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Sample records for pathway controlling mitotic

  1. Radiation-induced mitotic cell death and glioblastoma radioresistance: a new regulating pathway controlled by integrin-linked kinase, hypoxia-inducible factor 1 alpha and survivin in U87 cells.

    PubMed

    Lanvin, Olivia; Monferran, Sylvie; Delmas, Caroline; Couderc, Bettina; Toulas, Christine; Cohen-Jonathan-Moyal, Elizabeth

    2013-09-01

    We have previously shown that integrin-linked kinase (ILK) regulates U87 glioblastoma cell radioresistance by modulating the main radiation-induced cell death mechanism in solid tumours, the mitotic cell death. To decipher the biological pathways involved in these mechanisms, we constructed a U87 glioblastoma cell model expressing an inducible shRNA directed against ILK (U87shILK). We then demonstrated that silencing ILK enhanced radiation-induced centrosome overduplication, leading to radiation-induced mitotic cell death. In this model, ionising radiations induce hypoxia-inducible factor 1 alpha (HIF-1α) stabilisation which is inhibited by silencing ILK. Moreover, silencing HIF-1α in U87 cells reduced the surviving fraction after 2 Gy irradiation by increasing cell sensitivity to radiation-induced mitotic cell death and centrosome amplification. Because it is known that HIF-1α controls survivin expression, we then looked at the ILK silencing effect on survivin expression. We show that survivin expression is decreased in U87shILK cells. Furthermore, treating U87 cells with the specific survivin suppressor YM155 significantly increased the percentage of giant multinucleated cells, centrosomal overduplication and thus U87 cell radiosensitivity. In consequence, we decipher here a new pathway of glioma radioresistance via the regulation of radiation-induced centrosome duplication and therefore mitotic cell death by ILK, HIF-1α and survivin. This work identifies new targets in glioblastoma with the intention of radiosensitising these highly radioresistant tumours. PMID:23747271

  2. Analysis of the mitotic exit control system using locked levels of stable mitotic cyclin.

    PubMed

    Drapkin, Benjamin J; Lu, Ying; Procko, Andrea L; Timney, Benjamin L; Cross, Frederick R

    2009-01-01

    Cyclin-dependent kinase (Cdk) both promotes mitotic entry (spindle assembly and anaphase) and inhibits mitotic exit (spindle disassembly and cytokinesis), leading to an elegant quantitative hypothesis that a single cyclin oscillation can function as a ratchet to order these events. This ratchet is at the core of a published ODE model for the yeast cell cycle. However, the ratchet model requires appropriate cyclin dose-response thresholds. Here, we test the inhibition of mitotic exit in budding yeast using graded levels of stable mitotic cyclin (Clb2). In opposition to the ratchet model, stable levels of Clb2 introduced dose-dependent delays, rather than hard thresholds, that varied by mitotic exit event. The ensuing cell cycle was highly abnormal, suggesting a novel reason for cyclin degradation. Cdc14 phosphatase antagonizes Clb2-Cdk, and Cdc14 is released from inhibitory nucleolar sequestration independently of stable Clb2. Thus, Cdc14/Clb2 balance may be the appropriate variable for mitotic regulation. Although our results are inconsistent with the aforementioned ODE model, revision of the model to allow Cdc14/Clb2 balance to control mitotic exit corrects these discrepancies, providing theoretical support for our conclusions. PMID:19920813

  3. Genetic variation in mitotic regulatory pathway genes is associated with breast tumor grade.

    PubMed

    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

    2014-11-15

    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

  4. 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

  5. Targeting the Mitotic Catastrophe Signaling Pathway in Cancer

    PubMed Central

    Mc Gee, Margaret M.

    2015-01-01

    Mitotic catastrophe, as defined in 2012 by the International Nomenclature Committee on Cell Death, 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. Thus, failed mitotic catastrophe can promote the unrestrained growth of defective cells, thereby representing a major gateway to tumour development. Furthermore, the activation of mitotic catastrophe offers significant therapeutic advantage which has been exploited in the action of conventional and targeted anticancer agents. Yet, despite its importance in tumour prevention and treatment, the molecular mechanism of mitotic catastrophe is not well understood. A better understanding of the signals that determine cell fate following failed or defective mitosis will reveal new opportunities to selectively target and enhance the programme for therapeutic benefit and reveal biomarkers to predict patient response. This review is focused on the molecular mechanism of mitotic catastrophe induction and signalling and highlights current strategies to exploit the process in cancer therapy. PMID:26491220

  6. Mitotic Exit Control as an Evolved Complex System

    SciTech Connect

    Bosl, W; Li, R

    2005-04-25

    The exit from mitosis is the last critical decision a cell has to make during a division cycle. A complex regulatory system has evolved to evaluate the success of mitotic events and control this decision. Whereas outstanding genetic work in yeast has led to rapid discovery of a large number of interacting genes involved in the control of mitotic exit, it has also become increasingly difficult to comprehend the logic and mechanistic features embedded in the complex molecular network. Our view is that this difficulty stems in part from the attempt to explain mitotic exit control using concepts from traditional top-down engineering design, and that exciting new results from evolutionary engineering design applied to networks and electronic circuits may lend better insights. We focus on four particularly intriguing features of the mitotic exit control system: the two-stepped release of Cdc14; the self-activating nature of Tem1 GTPase; the spatial sensor associated with the spindle pole body; and the extensive redundancy in the mitotic exit network. We attempt to examine these design features from the perspective of evolutionary design and complex system engineering.

  7. Mitotic catenation is monitored and resolved by a PKCε-regulated pathway.

    PubMed

    Brownlow, Nicola; Pike, Tanya; Zicha, Daniel; Collinson, Lucy; Parker, Peter J

    2014-01-01

    Exit from mitosis is controlled by silencing of the spindle assembly checkpoint (SAC). It is important that preceding exit, all sister chromatid pairs are correctly bioriented, and that residual catenation is resolved, permitting complete sister chromatid separation in the ensuing anaphase. Here we determine that the metaphase response to catenation in mammalian cells operates through PKCε. The PKCε-controlled pathway regulates exit from the SAC only when mitotic cells are challenged by retained catenation and this delayed exit is characterized by BubR1-high and Mad2-low kinetochores. In addition, we show that this pathway is necessary to facilitate resolution of retained catenanes in mitosis. When delayed by catenation in mitosis, inhibition of PKCε results in premature entry into anaphase with PICH-positive strands and chromosome bridging. These findings demonstrate the importance of PKCε-mediated regulation in protection from loss of chromosome integrity in cells failing to resolve catenation in G2. PMID:25483024

  8. 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

  9. Defective control of mitotic and post-mitotic checkpoints in poly(ADP-ribose) polymerase-1(-/-) fibroblasts after mitotic spindle disruption.

    PubMed

    Halappanavar, Sabina S; Shah, Girish M

    2004-03-01

    Poly(ADP-ribose) polymerase-1 (PARP), a DNA damage-responsive nuclear enzyme present in higher eukaryotes, is well-known for its roles in protecting the genome after DNA damage. However, even without exogenous DNA damage, PARP may play a role in stabilizing the genome because cells or mice deficient in PARP exhibit various signs of genomic instability, such as tetraploidy, aneuploidy, chromosomal abnormalities and susceptibility to spontaneous carcinogenesis. Normally, cell cycle checkpoints ensure elimination of cells with genomic abnormalities. Therefore, we examined efficiency of mitotic and post-mitotic checkpoints in PARP-/- and PARP+/+ mouse embryonic fibroblasts treated with mitotic spindle disrupting agent colcemid. PARP+/+ cells, like most mammalian cells, eventually escaped from spindle disruption-induced mitotic checkpoint arrest by 60 h. In contrast, PARP-/- cells rapidly escaped from mitotic arrest within 24 h by downregulation of cyclin B1/CDK-1 kinase activity. After escaping from mitotic arrest; both the PARP genotypes arrive in G1 tetraploid state, where they face post-mitotic checkpoints which either induce apoptosis or prevent DNA endoreduplication. While all the G1 tetraploid PARP+/+ cells were eliminated by apoptosis, the majority of the G1 tetraploid PARP-/- cells became polyploid by resisting apoptosis and carrying out DNA endoreduplication. Introduction of PARP in PARP-/- fibroblasts partially increased the stringency of mitotic checkpoint arrest and fully restored susceptibility to G1 tetraploidy checkpoint-induced apoptosis; and thus prevented formation of polyploid cells. Our results suggest that PARP may serve as a guardian angel of the genome even without exogenous DNA damage through its role in mitotic and post-mitotic G1 tetraploidy checkpoints. PMID:14726664

  10. Semaphorin-Plexin Signaling Controls Mitotic Spindle Orientation during Epithelial Morphogenesis and Repair.

    PubMed

    Xia, Jingjing; Swiercz, Jakub M; Bañón-Rodríguez, Inmaculada; Matković, Ivana; Federico, Giuseppina; Sun, Tianliang; Franz, Timo; Brakebusch, Cord H; Kumanogoh, Atsushi; Friedel, Roland H; Martín-Belmonte, Fernando; Gröne, Hermann-Josef; Offermanns, Stefan; Worzfeld, Thomas

    2015-05-01

    Morphogenesis, homeostasis, and regeneration of epithelial tissues rely on the accurate orientation of cell divisions, which is specified by the mitotic spindle axis. To remain in the epithelial plane, symmetrically dividing epithelial cells align their mitotic spindle axis with the plane. Here, we show that this alignment depends on epithelial cell-cell communication via semaphorin-plexin signaling. During kidney morphogenesis and repair, renal tubular epithelial cells lacking the transmembrane receptor Plexin-B2 or its semaphorin ligands fail to correctly orient the mitotic spindle, leading to severe defects in epithelial architecture and function. Analyses of a series of transgenic and knockout mice indicate that Plexin-B2 controls the cell division axis by signaling through its GTPase-activating protein (GAP) domain and Cdc42. Our data uncover semaphorin-plexin signaling as a central regulatory mechanism of mitotic spindle orientation necessary for the alignment of epithelial cell divisions with the epithelial plane. PMID:25892012

  11. The Distribution of Active Force Generators Controls Mitotic Spindle Position

    NASA Astrophysics Data System (ADS)

    Grill, Stephan W.; Howard, Jonathon; Schäffer, Erik; Stelzer, Ernst H. K.; Hyman, Anthony A.

    2003-07-01

    During unequal cell divisions a mitotic spindle is eccentrically positioned before cell cleavage. To determine the basis of the net force imbalance that causes spindle displacement in one-cell Caenorhabditis elegans embryos, we fragmented centrosomes with an ultraviolet laser. Analysis of the mean and variance of fragment speeds suggests that the force imbalance is due to a larger number of force generators pulling on astral microtubules of the posterior aster relative to the anterior aster. Moreover, activation of heterotrimeric guanine nucleotide-binding protein (G protein) α subunits is required to generate these astral forces.

