; Dzhindzhev, Nikola S; Gottardo, Marco; Riparbelli, Maria Giovanna; Callaini, Giuliano; Glover, David M

2016-01-01

Centrioles are required to assemble centrosomes for cell division and cilia for motility and signalling. New centrioles assemble perpendicularly to pre-existing ones in G1-S and elongate throughout S and G2. Fully elongated daughter centrioles are converted into centrosomes during mitosis to be able to duplicate and organize pericentriolar material in the next cell cycle. Here we show that centriole-to-centrosome conversion requires sequential loading of Cep135, Ana1 (Cep295) and Asterless (Cep152) onto daughter centrioles during mitotic progression in both Drosophila melanogaster and human. This generates a molecular network spanning from the inner- to outermost parts of the centriole. Ana1 forms a molecular strut within the network, and its essential role can be substituted by an engineered fragment providing an alternative linkage between Asterless and Cep135. This conserved architectural framework is essential for loading Asterless or Cep152, the partner of the master regulator of centriole duplication, Plk4. Our study thus uncovers the molecular basis for centriole-to-centrosome conversion that renders daughter centrioles competent for motherhood.

Mitotic Regulation by NEK Kinase Networks

PubMed Central

Fry, Andrew M.; Bayliss, Richard; Roig, Joan

2017-01-01

Genetic studies in yeast and Drosophila led to identification of cyclin-dependent kinases (CDKs), Polo-like kinases (PLKs) and Aurora kinases as essential regulators of mitosis. These enzymes have since been found in the majority of eukaryotes and their cell cycle-related functions characterized in great detail. However, genetic studies in another fungal species, Aspergillus nidulans, identified a distinct family of protein kinases, the NEKs, that are also widely conserved and have key roles in the cell cycle, but which remain less well studied. Nevertheless, it is now clear that multiple NEK family members act in networks to regulate specific events of mitosis, including centrosome separation, spindle assembly and cytokinesis. Here, we describe our current understanding of how the NEK kinases contribute to these processes, particularly through targeted phosphorylation of proteins associated with the microtubule cytoskeleton. We also present the latest findings on molecular events that control the activation state of the NEKs and how these are revealing novel modes of enzymatic regulation relevant not only to other kinases but also to pathological mechanisms of disease. PMID:29250521

Vacuolar and cytoskeletal dynamics during elicitor-induced programmed cell death in tobacco BY-2 cells.

PubMed

Higaki, Takumi; Kadota, Yasuhiro; Goh, Tatsuaki; Hayashi, Teruyuki; Kutsuna, Natsumaro; Sano, Toshio; Hasezawa, Seiichiro; Kuchitsu, Kazuyuki

2008-09-01

Responses of plant cells to environmental stresses often involve morphological changes, differentiation and redistribution of various organelles and cytoskeletal network. Tobacco BY-2 cells provide excellent model system for in vivo imaging of these intracellular events. Treatment of the cell cycle-synchronized BY-2 cells with a proteinaceous oomycete elicitor, cryptogein, induces highly synchronous programmed cell death (PCD) and provide a model system to characterize vacuolar and cytoskeletal dynamics during the PCD. Sequential observation revealed dynamic reorganization of the vacuole and actin microfilaments during the execution of the PCD. We further characterized the effects cryptogein on mitotic microtubule organization in cell cycle-synchronized cells. Cryptogein treatment at S phase inhibited formation of the preprophase band, a cortical microtubule band that predicts the cell division site. Cortical microtubules kept their random orientation till their disruption that gradually occurred during the execution of the PCD twelve hours after the cryptogein treatment. Possible molecular mechanisms and physiological roles of the dynamic behavior of the organelles and cytoskeletal network in the pathogenic signal-induced PCD are discussed.