  12. 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

    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

  13. Novel chemical enhancers of heat shock increase thermal radiosensitization through a mitotic catastrophe pathway.

    PubMed

    Sekhar, Konjeti R; Sonar, Vijayakumar N; Muthusamy, Venkatraj; Sasi, Soumya; Laszlo, Andrei; Sawani, Jamil; Horikoshi, Nobuo; Higashikubo, Ryuji; Bristow, Robert G; Borrelli, Michael J; Crooks, Peter A; Lepock, James R; Roti Roti, Joseph L; Freeman, Michael L

    2007-01-15

    Radiation therapy combined with adjuvant hyperthermia has the potential to provide outstanding local-regional control for refractory disease. However, achieving therapeutic thermal dose can be problematic. In the current investigation, we used a chemistry-driven approach with the goal of designing and synthesizing novel small molecules that could function as thermal radiosensitizers. (Z)-(+/-)-2-(1-Benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol was identified as a compound that could lower the threshold for Hsf1 activation and thermal sensitivity. Enhanced thermal sensitivity was associated with significant thermal radiosensitization. We established the structural requirements for activity: the presence of an N-benzenesulfonylindole or N-benzylindole moiety linked at the indolic 3-position to a 2-(1-azabicyclo[2.2.2]octan-3-ol) or 2-(1-azabicyclo[2.2.2]octan-3-one) moiety. These small molecules functioned by exploiting the underlying biophysical events responsible for thermal sensitization. Thermal radiosensitization was characterized biochemically and found to include loss of mitochondrial membrane potential, followed by mitotic catastrophe. These studies identified a novel series of small molecules that represent a promising tool for the treatment of recurrent tumors by ionizing radiation. PMID:17234780

  14. Association of Mitotic Regulation Pathway Polymorphisms with Pancreatic Cancer Risk and Outcome

    PubMed Central

    Couch, Fergus J.; Wang, Xianshu; Bamlet, William R.; de Andrade, Mariza; Petersen, Gloria M.; McWilliams, Robert R.

    2009-01-01

    Background Mitosis is a highly regulated process that serves to ensure the fidelity of cell division. Disruption of mitotic regulators leading to aneuploidy and polyploidy is commonly observed in cancer cells. Single nucleotide polymorphisms (SNPs) in regulators of mitosis may promote chromosome mis-segregation and influence pancreatic cancer and/or survival. Methods Thirty four SNPs, previously associated with breast cancer risk, from 33 genes involved in regulation of mitosis, were investigated for associations with pancreatic cancer risk in 1,143 Caucasian patients with pancreatic adenocarcinoma and 1,097 unaffected controls from the Mayo Clinic. Associations with survival from pancreatic cancer were also assessed using 1,030 pancreatic cancer cases with known outcome. Results Two SNPs in the APC (rs2431238) and NIN (rs10145182) loci, out of 34 examined, were significantly associated with pancreatic cancer risk (p=0.035 and p=0.038, respectively). Further analyses of individuals categorized by smoking and BMI identified several SNPs displaying significant associations (p<0.05) with pancreatic cancer risk, including APC rs2431238 in individuals with high body mass index (BMI≥30) (p=0.031) and NIN rs10145182 in ever smokers (p=0.01). In addition, survival analyses detected significant associations between SNPs in EIF3S10 and overall survival (p=0.009), SNPs from five genes and survival in resected cancer cases (p<0.05), and SNPs from two other genes (p<0.05) and survival of locally advanced cancer cases. Conclusion Common variation in genes encoding regulators of mitosis may independently influence pancreatic cancer susceptibility and survival. PMID:20056645

  15. Robust control of mitotic spindle orientation in the developing epidermis

    PubMed Central

    Poulson, Nicholas D.

    2010-01-01

    Progenitor cells must balance self-amplification and production of differentiated progeny during development and homeostasis. In the epidermis, progenitors divide symmetrically to increase surface area and asymmetrically to promote stratification. In this study, we show that individual epidermal cells can undergo both types of division, and therefore, the balance is provided by the sum of individual cells’ choices. In addition, we define two control points for determining a cell’s mode of division. First is the expression of the mouse Inscuteable gene, which is sufficient to drive asymmetric cell division (ACD). However, there is robust control of division orientation as excessive ACDs are prevented by a change in the localization of NuMA, an effector of spindle orientation. Finally, we show that p63, a transcriptional regulator of stratification, does not control either of these processes. These data have uncovered two important regulatory points controlling ACD in the epidermis and allow a framework for analysis of how external cues control this important choice. PMID:21098114

  16. Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly

    PubMed Central

    Joseph, Nimesh; Cavazza, Tommaso; Vernos, Isabelle; Pfuhl, Mark; Gergely, Fanni; Bayliss, Richard

    2015-01-01

    The essential mammalian gene TACC3 is frequently mutated and amplified in cancers and its fusion products exhibit oncogenic activity in glioblastomas. TACC3 functions in mitotic spindle assembly and chromosome segregation. In particular, phosphorylation on S558 by the mitotic kinase, Aurora-A, promotes spindle recruitment of TACC3 and triggers the formation of a complex with ch-TOG-clathrin that crosslinks and stabilises kinetochore microtubules. Here we map the Aurora-A-binding interface in TACC3 and show that TACC3 potently activates Aurora-A through a domain centered on F525. Vertebrate cells carrying homozygous F525A mutation in the endogenous TACC3 loci exhibit defects in TACC3 function, namely perturbed localization, reduced phosphorylation and weakened interaction with clathrin. The most striking feature of the F525A cells however is a marked shortening of mitosis, at least in part due to rapid spindle assembly. F525A cells do not exhibit chromosome missegregation, indicating that they undergo fast yet apparently faithful mitosis. By contrast, mutating the phosphorylation site S558 to alanine in TACC3 causes aneuploidy without a significant change in mitotic duration. Our work has therefore defined a regulatory role for the Aurora-A-TACC3 interaction beyond the act of phosphorylation at S558. We propose that the regulatory relationship between Aurora-A and TACC3 enables the transition from the microtubule-polymerase activity of TACC3-ch-TOG to the microtubule-crosslinking activity of TACC3-ch-TOG-clathrin complexes as mitosis progresses. Aurora-A-dependent control of TACC3 could determine the balance between these activities, thereby influencing not only spindle length and stability but also the speed of spindle formation with vital consequences for chromosome alignment and segregation. PMID:26134678

  17. Mitotic control of kinetochore-associated dynein and spindle orientation by human Spindly

    PubMed Central

    Chan, Ying Wai; Fava, Luca L.; Uldschmid, Andreas; Schmitz, Michael H.A.; Gerlich, Daniel W.; Nigg, Erich A.

    2009-01-01

    Mitotic spindle formation and chromosome segregation depend critically on kinetochore–microtubule (KT–MT) interactions. A new protein, termed Spindly in Drosophila and SPDL-1 in C. elegans, was recently shown to regulate KT localization of dynein, but depletion phenotypes revealed striking differences, suggesting evolutionarily diverse roles of mitotic dynein. By characterizing the function of Spindly in human cells, we identify specific functions for KT dynein. We show that localization of human Spindly (hSpindly) to KTs is controlled by the Rod/Zw10/Zwilch (RZZ) complex and Aurora B. hSpindly depletion results in reduced inter-KT tension, unstable KT fibers, an extensive prometaphase delay, and severe chromosome misalignment. Moreover, depletion of hSpindly induces a striking spindle rotation, which can be rescued by co-depletion of dynein. However, in contrast to Drosophila, hSpindly depletion does not abolish the removal of MAD2 and ZW10 from KTs. Collectively, our data reveal hSpindly-mediated dynein functions and highlight a critical role of KT dynein in spindle orientation. PMID:19468067

  18. The Kinesin-8 motor, Kif18A, Suppresses Kinetochore Movements to Control Mitotic Chromosome Alignment

    PubMed Central

    Stumpff, Jason; Von Dassow, George; Wagenbach, Michael; Asbury, Charles; Wordeman, Linda

    2008-01-01

    SUMMARY During vertebrate cell division, chromosomes oscillate with periods of smooth motion interrupted by abrupt reversals in direction. These oscillations must be spatially constrained in order to align and segregate chromosomes with high fidelity, but the molecular mechanism for this activity is uncertain. We report here that the human kinesin-8, Kif18A, has a primary role in the control of chromosome oscillations. Kif18A accumulates as a gradient on kinetochore microtubules in a manner dependent on its motor activity. Quantitative analyses of kinetochore movements reveal that Kif18A reduces the amplitude of preanaphase oscillations and slows poleward movement during anaphase. Thus, the microtubule-depolymerizing kinesin, Kif18A, has the unexpected function of suppressing chromosome movements. Based on these findings we propose a molecular model in which Kif18A regulates kinetochore microtubule dynamics to control mitotic chromosome positioning. PMID:18267093

  19. Bcl-xL controls a switch between cell death modes during mitotic arrest

    PubMed Central

    Bah, N; Maillet, L; Ryan, J; Dubreil, S; Gautier, F; Letai, A; Juin, P; Barillé-Nion, S

    2014-01-01

    Antimitotic agents such as microtubule inhibitors (paclitaxel) are widely used in cancer therapy while new agents blocking mitosis onset are currently in development. All these agents impose a prolonged mitotic arrest in cancer cells that relies on sustained activation of the spindle assembly checkpoint and may lead to subsequent cell death by incompletely understood molecular events. We have investigated the role played by anti-apoptotic Bcl-2 family members in the fate of mitotically arrested mammary tumor cells treated with paclitaxel, or depleted in Cdc20, the activator of the anaphase promoting complex. Under these conditions, a weak and delayed mitotic cell death occurs that is caspase- and Bax/Bak-independent. Moreover, BH3 profiling assays indicate that viable cells during mitotic arrest are primed to die by apoptosis and that Bcl-xL is required to maintain mitochondrial integrity. Consistently, Bcl-xL depletion, or treatment with its inhibitor ABT-737 (but not with the specific Bcl-2 inhibitor ABT-199), during mitotic arrest converts cell response to antimitotics to efficient caspase and Bax-dependent apoptosis. Apoptotic priming under conditions of mitotic arrest relies, at least in part, on the phosphorylation on serine 62 of Bcl-xL, which modulates its interaction with Bax and its sensitivity to ABT-737. The phospho-mimetic S62D-Bcl-xL mutant is indeed less efficient than the corresponding phospho-deficient S62A-Bcl-xL mutant in sequestrating Bax and in protecting cancer cells from mitotic cell death or yeast cells from Bax-induced growth inhibition. Our results provide a rationale for combining Bcl-xL targeting to antimitotic agents to improve clinical efficacy of antimitotic strategy in cancer therapy. PMID:24922075

  20. A Late Mitotic Regulatory Network Controlling Cyclin Destruction in Saccharomyces cerevisiae

    PubMed Central

    Jaspersen, Sue L.; Charles, Julia F.; Tinker-Kulberg, Rachel L.; Morgan, David O.

    1998-01-01

    Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinase–cyclin complexes, primarily by ubiquitin-dependent cyclin proteolysis. Cyclin destruction is regulated by a ubiquitin ligase known as the anaphase-promoting complex (APC). In the budding yeast Saccharomyces cerevisiae, members of a large class of late mitotic mutants, including cdc15, cdc5, cdc14, dbf2, and tem1, arrest in anaphase with a phenotype similar to that of cells expressing nondegradable forms of mitotic cyclins. We addressed the possibility that the products of these genes are components of a regulatory network that governs cyclin proteolysis. We identified a complex array of genetic interactions among these mutants and found that the growth defect in most of the mutants is suppressed by overexpression of SPO12, YAK1, and SIC1 and is exacerbated by overproduction of the mitotic cyclin Clb2. When arrested in late mitosis, the mutants exhibit a defect in cyclin-specific APC activity that is accompanied by high Clb2 levels and low levels of the anaphase inhibitor Pds1. Mutant cells arrested in G1 contain normal APC activity. We conclude that Cdc15, Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC in late mitosis but are not required for maintenance of that activity in G1. PMID:9763445

  1. Nucleocytoplasmic transport in the midzone membrane domain controls yeast mitotic spindle disassembly

    PubMed Central

    Lucena, Rafael; Dephoure, Noah; Gygi, Steve P.; Kellogg, Douglas R.; Tallada, Victor A.

    2015-01-01

    During each cell cycle, the mitotic spindle is efficiently assembled to achieve chromosome segregation and then rapidly disassembled as cells enter cytokinesis. Although much has been learned about assembly, how spindles disassemble at the end of mitosis remains unclear. Here we demonstrate that nucleocytoplasmic transport at the membrane domain surrounding the mitotic spindle midzone, here named the midzone membrane domain (MMD), is essential for spindle disassembly in Schizosaccharomyces pombe cells. We show that, during anaphase B, Imp1-mediated transport of the AAA-ATPase Cdc48 protein at the MMD allows this disassembly factor to localize at the spindle midzone, thereby promoting spindle midzone dissolution. Our findings illustrate how a separate membrane compartment supports spindle disassembly in the closed mitosis of fission yeast. PMID:25963819

  2. Spindle assembly checkpoint is sufficient for complete Cdc20 sequestering in mitotic control.

    PubMed

    Ibrahim, Bashar

    2015-01-01

    The spindle checkpoint assembly (SAC) ensures genome fidelity by temporarily delaying anaphase onset, until all chromosomes are properly attached to the mitotic spindle. The SAC delays mitotic progression by preventing activation of the ubiquitin ligase anaphase-promoting complex (APC/C) or cyclosome; whose activation by Cdc20 is required for sister-chromatid separation marking the transition into anaphase. The mitotic checkpoint complex (MCC), which contains Cdc20 as a subunit, binds stably to the APC/C. Compelling evidence by Izawa and Pines (Nature 2014; 10.1038/nature13911) indicates that the MCC can inhibit a second Cdc20 that has already bound and activated the APC/C. Whether or not MCC per se is sufficient to fully sequester Cdc20 and inhibit APC/C remains unclear. Here, a dynamic model for SAC regulation in which the MCC binds a second Cdc20 was constructed. This model is compared to the MCC, and the MCC-and-BubR1 (dual inhibition of APC) core model variants and subsequently validated with experimental data from the literature. By using ordinary nonlinear differential equations and spatial simulations, it is shown that the SAC works sufficiently to fully sequester Cdc20 and completely inhibit APC/C activity. This study highlights the principle that a systems biology approach is vital for molecular biology and could also be used for creating hypotheses to design future experiments. PMID:25977749

  3. Spindle assembly checkpoint is sufficient for complete Cdc20 sequestering in mitotic control

    PubMed Central

    Ibrahim, Bashar

    2015-01-01

    The spindle checkpoint assembly (SAC) ensures genome fidelity by temporarily delaying anaphase onset, until all chromosomes are properly attached to the mitotic spindle. The SAC delays mitotic progression by preventing activation of the ubiquitin ligase anaphase-promoting complex (APC/C) or cyclosome; whose activation by Cdc20 is required for sister-chromatid separation marking the transition into anaphase. The mitotic checkpoint complex (MCC), which contains Cdc20 as a subunit, binds stably to the APC/C. Compelling evidence by Izawa and Pines (Nature 2014; 10.1038/nature13911) indicates that the MCC can inhibit a second Cdc20 that has already bound and activated the APC/C. Whether or not MCC per se is sufficient to fully sequester Cdc20 and inhibit APC/C remains unclear. Here, a dynamic model for SAC regulation in which the MCC binds a second Cdc20 was constructed. This model is compared to the MCC, and the MCC-and-BubR1 (dual inhibition of APC) core model variants and subsequently validated with experimental data from the literature. By using ordinary nonlinear differential equations and spatial simulations, it is shown that the SAC works sufficiently to fully sequester Cdc20 and completely inhibit APC/C activity. This study highlights the principle that a systems biology approach is vital for molecular biology and could also be used for creating hypotheses to design future experiments. PMID:25977749

  4. 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. PMID:26943585

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:26943585

  6. Targeting cannabinoid receptor-2 pathway by phenylacetylamide suppresses the proliferation of human myeloma cells through mitotic dysregulation and cytoskeleton disruption.

    PubMed

    Feng, Rentian; Tong, Qin; Xie, Zhaojun; Cheng, Haizi; Wang, Lirong; Lentzsch, Suzanne; Roodman, G David; Xie, Xiang-Qun

    2015-12-01

    Cannabinoid receptor-2 (CB2) is expressed dominantly in the immune system, especially on plasma cells. Cannabinergic ligands with CB2 selectivity emerge as a class of promising agents to treat CB2-expressing malignancies without psychotropic concerns. In this study, we found that CB2 but not CB1 was highly expressed in human multiple myeloma (MM) and primary CD138+ cells. A novel inverse agonist of CB2, phenylacetylamide but not CB1 inverse agonist SR141716, inhibited the proliferation of human MM cells (IC50 : 0.62 ∼ 2.5 μM) mediated by apoptosis induction, but exhibited minor cytotoxic effects on human normal mononuclear cells. CB2 gene silencing or pharmacological antagonism markedly attenuated phenylacetylamide's anti-MM effects. Phenylacetylamide triggered the expression of C/EBP homologous protein at the early treatment stage, followed by death receptor-5 upregulation, caspase activation, and β-actin/tubulin degradation. Cell cycle related protein cdc25C and mitotic regulator Aurora A kinase were inactivated by phenylacetylamide treatment, leading to an increase in the ratio inactive/active cdc2 kinase. As a result, phosphorylation of CDK substrates was decreased, and the MM cell mitotic division was largely blocked by treatment. Importantly, phenylacetylamide could overcome the chemoresistance of MM cells against dexamethasone or melphalan. Thus, targeting CB2 may represent an attractive approach to treat cancers of immune origin. PMID:25640641

  7. Caffeine inhibits adipogenesis through modulation of mitotic clonal expansion and the AKT/GSK3 pathway in 3T3-L1 adipocytes

    PubMed Central

    Kim, Hyo Jung; Yoon, Bo Kyung; Park, Hyounkyoung; Seok, Jo Woon; Choi, Hyeonjin; Yu, Jung Hwan; Choi, Yoonjeong; Song, Su Jin; Kim, Ara; Kim, Jae-woo

    2016-01-01

    Caffeine has been proposed to have several beneficial effects on obesity and its related metabolic diseases; however, how caffeine affects adipocyte differentiation has not been elucidated. In this study, we demonstrated that caffeine suppressed 3T3-L1 adipocyte differentiation and inhibited the expression of CCAAT/enhancer binding protein (C/EBP)α and peroxisome proliferator-activated receptor (PPAR)γ, two main adipogenic transcription factors. Anti-adipogenic markers, such as preadipocyte secreted factor (Pref)-1 and Krüppel-like factor 2, remained to be expressed in the presence of caffeine. Furthermore, 3T3-L1 cells failed to undergo typical mitotic clonal expansion in the presence of caffeine. Investigation of hormonal signaling revealed that caffeine inhibited the activation of AKT and glycogen synthase kinase (GSK) 3 in a dose-dependent manner, but not extracellular signal-regulated kinase (ERK). Our data show that caffeine is an anti-adipogenic bioactive compound involved in the modulation of mitotic clonal expansion during adipocyte differentiation through the AKT/GSK3 pathway. [BMB Reports 2016; 49(2): 111-115] PMID:26350746

  8. Misato Controls Mitotic Microtubule Generation by Stabilizing the Tubulin Chaperone Protein-1 Complex

    PubMed Central

    Palumbo, Valeria; Pellacani, Claudia; Heesom, Kate J.; Rogala, Kacper B.; Deane, Charlotte M.; Mottier-Pavie, Violaine; Gatti, Maurizio; Bonaccorsi, Silvia; Wakefield, James G.

    2015-01-01

    Summary Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers [1, 2]. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions [3, 4]. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies [5], but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs. PMID:26096973

  9. Mitotic Control of Planar Cell Polarity by Polo-like Kinase 1.

    PubMed

    Shrestha, Rezma; Little, Katherine A; Tamayo, Joel V; Li, Wenyang; Perlman, David H; Devenport, Danelle

    2015-06-01

    During cell division, polarized epithelial cells employ mechanisms to preserve cell polarity and tissue integrity. In dividing cells of the mammalian skin, planar cell polarity (PCP) is maintained through the bulk internalization, equal segregation, and polarized recycling of cortical PCP proteins. The dramatic redistribution of PCP proteins coincides precisely with cell-cycle progression, but the mechanisms coordinating PCP and mitosis are unknown. Here we identify Plk1 as a master regulator of PCP dynamics during mitosis. Plk1 interacts with core PCP component Celsr1 via a conserved polo-box domain (PBD)-binding motif, localizes to mitotic endosomes, and directly phosphorylates Celsr1. Plk1-dependent phosphorylation activates the endocytic motif specifically during mitosis, allowing bulk recruitment of Celsr1 into endosomes. Inhibiting Plk1 activity blocks PCP internalization and perturbs PCP asymmetry. Mimicking dileucine motif phosphorylation is sufficient to drive Celsr1 internalization during interphase. Thus, Plk1-mediated phosphorylation of Celsr1 ensures that PCP redistribution is precisely coordinated with mitotic entry. PMID:26004507

  10. Mitotic Control of Planar Cell Polarity by Polo-like Kinase 1

    PubMed Central

    Shrestha, Rezma; Little, Katherine A.; Tamayo, Joel V.; Li, Wenyang; Perlman, David H.; Devenport, Danelle

    2015-01-01

    SUMMARY During cell division, polarized epithelial cells employ mechanisms to preserve cell polarity and tissue integrity. In dividing cells of the mammalian skin, planar cell polarity (PCP) is maintained through the bulk internalization, equal segregation, and polarized recycling of cortical PCP proteins. The dramatic redistribution of PCP proteins coincides precisely with cell cycle progression, but the mechanisms coordinating PCP and mitosis are unknown. Here we identify Plk1 as a master regulator of PCP dynamics during mitosis. Plk1 interacts with core PCP component, Celsr1, via a conserved polo-box domain (PBD) binding motif, localizes to mitotic endosomes and directly phosphorylates Celsr1. Plk1-dependent phosphorylation activates the endocytic motif specifically during mitosis, allowing bulk recruitment of Celsr1 into endosomes. Inhibiting Plk1 activity blocks PCP internalization and perturbs PCP asymmetry. Mimicking dileucine motif phosphorylation is sufficient to drive Celsr1 internalization during interphase. Thus, Plk1-mediated phosphorylation of Celsr1 ensures PCP redistribution is precisely coordinated with mitotic entry. PMID:26004507

  11. The S. pombe “cytokinesis” NDR kinase Sid2 activates Fin1 NIMA kinase to control mitotic commitment via Pom1/Wee1

    PubMed Central

    Grallert, Agnes; Connolly, Yvonne; Smith, Duncan L.; Simanis, Viesturs; Hagan, Iain M.

    2014-01-01

    Mitotic exit integrates the reversal of the phosphorylation events initiated by mitotic kinases with a controlled cytokinesis event that cleaves the cell in two. The Mitotic Exit Network (MEN) of budding yeast regulates both processes, while the fission yeast equivalent, the Septum Initiation Network (SIN), only controls the execution of cytokinesis. The components and architecture of the SIN and MEN are highly conserved1. It is currently assumed that the functions of the core SIN/MEN components are restricted to their characterised roles at the end of mitosis. We now show that the NDR kinase component of the fission yeast SIN, Sid2/Mob1, acts independently of the other known SIN components in G2 phase of the cell cycle to control the timing of mitotic commitment. Sid2/Mob1 promotes mitotic commitment by directly activating the NIMA related kinase Fin1. Fin1’s activation promotes its own destruction, thereby making Fin1 activation a transient feature of G2 phase. This spike of Fin1 activation modulates the activity of the Pom1/Cdr1/Cdr2 geometry network towards Wee1. PMID:22684255

  12. Loss of MAPK Pathway Activation in Post-Mitotic Retinal Cells as Mechanism in MEK Inhibition-Related Retinopathy in Cancer Patients

    PubMed Central

    van Dijk, Elon H. C.; Duits, Danique E. M.; Versluis, Mieke; Luyten, Gregrorius P. M.; Bergen, Arthur A. B.; Kapiteijn, Ellen W.; de Lange, Mark J.; Boon, Camiel J. F.; van der Velden, Pieter A.