Modeling Cytoskeletal Active Matter Systems

NASA Astrophysics Data System (ADS)

Blackwell, Robert

Active networks of filamentous proteins and crosslinking motor proteins play a critical role in many important cellular processes. One of the most important microtubule-motor protein assemblies is the mitotic spindle, a self-organized active liquid-crystalline structure that forms during cell division and that ultimately separates chromosomes into two daughter cells. Although the spindle has been intensively studied for decades, the physical principles that govern its self-organization and function remain mysterious. To evolve a better understanding of spindle formation, structure, and dynamics, I investigate course-grained models of active liquid-crystalline networks composed of microtubules, modeled as hard spherocylinders, in diffusive equilibrium with a reservoir of active crosslinks, modeled as hookean springs that can adsorb to microtubules and and translocate at finite velocity along the microtubule axis. This model is investigated using a combination of brownian dynamics and kinetic monte carlo simulation. I have further refined this model to simulate spindle formation and kinetochore capture in the fission yeast S. pombe. I then make predictions for experimentally realizable perturbations in motor protein presence and function in S. pombe.

Adolescents Exiting Homelessness Over Two Years: The Risk Amplification and Abatement Model

PubMed Central

Milburn, Norweeta G.; Rice, Eric; Rotheram-Borus, Mary Jane; Mallett, Shelley; Rosenthal, Doreen; Batterham, Phillip; May, Susanne J.; Witkin, Andrea; Duan, Naihua

2014-01-01

The Risk Amplification and Abatement Model (RAAM), demonstrates that negative contact with socializing agents amplify risk, while positive contact abates risk for homeless adolescents. To test this model, the likelihood of exiting homelessness and returning to familial housing at 2 years and stably exiting over time are examined with longitudinal data collected from 183 newly homeless adolescents followed over 2 years in Los Angeles, CA. In support of RAAM, unadjusted odds of exiting at 2 years and stably exiting over2 years revealed that engagement with pro-social peers, maternal social support, and continued school attendance all promoted exiting behaviors. Simultaneously, exposure to family violence and reliance on shelter services discouraged stably exiting behaviors. Implications for family-based interventions are proposed. PMID:25067896

Condensins under the microscope.

PubMed

Maeshima, Kazuhiro; Hibino, Kayo; Hudson, Damien F

2018-04-30

Condensins are key players in mitotic chromosome condensation. Using an elegant combination of state-of-the-art imaging techniques, Walther et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201801048) counted the number of Condensins, examined their behaviors on human mitotic chromosomes, and integrated the quantitative data to propose a new mechanistic model for chromosome condensation. © 2018 Maeshima et al.

DREAMs make plant cells to cycle or to become quiescent.

PubMed

Magyar, Zoltán; Bögre, László; Ito, Masaki

2016-12-01

Cell cycle phase specific oscillation of gene transcription has long been recognized as an underlying principle for ordered processes during cell proliferation. The G1/S-specific and G2/M-specific cohorts of genes in plants are regulated by the E2F and the MYB3R transcription factors. Mutant analysis suggests that activator E2F functions might not be fully required for cell cycle entry. In contrast, the two activator-type MYB3Rs are part of positive feedback loops to drive the burst of mitotic gene expression, which is necessary at least to accomplish cytokinesis. Repressor MYB3Rs act outside the mitotic time window during cell cycle progression, and are important for the shutdown of mitotic genes to impose quiescence in mature organs. The two distinct classes of E2Fs and MYB3Rs together with the RETINOBLATOMA RELATED are part of multiprotein complexes that may be evolutionary related to what is known as DREAM complex in animals. In plants, there are multiple such complexes with distinct compositions and functions that may be involved in the coordinated cell cycle and developmental regulation of E2F targets and mitotic genes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of 60-Hz magnetic fields on ultraviolet light-induced mutation and mitotic recombination in Saccharomyces cerevisiae.