    2016-01-01

    Abstract Recently, treatment with MEK inhibitors has been shown to be an effective treatment option for metastatic melanoma. Treatment efficacy is dependent on inhibition of MAPK-related melanoma proliferation. However, targeting of MEK can be accompanied by a time-dependent and reversible serous retinopathy of unknown origin. We analyzed the molecular mechanism by which the MEK inhibitor binimetinib may lead to retinopathy, using neuroretina and cell models of retinal pigment epithelium (RPE). Binimetinib inhibited the MAPK pathway while discontinuation of treatment resulted in reactivation. However, cell proliferation was not inhibited correspondingly during binimetinib treatment of ARPE19 cells. Remarkably, post-mitotic neuroretinal tissue displayed a strong MAPK activation that was lost after binimetinib treatment. We propose that binimetinib-associated retinopathy is correlated with inhibition of the MAPK pathway in multiple retinal components. Retinal cells are able to regain the activation after binimetinib treatment, mimicking the reversibility of the retinopathy. As most retinal cells are nonregenerating, other mechanisms than stimulation of proliferation must be involved. PMID:27149444

  13. Role for E2F in Control of Both DNA Replication and Mitotic Functions as Revealed from DNA Microarray Analysis

    PubMed Central

    Ishida, Seiichi; Huang, Erich; Zuzan, Harry; Spang, Rainer; Leone, Gustavo; West, Mike; Nevins, Joseph R.

    2001-01-01

    We have used high-density DNA microarrays to provide an analysis of gene regulation during the mammalian cell cycle and the role of E2F in this process. Cell cycle analysis was facilitated by a combined examination of gene control in serum-stimulated fibroblasts and cells synchronized at G1/S by hydroxyurea block that were then released to proceed through the cell cycle. The latter approach (G1/S synchronization) is critical for rigorously maintaining cell synchrony for unambiguous analysis of gene regulation in later stages of the cell cycle. Analysis of these samples identified seven distinct clusters of genes that exhibit unique patterns of expression. Genes tend to cluster within these groups based on common function and the time during the cell cycle that the activity is required. Placed in this context, the analysis of genes induced by E2F proteins identified genes or expressed sequence tags not previously described as regulated by E2F proteins; surprisingly, many of these encode proteins known to function during mitosis. A comparison of the E2F-induced genes with the patterns of cell growth-regulated gene expression revealed that virtually all of the E2F-induced genes are found in only two of the cell cycle clusters; one group was regulated at G1/S, and the second group, which included the mitotic activities, was regulated at G2. The activation of the G2 genes suggests a broader role for E2F in the control of both DNA replication and mitotic activities. PMID:11416145

  14. PP1-mediated dephosphorylation of phosphoproteins at mitotic exit is controlled by inhibitor-1 and PP1 phosphorylation

    PubMed Central

    Wu, Judy Qiju; Guo, Jessie Yanxiang; Tang, Wanli; Yang, Chih-Sheng; Freel, Christopher D.; Chen, Chen; Nairn, Angus C.; Kornbluth, Sally

    2009-01-01

    Loss of Cdc2 activity following Cyclin B degradation is necessary, but not sufficient, for mitotic exit. Proteins phosphorylated by Cdc2 and downstream mitotic kinases must also be dephosphorylated. We report here that protein phosphatase-1 (PP1) is the major catalyst of mitotic phosphoprotein dephosphorylation. Suppression of PP1 during early mitosis is maintained through the dual inhibition of PP1 by Cdc2 phosphorylation and the binding of Inhibitor-1 (I1), which is facilitated by PKA-mediated I1 phosphorylation. As Cdc2 levels drop following Cyclin B degradation, autodephosphorylation of PP1 at the site of Cdc2 phosphorylation (T320) allows partial PP1 activation. This promotes PP1-regulated dephosphorylation of I1 at its activating site (T35), dissociation of the I1-PP1 complex, and full PP1 activation to promote mitotic exit. Thus, Cdc2 both phosphorylates multiple mitotic substrates and inhibits their PP1-mediated dephosphorylation. PMID:19396163

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

    PubMed Central

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

    2016-01-01

    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. PMID:26769847

  16. The transcription factor ERG recruits CCR4-NOT to control mRNA decay and mitotic progression.

    PubMed

    Rambout, Xavier; Detiffe, Cécile; Bruyr, Jonathan; Mariavelle, Emeline; Cherkaoui, Majid; Brohée, Sylvain; Demoitié, Pauline; Lebrun, Marielle; Soin, Romuald; Lesage, Bart; Guedri, Katia; Beullens, Monique; Bollen, Mathieu; Farazi, Thalia A; Kettmann, Richard; Struman, Ingrid; Hill, David E; Vidal, Marc; Kruys, Véronique; Simonis, Nicolas; Twizere, Jean-Claude; Dequiedt, Franck

    2016-07-01

    Control of mRNA levels, a fundamental aspect in the regulation of gene expression, is achieved through a balance between mRNA synthesis and decay. E26-related gene (Erg) proteins are canonical transcription factors whose previously described functions are confined to the control of mRNA synthesis. Here, we report that ERG also regulates gene expression by affecting mRNA stability and identify the molecular mechanisms underlying this function in human cells. ERG is recruited to mRNAs via interaction with the RNA-binding protein RBPMS, and it promotes mRNA decay by binding CNOT2, a component of the CCR4-NOT deadenylation complex. Transcriptome-wide mRNA stability analysis revealed that ERG controls the degradation of a subset of mRNAs highly connected to Aurora signaling, whose decay during S phase is necessary for mitotic progression. Our data indicate that control of gene expression by mammalian transcription factors may follow a more complex scheme than previously anticipated, integrating mRNA synthesis and degradation. PMID:27273514

  17. Cell Size Checkpoint Control by the Retinoblastoma Tumor Suppressor Pathway

    PubMed Central

    Fang, Su-Chiung; de los Reyes, Chris; Umen, James G

    2006-01-01

    Size control is essential for all proliferating cells, and is thought to be regulated by checkpoints that couple cell size to cell cycle progression. The aberrant cell-size phenotypes caused by mutations in the retinoblastoma (RB) tumor suppressor pathway are consistent with a role in size checkpoint control, but indirect effects on size caused by altered cell cycle kinetics are difficult to rule out. The multiple fission cell cycle of the unicellular alga Chlamydomonas reinhardtii uncouples growth from division, allowing direct assessment of the relationship between size phenotypes and checkpoint function. Mutations in the C. reinhardtii RB homolog encoded by MAT3 cause supernumerous cell divisions and small cells, suggesting a role for MAT3 in size control. We identified suppressors of an mat3 null allele that had recessive mutations in DP1 or dominant mutations in E2F1, loci encoding homologs of a heterodimeric transcription factor that is targeted by RB-related proteins. Significantly, we determined that the dp1 and e2f1 phenotypes were caused by defects in size checkpoint control and were not due to a lengthened cell cycle. Despite their cell division defects, mat3, dp1, and e2f1 mutants showed almost no changes in periodic transcription of genes induced during S phase and mitosis, many of which are conserved targets of the RB pathway. Conversely, we found that regulation of cell size was unaffected when S phase and mitotic transcription were inhibited. Our data provide direct evidence that the RB pathway mediates cell size checkpoint control and suggest that such control is not directly coupled to the magnitude of periodic cell cycle transcription. PMID:17040130

  18. Glycosyltransferase efficiently controls phenylpropanoid pathway

    PubMed Central

    Aksamit-Stachurska, Anna; Korobczak-Sosna, Alina; Kulma, Anna; Szopa, Jan

    2008-01-01

    Background In a previous study, anthocyanin levels in potato plants were increased by manipulating genes connected with the flavonoid biosynthesis pathway. However, starch content and tuber yield were dramatically reduced in the transgenic plants, which over-expressed dihydroflavonol reductase (DFR). Results Transgenic plants over-expressing dihydroflavonol reductase (DFR) were subsequently transformed with the cDNA coding for the glycosyltransferase (UGT) of Solanum sogarandinum in order to obtain plants with a high anthocyanin content without reducing tuber yield and quality. Based on enzyme studies, the recombinant UGT is a 7-O-glycosyltransferase whose natural substrates include both anthocyanidins and flavonols such as kaempferol and quercetin. In the super-transformed plants, tuber production was much higher than in the original transgenic plants bearing only the transgene coding for DFR, and was almost the same as in the control plants. The anthocyanin level was lower than in the initial plants, but still higher than in the control plants. Unexpectedly, the super-transformed plants also produced large amounts of kaempferol, chlorogenic acid, isochlorogenic acid, sinapic acid and proanthocyanins. Conclusion In plants over-expressing both the transgene for DFR and the transgene for UGT, the synthesis of phenolic acids was diverted away from the anthocyanin branch. This represents a novel approach to manipulating phenolic acids synthesis in plants. PMID:18321380

  19. TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways

    PubMed Central

    De Conti, Laura; Akinyi, Maureen V.; Mendoza-Maldonado, Ramiro; Romano, Maurizio; Baralle, Marco; Buratti, Emanuele

    2015-01-01

    In recent times, high-throughput screening analyses have broadly defined the RNA cellular targets of TDP-43, a nuclear factor involved in neurodegeneration. A common outcome of all these studies is that changing the expression levels of this protein can alter the expression of several hundred RNAs within cells. What still remains to be clarified is which changes represent direct cellular targets of TDP-43 or just secondary variations due to the general role played by this protein in RNA metabolism. Using an HTS-based splicing junction analysis we identified at least six bona fide splicing events that are consistent with being controlled by TDP-43. Validation of the data, both in neuronal and non-neuronal cell lines demonstrated that TDP-43 substantially alters the levels of isoform expression in four genes potentially important for neuropathology: MADD/IG20, STAG2, FNIP1 and BRD8. For MADD/IG20 and STAG2, these changes could also be confirmed at the protein level. These alterations were also observed in a cellular model that successfully mimics TDP-43 loss of function effects following its aggregation. Most importantly, our study demonstrates that cell cycle alterations induced by TDP-43 knockdown can be recovered by restoring the STAG2, an important component of the cohesin complex, normal splicing profile. PMID:26261209

  20. The cyclin-dependent kinase inhibitor seliciclib (R-roscovitine; CYC202) decreases the expression of mitotic control genes and prevents entry into mitosis.

    PubMed

    Whittaker, Steven R; Te Poele, Robert H; Chan, Florence; Linardopoulos, Spiros; Walton, Michael I; Garrett, Michelle D; Workman, Paul

    2007-12-15

    The cyclin-dependent kinase (CDK) inhibitor seliciclib (R-roscovitine, CYC202) shows promising antitumor activity in preclinical models and is currently undergoing phase II clinical trials. Inhibition of the CDKs by seliciclib could contribute to cell cycle arrest and apoptosis seen with the drug. However, it is common for drugs to exert multiple effects on gene expression and biochemical pathways. To further our understanding of the molecular pharmacology of seliciclib, we employed cDNA microarrays to determine changes in gene expression profiles induced by the drug in HT29 human colon cancer cells. Concentrations of seliciclib were used that inhibited RB phosphorylation and cell proliferation. An increase in the mRNA expression for CJUN and EGR1 was confirmed by Western blotting, consistent with activation of the ERK1/2 MAPK pathway by seliciclib. Transcripts of key genes required for the progression through mitosis showed markedly reduced expression, including Aurora-A/B (AURK-A/B), Polo-like kinase (PLK), cyclin B2 (CCNB2), WEE1 and CDC25C. Reduced expression of these mitotic genes was also seen at the protein level. siRNA-mediated depletion of Aurora-A protein led to an arrest of cells in the G(2)/M phase, consistent with the effects of seliciclib treatment. Inhibition of mitotic entry following seliciclib treatment was indicated by a reduction of histone H3 phosphorylation, which is catalyzed by Aurora-B, and by decreased expression of mitotic markers, including phospho-protein phosphatase 1 alpha. The results indicate a potential mechanism through which seliciclib prevents entry into mitosis. Gene expression profiling has generated hypotheses that led to an increase in our knowledge of the cellular effects of seliciclib and could provide potential pharmacodynamic or response biomarkers for use in animal models and clinical trials. PMID:18075315

  1. p21 as a Transcriptional Co-Repressor of S-Phase and Mitotic Control Genes

    PubMed Central

    Ferrándiz, Nuria; Caraballo, Juan M.; García-Gutierrez, Lucía; Devgan, Vikram; Rodriguez-Paredes, Manuel; Lafita, M. Carmen; Bretones, Gabriel; Quintanilla, Andrea; Muñoz-Alonso, M. Jose; Blanco, Rosa; Reyes, Jose C.; Agell, Neus; Delgado, M. Dolores; Dotto, G. Paolo; León, Javier

    2012-01-01

    It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes. PMID:22662213

  2. p21 as a transcriptional co-repressor of S-phase and mitotic control genes.

    PubMed

    Ferrándiz, Nuria; Caraballo, Juan M; García-Gutierrez, Lucía; Devgan, Vikram; Rodriguez-Paredes, Manuel; Lafita, M Carmen; Bretones, Gabriel; Quintanilla, Andrea; Muñoz-Alonso, M Jose; Blanco, Rosa; Reyes, Jose C; Agell, Neus; Delgado, M Dolores; Dotto, G Paolo; León, Javier

    2012-01-01

    It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes. PMID:22662213

  3. Myosin-10 independently influences mitotic spindle structure and mitotic progression.

    PubMed

    Sandquist, Joshua C; Larson, Matthew E; Hine, Ken J

    2016-06-01

    The iconic bipolar structure of the mitotic spindle is of extreme importance to proper spindle function. At best, spindle abnormalities result in a delayed mitosis, while worse outcomes include cell death or disease. Recent work has uncovered an important role for the actin-based motor protein myosin-10 in the regulation of spindle structure and function. Here we examine the contribution of the myosin tail homology 4 (MyTH4) domain of the myosin-10 tail to the protein's spindle functions. The MyTH4 domain is known to mediate binding to microtubules and we verify the suspicion that this domain contributes to myosin-10's close association with the spindle. More surprisingly, our data demonstrate that some but not all of myosin-10's spindle functions require microtubule binding. In particular, myosin-10's contribution to spindle pole integrity requires microtubule binding, whereas its contribution to normal mitotic progression does not. This is demonstrated by the observation that dominant negative expression of the wild-type MyTH4 domain produces multipolar spindles and an increased mitotic index, whereas overexpression of a version of the MyTH4 domain harboring point mutations that abrogate microtubule binding results in only the mitotic index phenotype. Our data suggest that myosin-10 helps to control the metaphase to anaphase transition in cells independent of microtubule binding. © 2016 Wiley Periodicals, Inc. PMID:27220038

  4. Mechanisms of Mitotic Spindle Assembly

    PubMed Central

    Petry, Sabine

    2016-01-01

    Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ~200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes. PMID:27145846

  5. Non-centrosomal nucleation mediated by augmin organizes microtubules in post-mitotic neurons and controls axonal microtubule polarity.

    PubMed

    Sánchez-Huertas, Carlos; Freixo, Francisco; Viais, Ricardo; Lacasa, Cristina; Soriano, Eduardo; Lüders, Jens

    2016-01-01

    Neurons display a highly polarized microtubule network that mediates trafficking throughout the extensive cytoplasm and is crucial for neuronal differentiation and function. In newborn migrating neurons, the microtubule network is organized by the centrosome. During neuron maturation, however, the centrosome gradually loses this activity, and how microtubules are organized in more mature neurons remains poorly understood. Here, we demonstrate that microtubule organization in post-mitotic neurons strongly depends on non-centrosomal nucleation mediated by augmin and by the nucleator γTuRC. Disruption of either complex not only reduces microtubule density but also microtubule bundling. These microtubule defects impair neurite formation, interfere with axon specification and growth, and disrupt axonal trafficking. In axons augmin does not merely mediate nucleation of microtubules but ensures their uniform plus end-out orientation. Thus, the augmin-γTuRC module, initially identified in mitotic cells, may be commonly used to generate and maintain microtubule configurations with specific polarity. PMID:27405868

  6. Non-centrosomal nucleation mediated by augmin organizes microtubules in post-mitotic neurons and controls axonal microtubule polarity

    PubMed Central

    Sánchez-Huertas, Carlos; Freixo, Francisco; Viais, Ricardo; Lacasa, Cristina; Soriano, Eduardo; Lüders, Jens

    2016-01-01

    Neurons display a highly polarized microtubule network that mediates trafficking throughout the extensive cytoplasm and is crucial for neuronal differentiation and function. In newborn migrating neurons, the microtubule network is organized by the centrosome. During neuron maturation, however, the centrosome gradually loses this activity, and how microtubules are organized in more mature neurons remains poorly understood. Here, we demonstrate that microtubule organization in post-mitotic neurons strongly depends on non-centrosomal nucleation mediated by augmin and by the nucleator γTuRC. Disruption of either complex not only reduces microtubule density but also microtubule bundling. These microtubule defects impair neurite formation, interfere with axon specification and growth, and disrupt axonal trafficking. In axons augmin does not merely mediate nucleation of microtubules but ensures their uniform plus end-out orientation. Thus, the augmin-γTuRC module, initially identified in mitotic cells, may be commonly used to generate and maintain microtubule configurations with specific polarity. PMID:27405868

  7. Arabidopsis COPPER MODIFIED RESISTANCE1/PATRONUS1 is essential for growth adaptation to stress and required for mitotic onset control.

    PubMed

    Juraniec, Michal; Heyman, Jefri; Schubert, Veit; Salis, Pietrino; De Veylder, Lieven; Verbruggen, Nathalie

    2016-01-01

    The mitotic checkpoint (MC) guards faithful sister chromatid segregation by monitoring the attachment of spindle microtubules to the kinetochores. When chromosome attachment errors are detected, MC delays the metaphase-to-anaphase transition through the inhibition of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. In contrast to yeast and mammals, our knowledge on the proteins involved in MC in plants is scarce. Transient synchronization of root tips as well as promoter-reporter gene fusions were performed to analyze temporal and spatial expression of COPPER MODIFIED RESISTANCE1/PATRONUS1 (CMR1/PANS1) in developing Arabidopsis thaliana seedlings. Functional analysis of the gene was carried out, including CYCB1;2 stability in CMR1/PANS1 knockout and overexpressor background as well as metaphase-anaphase chromosome status. CMR1/PANS1 is transcriptionally active during M phase. Its deficiency provokes premature cell cycle exit and in consequence a rapid consumption of the number of meristematic cells in particular under stress conditions that are known to affect spindle microtubules. Root growth impairment is correlated with a failure to delay the onset of anaphase, resulting in anaphase bridges and chromosome missegregation. CMR1/PANS1 overexpression stabilizes the mitotic CYCB1;2 protein. Likely, CMR1/PANS1 coordinates mitotic cell cycle progression by acting as an APC/C inhibitor and plays a key role in growth adaptation to stress. PMID:26261921

  8. Role of senescence and mitotic catastrophe in cancer therapy

    PubMed Central

    2010-01-01

    Senescence and mitotic catastrophe (MC) are two distinct crucial non-apoptotic mechanisms, often triggered in cancer cells and tissues in response to anti-cancer drugs. Chemotherapeuticals and myriad other factors induce cell eradication via these routes. While senescence drives the cells to a state of quiescence, MC drives the cells towards death during the course of mitosis. The senescent phenotype distinguishes tumor cells that survived drug exposure but lost the ability to form colonies from those that recover and proliferate after treatment. Although senescent cells do not proliferate, they are metabolically active and may secrete proteins with potential tumor-promoting activities. The other anti-proliferative response of tumor cells is MC that is a form of cell death that results from abnormal mitosis and leads to the formation of interphase cells with multiple micronuclei. Different classes of cytotoxic agents induce MC, but the pathways of abnormal mitosis differ depending on the nature of the inducer and the status of cell-cycle checkpoints. In this review, we compare the two pathways and mention that they are activated to curb the growth of tumors. Altogether, we have highlighted the possibilities of the use of senescence targeting drugs, mitotic kinases and anti-mitotic agents in fabricating novel strategies in cancer control. PMID:20205872

  9. Genetic dissection of cardiac growth control pathways

    NASA Technical Reports Server (NTRS)

    MacLellan, W. R.; Schneider, M. D.

    2000-01-01

    Cardiac muscle cells exhibit two related but distinct modes of growth that are highly regulated during development and disease. Cardiac myocytes rapidly proliferate during fetal life but exit the cell cycle irreversibly soon after birth, following which the predominant form of growth shifts from hyperplastic to hypertrophic. Much research has focused on identifying the candidate mitogens, hypertrophic agonists, and signaling pathways that mediate these processes in isolated cells. What drives the proliferative growth of embryonic myocardium in vivo and the mechanisms by which adult cardiac myocytes hypertrophy in vivo are less clear. Efforts to answer these questions have benefited from rapid progress made in techniques to manipulate the murine genome. Complementary technologies for gain- and loss-of-function now permit a mutational analysis of these growth control pathways in vivo in the intact heart. These studies have confirmed the importance of suspected pathways, have implicated unexpected pathways as well, and have led to new paradigms for the control of cardiac growth.