PubMed

Ager, D D; Radul, J A

1992-12-01

The purpose of this study was to examine the effect of extremely low frequency (ELF) magnetic fields on the induction of genetic damage. In general, mutational studies involving ELF magnetic fields have proven negative. However, studies examining sister-chromatid exchange and chromosome aberrations have yielded conflicting results. In this study, we have examined whether 60-Hz magnetic fields are capable of inducing mutation or mitotic recombination in the yeast Saccharomyces cerevisiae. In addition we determined whether magnetic fields were capable of altering the genetic response of S. cerevisiae to UV (254 nm). We measured the frequencies of induced mutation, gene conversion and reciprocal mitotic crossing-over for exposures to magnetic fields alone (1 mT) or in combination with various UV exposures (2-50 J/m2). These experiments were performed using a repair-proficient strain (RAD+), as well as a strain of yeast (rad3) which is incapable of excising UV-induced thymine dimers. Magnetic field exposures did not induce mutation, gene conversion or reciprocal mitotic crossing-over in either of these strains, nor did the fields influence the frequencies of UV-induced genetic events.

Epigenome analysis of pluripotent stem cells

PubMed Central

Ricupero, Christopher L.; Swerdel, Mavis R.; Hart, Ronald P.

2015-01-01

Summary Mis-regulation of gene expression due to epigenetic abnormalities has been linked with complex genetic disorders, psychiatric illness and cancer. In addition, the dynamic epigenetic changes that occur in pluripotent stem cells are believed to impact regulatory networks essential for proper lineage development. Chromatin immunoprecipitation (ChIP) is a technique used to isolate and enrich chromatin fragments using antibodies against specific chromatin modifications, such as DNA binding proteins or covalent histone modifications. Until recently, many ChIP protocols required millions of cells for each immunoprecipitation. This severely limited analysis of rare cell populations or post-mitotic, differentiated cell lines. Here, we describe a low cell number ChIP protocol with next generation sequencing and analysis, that has the potential to uncover novel epigenetic regulatory pathways that were previously difficult or impossible to obtain. PMID:23546758

DOE Office of Scientific and Technical Information (OSTI.GOV)

Breault, Ronald W.; Monazam, Esmail R.; Shadle, Lawrence J.

Riser hydrodynamics are a function of the flow rates of gas and solids as well as the exit geometry, particularly when operated above the upper transport velocity. This work compares the exit voidage for multiple geometries and two different solids: Geldart group A glass beads and Geldart group B coke. Geometries were changed by modifying the volume of an abrupt T-shaped exit above the lateral riser exit. This was accomplished by positioning a plunger at various heights above the exit from zero to 0.38 m. A dimensionless expression used to predict smooth exit voidage was modified to account for themore » effect of the depth of the blind-T. The new correlation contains the solids-gas load ratio, solids-to-gas density ratio, bed-to-particle diameter ratio, gas Reynolds Number, as well as a term for the exit geometry. This study also found that there was a minimum riser roof height above the blind-T exit beyond which the riser exit voidage was not affected by the exit geometry. A correlation for this minimum riser roof height has also been developed in this study. This study covered riser superficial gas velocities of 4.35 to 7.7 m/s and solids circulation rates of 1.3 to 11.5 kg/s.« less

Influence of drill helical direction on exit damage development in drilling carbon fiber reinforced plastic

NASA Astrophysics Data System (ADS)

Bai, Y.; Jia, Z. Y.; Wang, F. J.; Fu, R.; Guo, H. B.; Cheng, D.; Zhang, B. Y.

2017-06-01

Drilling is inevitable for CFRP components’ assembling process in the aviation industry. The exit damage frequently occurs and affects the load carrying capacity of components. Consequently, it is of great urgency to enhance drilling exit quality on CFRP components. The article aims to guide the reasonable choice of drill helical direction and effectively reduce exit damage. Exit observation experiments are carried out with left-hand helical, right-hand helical and straight one-shot drill drilling T800S CFRP laminates separately. The development rules of exit damage and delamination factor curves are obtained. Combined with loading conditions and fracture modes of push-out burrs, and thrust force curves, the influence of drill helical direction on exit damage development is derived. It is found that the main fracture modes for left-hand helical, right-hand helical, and straight one-shot drill are mode I, extrusive fracture, mode III respectively. Among them, mode III has the least effect on exit damage development. Meanwhile, the changing rate of thrust force is relative slow for right-hand helical and straight one-shot drill in the thrust force increasing phase of stage II, which is disadvantaged for exit damage development. Therefore, straight one-shot drill’s exit quality is the best.