  10. Plk1-dependent recruitment of gamma-tubulin complexes to mitotic centrosomes involves multiple PCM components.

    PubMed

    Haren, Laurence; Stearns, Tim; Lüders, Jens

    2009-01-01

    The nucleation of microtubules requires protein complexes containing gamma-tubulin, which are present in the cytoplasm and associate with the centrosome and with the mitotic spindle. We have previously shown that these interactions require the gamma-tubulin targeting factor GCP-WD/NEDD1, which has an essential role in spindle formation. The recruitment of additional gamma-tubulin to the centrosomes occurs during centrosome maturation at the G2/M transition and is regulated by the mitotic kinase Plk1. However, the molecular details of this important pathway are unknown and a Plk1 substrate that controls gamma-tubulin recruitment has not been identified. Here we show that Plk1 associates with GCP-WD in mitosis and Plk1 activity contributes to phosphorylation of GCP-WD. Plk1 depletion or inhibition prevents accumulation of GCP-WD at mitotic centrosomes, but GCP-WD mutants that are defective in Plk1-binding and -phosphorylation still accumulate at mitotic centrosomes and recruit gamma-tubulin. Moreover, Plk1 also controls the recruitment of other PCM proteins implicated in centrosomal gamma-tubulin attachment (Cep192/hSPD2, pericentrin, Cep215/Cdk5Rap2). Our results support a model in which Plk1-dependent recruitment of gamma-tubulin to mitotic centrosomes is regulated upstream of GCP-WD, involves multiple PCM proteins and therefore potentially multiple Plk1 substrates. PMID:19543530

  11. Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression

    PubMed Central

    Lim, Jung Mi; Lee, Kyung S.; Woo, Hyun Ae

    2015-01-01

    Proteins associated with the centrosome play key roles in mitotic progression in mammalian cells. The activity of Cdk1-opposing phosphatases at the centrosome must be inhibited during early mitosis to prevent premature dephosphorylation of Cdh1—an activator of the ubiquitin ligase anaphase-promoting complex/cyclosome—and the consequent premature degradation of mitotic activators. In this paper, we show that reversible oxidative inactivation of centrosome-bound protein phosphatases such as Cdc14B by H2O2 is likely responsible for this inhibition. The intracellular concentration of H2O2 increases as the cell cycle progresses. Whereas the centrosome is shielded from H2O2 through its association with the H2O2-eliminating enzyme peroxiredoxin I (PrxI) during interphase, the centrosome-associated PrxI is selectively inactivated through phosphorylation by Cdk1 during early mitosis, thereby exposing the centrosome to H2O2 and facilitating inactivation of centrosome-bound phosphatases. Dephosphorylation of PrxI by okadaic acid–sensitive phosphatases during late mitosis again shields the centrosome from H2O2 and thereby allows the reactivation of Cdk1-opposing phosphatases at the organelle. PMID:26150388

  12. An evolutionarily conserved pathway controls proteasome homeostasis.

    PubMed

    Rousseau, Adrien; Bertolotti, Anne

    2016-08-11

    The proteasome is essential for the selective degradation of most cellular proteins, but how cells maintain adequate amounts of proteasome is unclear. Here we show that there is an evolutionarily conserved signalling pathway controlling proteasome homeostasis. Central to this pathway is TORC1, the inhibition of which induced all known yeast 19S regulatory particle assembly-chaperones (RACs), as well as proteasome subunits. Downstream of TORC1 inhibition, the yeast mitogen-activated protein kinase, Mpk1, acts to increase the supply of RACs and proteasome subunits under challenging conditions in order to maintain proteasomal degradation and cell viability. This adaptive pathway was evolutionarily conserved, with mTOR and ERK5 controlling the levels of the four mammalian RACs and proteasome abundance. Thus, the central growth and stress controllers, TORC1 and Mpk1/ERK5, endow cells with a rapid and vital adaptive response to adjust proteasome abundance in response to the rising needs of cells. Enhancing this pathway may be a useful therapeutic approach for diseases resulting from impaired proteasomal degradation. PMID:27462806

  13. Post-slippage multinucleation renders cytotoxic variation in anti-mitotic drugs that target the microtubules or mitotic spindle.

    PubMed

    Zhu, Yanting; Zhou, Yuan; Shi, Jue

    2014-01-01

    One common cancer chemotherapeutic strategy is to perturb cell division with anti-mitotic drugs. Paclitaxel, the classic microtubule-targeting anti-mitotic drug, so far still outperforms the newer, more spindle-specific anti-mitotics in the clinic, but the underlying cellular mechanism is poorly understood. In this study we identified post-slippage multinucleation, which triggered extensive DNA damage and apoptosis after drug-induced mitotic slippage, contributes to the extra cytotoxicity of paclitaxel in comparison to the spindle-targeting drug, Kinesin-5 inhibitor. Based on quantitative single-cell microscopy assays, we showed that attenuation of the degree of post-slippage multinucleation significantly reduced DNA damage and apoptosis in response to paclitaxel, and that post-slippage apoptosis was likely mediated by the p53-dependent DNA damage response pathway. Paclitaxel appeared to act as a double-edge sword, capable of killing proliferating cancer cells both during mitotic arrest and after mitotic slippage by inducing DNA damage. Our results thus suggest that to predict drug response to paclitaxel and anti-mitotics in general, 2 distinct sets of bio-markers, which regulate mitotic and post-slippage cytotoxicity, respectively, may need to be considered. Our findings provide important new insight not only for elucidating the cytotoxic mechanisms of paclitaxel, but also for understanding the variable efficacy of different anti-mitotic chemotherapeutics. PMID:24694730

  14. Optogenetic control of intracellular signaling pathways

    PubMed Central

    Zhang, Kai; Cui, Bianxiao

    2014-01-01

    Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, though useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open up exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways, and discuss future prospects for the field, including integration of new genetic approaches into optogenetics. PMID:25529484

  15. Prolonged mitotic arrest induces a caspase-dependent DNA damage response at telomeres that determines cell survival.

    PubMed

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

    2016-01-01

    A delay in the completion of metaphase induces a stress response that inhibits further cell proliferation or induces apoptosis. This response is thought to protect against genomic instability and is important for the effects of anti-mitotic cancer drugs. Here, we show that mitotic arrest induces a caspase-dependent DNA damage response (DDR) at telomeres in non-apoptotic cells. This pathway is under the control of Mcl-1 and other Bcl-2 family proteins and requires caspase-9, caspase-3/7 and the endonuclease CAD/DFF40. The gradual caspase-dependent loss of the shelterin complex protein TRF2 from telomeres promotes a DDR that involves DNA-dependent protein kinase (DNA-PK). Suppression of mitotic telomere damage by enhanced expression of TRF2, or the inhibition of either caspase-3/7 or DNA-PK during mitotic arrest, promotes subsequent cell survival. Thus, we demonstrate that mitotic stress is characterised by the sub-apoptotic activation of a classical caspase pathway, which promotes telomere deprotection, activates DNA damage signalling, and determines cell fate in response to a prolonged delay in mitosis. PMID:27230693

  16. Prolonged mitotic arrest induces a caspase-dependent DNA damage response at telomeres that determines cell survival

    PubMed Central

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

    2016-01-01

    A delay in the completion of metaphase induces a stress response that inhibits further cell proliferation or induces apoptosis. This response is thought to protect against genomic instability and is important for the effects of anti-mitotic cancer drugs. Here, we show that mitotic arrest induces a caspase-dependent DNA damage response (DDR) at telomeres in non-apoptotic cells. This pathway is under the control of Mcl-1 and other Bcl-2 family proteins and requires caspase-9, caspase-3/7 and the endonuclease CAD/DFF40. The gradual caspase-dependent loss of the shelterin complex protein TRF2 from telomeres promotes a DDR that involves DNA-dependent protein kinase (DNA-PK). Suppression of mitotic telomere damage by enhanced expression of TRF2, or the inhibition of either caspase-3/7 or DNA-PK during mitotic arrest, promotes subsequent cell survival. Thus, we demonstrate that mitotic stress is characterised by the sub-apoptotic activation of a classical caspase pathway, which promotes telomere deprotection, activates DNA damage signalling, and determines cell fate in response to a prolonged delay in mitosis. PMID:27230693

  17. Mitotic bookmarking by transcription factors

    PubMed Central

    2013-01-01

    Mitosis is accompanied by dramatic changes in chromatin organization and nuclear architecture. Transcription halts globally and most sequence-specific transcription factors and co-factors are ejected from mitotic chromatin. How then does the cell maintain its transcriptional identity throughout the cell division cycle? It has become clear that not all traces of active transcription and gene repression are erased within mitotic chromatin. Many histone modifications are stable or only partially diminished throughout mitosis. In addition, some sequence-specific DNA binding factors have emerged that remain bound to select sites within mitotic chromatin, raising the possibility that they function to transmit regulatory information through the transcriptionally silent mitotic phase, a concept that has been termed “mitotic bookmarking.” Here we review recent approaches to studying potential bookmarking factors with regards to their mitotic partitioning, and summarize emerging ideas concerning the in vivo functions of mitotically bound nuclear factors. PMID:23547918

  18. Mitotic regulation by NIMA-related kinases

    PubMed Central

    O'Regan, Laura; Blot, Joelle; Fry, Andrew M

    2007-01-01

    The NIMA-related kinases represent a family of serine/threonine kinases implicated in cell cycle control. The founding member of this family, the NIMA kinase of Aspergillus nidulans, as well as the fission yeast homologue Fin1, contribute to multiple aspects of mitotic progression including the timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Mammals contain a large family of eleven NIMA-related kinases, named Nek1 to Nek11. Of these, there is now substantial evidence that Nek2, Nek6, Nek7 and Nek9 also regulate mitotic events. At least three of these kinases, as well as NIMA and Fin1, have been localized to the microtubule organizing centre of their respective species, namely the centrosome or spindle pole body. Here, they have important functions in microtubule organization and mitotic spindle assembly. Other Nek kinases have been proposed to play microtubule-dependent roles in non-dividing cells, most notably in regulating the axonemal microtubules of cilia and flagella. In this review, we discuss the evidence that NIMA-related kinases make a significant contribution to the orchestration of mitotic progression and thereby protect cells from chromosome instability. Furthermore, we highlight their potential as novel chemotherapeutic targets. PMID:17727698

  19. Chemically diverse microtubule stabilizing agents initiate distinct mitotic defects and dysregulated expression of key mitotic kinases.

    PubMed

    Rohena, Cristina C; Peng, Jiangnan; Johnson, Tyler A; Crews, Phillip; Mooberry, Susan L

    2013-04-15

    Microtubule stabilizers are some of the most successful drugs used in the treatment of adult solid tumors and yet the molecular events responsible for their antimitotic actions are not well defined. The mitotic events initiated by three structurally and biologically diverse microtubule stabilizers; taccalonolide AJ, laulimalide/fijianolide B and paclitaxel were studied. These microtubule stabilizers cause the formation of aberrant, but structurally distinct mitotic spindles leading to the hypothesis that they differentially affect mitotic signaling. Each microtubule stabilizer initiated different patterns of expression of key mitotic signaling proteins. Taccalonolide AJ causes centrosome separation and disjunction failure to a much greater extent than paclitaxel or laulimalide, which is consistent with the distinct defects in expression and activation of Plk1 and Eg5 caused by each stabilizer. Localization studies revealed that TPX2 and Aurora A are associated with each spindle aster formed by each stabilizer. This suggests a common mechanism of aster formation. However, taccalonolide AJ also causes pericentrin accumulation on every spindle aster. The presence of pericentrin at every spindle aster initiated by taccalonolide AJ might facilitate the maintenance and stability of the highly focused asters formed by this stabilizer. Laulimalide and paclitaxel cause completely different patterns of expression and activation of these proteins, as well as phenotypically different spindle phenotypes. Delineating how diverse microtubule stabilizers affect mitotic signaling pathways could identify key proteins involved in modulating sensitivity and resistance to the antimitotic actions of these compounds. PMID:23399639

  20. Depletion of pre-mRNA splicing factor Cdc5L inhibits mitotic progression and triggers mitotic catastrophe

    PubMed Central

    Mu, R; Wang, Y-B; Wu, M; Yang, Y; Song, W; Li, T; Zhang, W-N; Tan, B; Li, A-L; Wang, N; Xia, Q; Gong, W-L; Wang, C-G; Zhou, T; Guo, N; Sang, Z-H; Li, H-Y