Prognosis after surgical excision of canine fibrous connective tissue sarcomas.

PubMed

Bostock, D E; Dye, M T

1980-09-01

One hundred eighty seven dogs from which fibrous connective tissue sarcomas had been excised were studied until death or for at least 3 years after surgery. Dogs with a skin fibrosarcoma had a median survival time of 80 weeks, compared with 140 weeks for animals with haemangiopericytoma in similar sites, this difference being statistically significant. However, the difference in survival time between the two histologic types disappeared when tumours with a similar mitotic index were compared. Dogs with a tumour of mitotic index 9 or more had a median survival time of 49 weeks, compared with 118 weeks for those with a tumour of mitotic index less than 9, regardless of tumour morphology. Tumour recurrence rates of 62% and 25% respectively for the two groups were also significantly different.

Endocycles: a recurrent evolutionary innovation for post-mitotic cell growth.

PubMed

Edgar, Bruce A; Zielke, Norman; Gutierrez, Crisanto

2014-03-01

In endoreplication cell cycles, known as endocycles, cells successively replicate their genomes without segregating chromosomes during mitosis and thereby become polyploid. Such cycles, for which there are many variants, are widespread in protozoa, plants and animals. Endocycling cells can achieve ploidies of >200,000 C (chromatin-value); this increase in genomic DNA content allows a higher genomic output, which can facilitate the construction of very large cells or enhance macromolecular secretion. These cells execute normal S phases, using a G1-S regulatory apparatus similar to the one used by mitotic cells, but their capability to segregate chromosomes has been suppressed, typically by downregulation of mitotic cyclin-dependent kinase activity. Endocycles probably evolved many times, and the various endocycle mechanisms found in nature highlight the versatility of the cell cycle control machinery.

Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy

PubMed Central

Myers, Stephanie M.; Collins, Ian

2016-01-01

The kinesin class of microtubule-associated motor proteins present attractive anti-cancer targets owing to their roles in key functions in dividing cells. Two interpolar mitotic kinesins Eg5 and HSET have opposing motor functions in mitotic spindle assembly with respect to microtubule movement, but both offer opportunities to develop cancer selective therapeutic agents. Here, we summarize the progress to date in developing inhibitors of Eg5 and HSET, with an emphasis on structural biology insights into the binding modes of allosteric inhibitors, compound selectivity and mechanisms of action of different chemical scaffolds. We discuss translation of preclinical studies to clinical experience with Eg5 inhibitors, recent findings on potential resistance mechanisms, and explore the implications for future anticancer drug development against these targets. PMID:26976726

Brownian dynamics simulation of fission yeast mitotic spindle formation

NASA Astrophysics Data System (ADS)

Edelmaier, Christopher

2014-03-01

The mitotic spindle segregates chromosomes during mitosis. The dynamics that establish bipolar spindle formation are not well understood. We have developed a computational model of fission-yeast mitotic spindle formation using Brownian dynamics and kinetic Monte Carlo methods. Our model includes rigid, dynamic microtubules, a spherical nuclear envelope, spindle pole bodies anchored in the nuclear envelope, and crosslinkers and crosslinking motor proteins. Crosslinkers and crosslinking motor proteins attach and detach in a grand canonical ensemble, and exert forces and torques on the attached microtubules. We have modeled increased affinity for crosslinking motor attachment to antiparallel microtubule pairs, and stabilization of microtubules in the interpolar bundle. We study parameters controlling the stability of the interpolar bundle and assembly of a bipolar spindle from initially adjacent spindle-pole bodies.