    2014-01-01

    Disturbing mitotic progression via targeted anti-mitotic therapy is an attractive strategy for cancer treatment. Therefore, the exploration and elucidation of molecular targets and pathways in mitosis are critical for the development of anti-mitotic drugs. Here, we show that cell division cycle 5-like (Cdc5L), a pre-mRNA splicing factor, is a regulator of mitotic progression. Depletion of Cdc5L causes dramatic mitotic arrest, chromosome misalignments and sustained activation of spindle assembly checkpoint, eventually leading to mitotic catastrophe. Moreover, these defects result from severe impairment of kinetochore-microtubule attachment and serious DNA damage. Genome-wide gene expression analysis reveals that Cdc5L modulates the expression of a set of genes involved in the mitosis and the DNA damage response. We further found that the pre-mRNA splicing efficiency of these genes were impaired when Cdc5L was knocked down. Interestingly, Cdc5L is highly expressed in cervical tumors and osteosarcoma. Finally, we demonstrate that downregulation of Cdc5L decreases the cell viability of related tumor cells. These results suggest that Cdc5L is a key regulator of mitotic progression and highlight the potential of Cdc5L as a target for cancer therapy. PMID:24675469

  1. Depletion of pre-mRNA splicing factor Cdc5L inhibits mitotic progression and triggers mitotic catastrophe.

    PubMed

    Mu, R; Wang, Y-B; Wu, M; Yang, Y; Song, W; Li, T; Zhang, W-N; Tan, B; Li, A-L; Wang, N; Xia, Q; Gong, W-L; Wang, C-G; Zhou, T; Guo, N; Sang, Z-H; Li, H-Y

    2014-01-01

    Disturbing mitotic progression via targeted anti-mitotic therapy is an attractive strategy for cancer treatment. Therefore, the exploration and elucidation of molecular targets and pathways in mitosis are critical for the development of anti-mitotic drugs. Here, we show that cell division cycle 5-like (Cdc5L), a pre-mRNA splicing factor, is a regulator of mitotic progression. Depletion of Cdc5L causes dramatic mitotic arrest, chromosome misalignments and sustained activation of spindle assembly checkpoint, eventually leading to mitotic catastrophe. Moreover, these defects result from severe impairment of kinetochore-microtubule attachment and serious DNA damage. Genome-wide gene expression analysis reveals that Cdc5L modulates the expression of a set of genes involved in the mitosis and the DNA damage response. We further found that the pre-mRNA splicing efficiency of these genes were impaired when Cdc5L was knocked down. Interestingly, Cdc5L is highly expressed in cervical tumors and osteosarcoma. Finally, we demonstrate that downregulation of Cdc5L decreases the cell viability of related tumor cells. These results suggest that Cdc5L is a key regulator of mitotic progression and highlight the potential of Cdc5L as a target for cancer therapy. PMID:24675469

  2. Mitotic Exit and Separation of Mother and Daughter Cells

    PubMed Central

    Weiss, Eric L.

    2012-01-01

    Productive cell proliferation involves efficient and accurate splitting of the dividing cell into two separate entities. This orderly process reflects coordination of diverse cytological events by regulatory systems that drive the cell from mitosis into G1. In the budding yeast Saccharomyces cerevisiae, separation of mother and daughter cells involves coordinated actomyosin ring contraction and septum synthesis, followed by septum destruction. These events occur in precise and rapid sequence once chromosomes are segregated and are linked with spindle organization and mitotic progress by intricate cell cycle control machinery. Additionally, critical parts of the mother/daughter separation process are asymmetric, reflecting a form of fate specification that occurs in every cell division. This chapter describes central events of budding yeast cell separation, as well as the control pathways that integrate them and link them with the cell cycle. PMID:23212898

  3. Regulation of midbody formation and function by mitotic kinases.

    PubMed

    D'Avino, Pier Paolo; Capalbo, Luisa

    2016-05-01

    Cytokinesis is the final phase of cell division and safeguards the correct distribution of genomic and cytoplasmic materials between the two nascent daughter cells. The final separation, or abscission, of the daughter cells depends on the proper assembly of an organelle at the intercellular bridge, the midbody, which acts as a platform for the recruitment and organisation of various proteins involved in both the control and execution of the abscission process. Recent studies have led to the identification of the mechanisms, signalling pathways and molecules that control the two tightly linked processes of midbody formation and abscission. Here we review our current knowledge of the role that mitotic kinases play in these processes and offer our perspectives on the potential future challenges that await researchers in the field. PMID:26802517

  4. Green synthesis of bacterial mediated anti-proliferative gold nanoparticles: inducing mitotic arrest (G2/M phase) and apoptosis (intrinsic pathway)

    NASA Astrophysics Data System (ADS)

    Ganesh Kumar, C.; Poornachandra, Y.; Chandrasekhar, Cheemalamarri

    2015-11-01

    The physiochemical and biological properties of microbial derived gold nanoparticles have potential applications in various biomedical domains as well as in cancer therapy. We have fabricated anti-proliferative bacterial mediated gold nanoparticles (b-Au NPs) using a culture supernatant of Streptomyces clavuligerus and later characterized them by UV-visible, TEM, DLS, XRD and FT-IR spectroscopic techniques. The capping agent responsible for the nanoparticle formation was characterized based on SDS-PAGE and MALDI-TOF-MS analyses. They were tested for anticancer activity in A549, HeLa and DU145 cell lines. The biocompatibility and non-toxic nature of the nanoparticles were tested on normal human lung cell line (MRC-5). The b-Au NPs induced the cell cycle arrest in G2/M phase and also inhibited the microtubule assembly in DU145 cells. Mechanistic studies, such as ROS, MMP, Cyt-c, GSH, caspases 9, 8 and 3 activation and the Annexin V-FITC staining, along with the above parameters tested provided sufficient evidence that the b-Au NPs induced apoptosis through the intrinsic pathway. The results supported the use of b-Au NPs for future therapeutic application in cancer therapy and other biomedical applications.The physiochemical and biological properties of microbial derived gold nanoparticles have potential applications in various biomedical domains as well as in cancer therapy. We have fabricated anti-proliferative bacterial mediated gold nanoparticles (b-Au NPs) using a culture supernatant of Streptomyces clavuligerus and later characterized them by UV-visible, TEM, DLS, XRD and FT-IR spectroscopic techniques. The capping agent responsible for the nanoparticle formation was characterized based on SDS-PAGE and MALDI-TOF-MS analyses. They were tested for anticancer activity in A549, HeLa and DU145 cell lines. The biocompatibility and non-toxic nature of the nanoparticles were tested on normal human lung cell line (MRC-5). The b-Au NPs induced the cell cycle arrest in G2

  5. Green synthesis of bacterial mediated anti-proliferative gold nanoparticles: inducing mitotic arrest (G2/M phase) and apoptosis (intrinsic pathway).

    PubMed

    Kumar, C Ganesh; Poornachandra, Y; Chandrasekhar, Cheemalamarri

    2015-11-28

    The physiochemical and biological properties of microbial derived gold nanoparticles have potential applications in various biomedical domains as well as in cancer therapy. We have fabricated anti-proliferative bacterial mediated gold nanoparticles (b-Au NPs) using a culture supernatant of Streptomyces clavuligerus and later characterized them by UV-visible, TEM, DLS, XRD and FT-IR spectroscopic techniques. The capping agent responsible for the nanoparticle formation was characterized based on SDS-PAGE and MALDI-TOF-MS analyses. They were tested for anticancer activity in A549, HeLa and DU145 cell lines. The biocompatibility and non-toxic nature of the nanoparticles were tested on normal human lung cell line (MRC-5). The b-Au NPs induced the cell cycle arrest in G2/M phase and also inhibited the microtubule assembly in DU145 cells. Mechanistic studies, such as ROS, MMP, Cyt-c, GSH, caspases 9, 8 and 3 activation and the Annexin V-FITC staining, along with the above parameters tested provided sufficient evidence that the b-Au NPs induced apoptosis through the intrinsic pathway. The results supported the use of b-Au NPs for future therapeutic application in cancer therapy and other biomedical applications. PMID:26503300

  6. The Proliferation-Quiescence Decision Is Controlled by a Bifurcation in CDK2 Activity at Mitotic Exit

    PubMed Central

    Spencer, Sabrina L.; Cappell, Steven D.; Tsai, Feng-Chiao; Overton, K. Wesley; Wang, Clifford L.; Meyer, Tobias

    2014-01-01

    SUMMARY Tissue homeostasis in metazoans is regulated by transitions of cells between quiescence and proliferation. The hallmark of proliferating populations is progression through the cell cycle, which is driven by cyclin-dependent kinase (CDK) activity. Here, we introduce a live-cell sensor for CDK2 activity and unexpectedly found that proliferating cells bifurcate into two populations as they exit mitosis. Many cells immediately commit to the next cell cycle by building up CDK2 activity from an intermediate level, while other cells lack CDK2 activity and enter a transient state of quiescence. This bifurcation is directly controlled by the CDK inhibitor p21 and is regulated by mitogens during a restriction window at the end of the previous cell cycle. Thus, cells decide at the end of mitosis to either start the next cell cycle by immediately building up CDK2 activity or to enter a transient G0-like state by suppressing CDK2 activity. PMID:24075009

  7. The Basic Leucine Zipper Domain Transcription Factor Atf1 Directly Controls Cdc13 Expression and Regulates Mitotic Entry Independently of Wee1 and Cdc25 in Schizosaccharomyces pombe

    PubMed Central

    Bandyopadhyay, Sushobhana; Dey, Isha; Suresh, Megalakshmi

    2014-01-01

    Progression into mitosis is a major point of regulation in the Schizosaccharomyces pombe cell cycle, and its proper control is essential for maintenance of genomic stability. Investigation of the G2/M progression event in S. pombe has revealed the existence of a complex regulatory process that is responsible for making the decision to enter mitosis. Newer aspects of this regulation are still being revealed. In this paper, we report the discovery of a novel mode of regulation of G2/M progression in S. pombe. We show that the mitogen-activated protein kinase (MAPK)-regulated transcription factor Atf1 is a regulator of Cdc13 (mitotic cyclin) transcription and is therefore a prominent player in the regulation of mitosis in S. pombe. We have used genetic approaches to study the effect of overexpression or deletion of Atf1 on the cell length and G2/M progression of S. pombe cells. Our results clearly show that Atf1 overexpression accelerates mitosis, leading to an accumulation of cells with shorter lengths. The previously known major regulators of entry into mitosis are the Cdc25 phosphatase and the Wee1 kinase, which modulate cyclin-dependent kinase (CDK) activity. The significantly striking aspect of our discovery is that Atf1-mediated G2/M progression is independent of both Cdc25 and Wee1. We have shown that Atf1 binds to the Cdc13 promoter, leading to activation of Cdc13 expression. This leads to enhanced nuclear localization of CDK Cdc2, thereby promoting the G2/M transition. PMID:24728197

  8. Ssp1 CaMKK: A Sensor of Actin Polarization That Controls Mitotic Commitment through Srk1 in Schizosaccharomyces pombe

    PubMed Central

    Giménez-Zaragoza, David; López-Avilés, Sandra; Yance-Chávez, Tula; Montserrat, Marta; Pujol, M. Jesús; Bachs, Oriol; Aligue, Rosa

    2015-01-01

    Background Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is required for diverse cellular functions. Mammalian CaMKK activates CaMKs and also the evolutionarily-conserved AMP-activated protein kinase (AMPK). The fission yeast Schizosaccharomyces pombe CaMKK, Ssp1, is required for tolerance to limited glucose through the AMPK, Ssp2, and for the integration of cell growth and division through the SAD kinase Cdr2. Results Here we report that Ssp1 controls the G2/M transition by regulating the activity of the CaMK Srk1. We show that inhibition of Cdc25 by Srk1 is regulated by Ssp1; and also that restoring growth polarity and actin localization of ssp1-deleted cells by removing the actin-monomer-binding protein, twinfilin, is sufficient to suppress the ssp1 phenotype. Conclusions These findings demonstrate that entry into mitosis is mediated by a network of proteins, including the Ssp1 and Srk1 kinases. Ssp1 connects the network of components that ensures proper polarity and cell size with the network of proteins that regulates Cdk1-cyclin B activity, in which Srk1 plays an inhibitory role. PMID:26575035

  9. Pathway Controlled Penetration (PcP)

    SciTech Connect

    Knight, Earl E.; Rougier, Esteban; Zubelewicz, Aleksander

    2012-08-29

    The technical approach employs advanced computational simulation tools to demonstrate how current assets can destroy RWK-RFI-12-0001's HDBT, a tunnel complex with two portals built into the base of a granite mountain. The granite over layer is assumed to be 60 meters thick over both portals and 80 meters over the facility's mission space. Key S&T is the completed development of a highly innovative viscoplastic fracture material model, 3D parallel gas-fracture capabilities into FDEM, and a stochastic handling of the material properties. Phase I - Develop and validate code simulation tools: (1) develop, incorporate and validate AZ-Frac material model for granite; and (2) Develop and incorporate gas-driven-fracture modeling into LANL's FDEM MUNROU code; (3) Develop and incorporate stochastic features into FDEM modeling. Phase II - Conduct PcP analysis on above HDBT: (1) Acquire HDBT design data, develop simulation model; and (2) Evaluate and select most promising defeat alternative. Phase III - Deliver code, train Service target analysts, and conduct simulations against real world HDBTs. PcP uses advanced computer simulations to enhance HDBT functional defeat efforts. Newly developed material models that account for fractural energy coupled with the finite discrete element methodology (FDEM) will provide targeting packages that will create penetration avenues for current or future lethality options. This novel computational approach requires full 3D geologic and structure characterization as well as significant high performance computing capabilities. The goal is to distinctively alter the targeting paradigm by leveraging critical DoD assets along with insitu geologic strata. In other words, assets will utilize underground rock structure to their benefit by creating rubbilization zones that will allow pathway controlled penetration.

  10. Erythrocytosis: the HIF pathway in control.

    PubMed

    Franke, Kristin; Gassmann, Max; Wielockx, Ben

    2013-08-15

    Organisms living under aerobic conditions need oxygen for the metabolic conversion of nutrition into energy. With the appearance of increasingly complex animals, a specialized transport system (erythrocytes) arose during evolution to provide oxygen to virtually every single cell in the body. Moreover, in case of low environmental partial pressure of oxygen, the number of erythrocytes automatically increases to preserve sustained oxygen delivery. This process relies predominantly on the cytokine erythropoietin (Epo) and its transcription factor hypoxia inducible factor (HIF), whereas the von Hippel-Lindau (VHL) ubiquitin ligase as well as the oxygen-sensitive prolyl hydroxylases (PHDs) represent essential regulators of this oxygen-sensing system. Deregulation of particular members of this pathway (eg, PHD2, HIF2α, VHL) lead to disorders in blood homeostasis as a result of insufficient (anemia) or excessive (erythrocytosis) red blood cell production. PMID:23733342

  11. RB/PLK1-dependent induced pathway by SLAMF3 expression inhibits mitosis and control hepatocarcinoma cell proliferation.

    PubMed

    Bouhlal, Hicham; Ouled-Haddou, Hakim; Debuysscher, Véronique; Singh, Amrathlal Rabbind; Ossart, Christèle; Reignier, Aline; Hocini, Hakim; Fouquet, Gregory; Al Baghami, Mohammed; Eugenio, Mélanie Simoes; Nguyen-Khac, Eric; Regimbeau, Jean-Marc; Marcq, Ingrid

    2016-03-01

    Polo-like kinase PLK1 is a cell cycle protein that plays multiple roles in promoting cell cycle progression. Among the many roles, the most prominent role of PLK1 is to regulate the mitotic spindle formation checkpoint at the M-phase. Recently we reported the expression of SLAMF3 in Hepatocytes and show that it is down regulated in tumor cells of hepatocellular carcinoma (HCC). We also show that the forced high expression level of SLAMF3 in HCC cells controls proliferation by inhibiting the MAPK ERK/JNK and the mTOR pathways. In the present study, we provide evidence that the inhibitory effect of SLAMF3 on HCC proliferation occurs through Retinoblastoma (RB) factor and PLK1-dependent pathway. In addition to the inhibition of MAPK ERK/JNK and the mTOR pathways, expression of SLAMF3 in HCC retains RB factor in its hypophosphorylated active form, which in turn inactivates E2F transcription factor, thereby repressing the expression and activation of PLK1. A clear inverse correlation was also observed between SLAMF3 and PLK expression in patients with HCC. In conclusion, the results presented here suggest that the tumor suppressor potential of SLAMF3 occurs through activation of RB that represses PLK1. We propose that the induction of a high expression level of SLAMF3 in cancerous cells could control cellular mitosis and block tumor progression. PMID:26799423

  12. RB/PLK1-dependent induced pathway by SLAMF3 expression inhibits mitosis and control hepatocarcinoma cell proliferation

    PubMed Central

    Bouhlal, Hicham; Singh, Amrathlal Rabbind; Ossart, Christèle; Reignier, Aline; Hocini, Hakim; Fouquet, Gregory; Baghami, Mohammed Al; Eugenio, Mélanie Simoes; Nguyen-Khac, Eric; Regimbeau, Jean-Marc; Marcq, Ingrid

    2016-01-01

    Polo-like kinase PLK1 is a cell cycle protein that plays multiple roles in promoting cell cycle progression. Among the many roles, the most prominent role of PLK1 is to regulate the mitotic spindle formation checkpoint at the M-phase. Recently we reported the expression of SLAMF3 in Hepatocytes and show that it is down regulated in tumor cells of hepatocellular carcinoma (HCC). We also show that the forced high expression level of SLAMF3 in HCC cells controls proliferation by inhibiting the MAPK ERK/JNK and the mTOR pathways. In the present study, we provide evidence that the inhibitory effect of SLAMF3 on HCC proliferation occurs through Retinoblastoma (RB) factor and PLK1-dependent pathway. In addition to the inhibition of MAPK ERK/JNK and the mTOR pathways, expression of SLAMF3 in HCC retains RB factor in its hypophosphorylated active form, which in turn inactivates E2F transcription factor, thereby repressing the expression and activation of PLK1. A clear inverse correlation was also observed between SLAMF3 and PLK expression in patients with HCC. In conclusion, the results presented here suggest that the tumor suppressor potential of SLAMF3 occurs through activation of RB that represses PLK1. We propose that the induction of a high expression level of SLAMF3 in cancerous cells could control cellular mitosis and block tumor progression. PMID:26799423

  13. 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.

  14. Theory of Mitotic Spindle Oscillations

    NASA Astrophysics Data System (ADS)

    Grill, Stephan W.; Kruse, Karsten; Jülicher, Frank

    2005-03-01

    During unequal cell division the mitotic spindle is positioned away from the center of the cell before cell cleavage. In many biological systems this repositioning is accompanied by oscillatory movements of the spindle. We present a theoretical description for mitotic spindle oscillations. We show that the cooperative attachment and detachment of cortical force generators to astral microtubules leads to spontaneous oscillations beyond a critical number of force generators. This mechanism can quantitatively describe the spindle oscillations observed during unequal division of the one cell stage Caenorhabditis elegans embryo.

  15. The Exon Junction Complex Controls the Efficient and Faithful Splicing of a Subset of Transcripts Involved in Mitotic Cell-Cycle Progression

    PubMed Central

    Fukumura, Kazuhiro; Wakabayashi, Shunichi; Kataoka, Naoyuki; Sakamoto, Hiroshi; Suzuki, Yutaka; Nakai, Kenta; Mayeda, Akila; Inoue, Kunio

    2016-01-01

    The exon junction complex (EJC) that is deposited onto spliced mRNAs upstream of exon–exon junctions plays important roles in multiple post-splicing gene expression events, such as mRNA export, surveillance, localization, and translation. However, a direct role for the human EJC in pre-mRNA splicing has not been fully understood. Using HeLa cells, we depleted one of the EJC core components, Y14, and the resulting transcriptome was analyzed by deep sequencing (RNA-Seq) and confirmed by RT–PCR. We found that Y14 is required for efficient and faithful splicing of a group of transcripts that is enriched in short intron-containing genes involved in mitotic cell-cycle progression. Tethering of EJC core components (Y14, eIF4AIII or MAGOH) to a model reporter pre-mRNA harboring a short intron showed that these core components are prerequisites for the splicing activation. Taken together, we conclude that the EJC core assembled on pre-mRNA is critical for efficient and faithful splicing of a specific subset of short introns in mitotic cell cycle-related genes. PMID:27490541

  16. The Exon Junction Complex Controls the Efficient and Faithful Splicing of a Subset of Transcripts Involved in Mitotic Cell-Cycle Progression.

    PubMed

    Fukumura, Kazuhiro; Wakabayashi, Shunichi; Kataoka, Naoyuki; Sakamoto, Hiroshi; Suzuki, Yutaka; Nakai, Kenta; Mayeda, Akila; Inoue, Kunio

    2016-01-01

    The exon junction complex (EJC) that is deposited onto spliced mRNAs upstream of exon-exon junctions plays important roles in multiple post-splicing gene expression events, such as mRNA export, surveillance, localization, and translation. However, a direct role for the human EJC in pre-mRNA splicing has not been fully understood. Using HeLa cells, we depleted one of the EJC core components, Y14, and the resulting transcriptome was analyzed by deep sequencing (RNA-Seq) and confirmed by RT-PCR. We found that Y14 is required for efficient and faithful splicing of a group of transcripts that is enriched in short intron-containing genes involved in mitotic cell-cycle progression. Tethering of EJC core components (Y14, eIF4AIII or MAGOH) to a model reporter pre-mRNA harboring a short intron showed that these core components are prerequisites for the splicing activation. Taken together, we conclude that the EJC core assembled on pre-mRNA is critical for efficient and faithful splicing of a specific subset of short introns in mitotic cell cycle-related genes. PMID:27490541

  17. Crosstalk between pathways enhances the controllability of signalling networks.

    PubMed

    Wang, Dingjie; Jin, Suoqin; Zou, Xiufen

    2016-02-01

    The control of complex networks is one of the most challenging problems in the fields of biology and engineering. In this study, the authors explored the controllability and control energy of several signalling networks, which consisted of many interconnected pathways, including networks with a bow-tie architecture. On the basis of the theory of structure controllability, they revealed that biological mechanisms, such as cross-pathway interactions, compartmentalisation and so on make the networks easier to fully control. Furthermore, using numerical simulations for two realistic examples, they demonstrated that the control energy of normal networks with crosstalk is lower than in networks without crosstalk. These results indicate that the biological networks are optimally designed to achieve their normal functions from the viewpoint of the control theory. The authors' work provides a comprehensive understanding of the impact of network structures and properties on controllability. PMID:26816393

  18. Influence of the circadian rhythm in cell division on radiation-induced mitotic delay in vivo

    SciTech Connect

    Rubin, N.H.

    1982-01-01

    Mitotic delay is described as a classical response to radiation; however, circadian rhythmicity in cell division in vivo has not been considered by many authors. The present study investigated the relation between fluctuations reported as mitotic delay and recovery in vivo and circadian oscillations in mitotic index in mouse corneal epithelium. One aspect involved single doses (approximately 600 rad) given to mice at different circadian stages. The normal circadian rhythm in cell division was never obliterated. Inhibition of mitosis was evident but unpredictable, ranging from 6 to 15 hr after irradiation. Recovery was evident only during the daily increase in mitotic index of controls. The classical interpretation of recovery from mitotic delay may be in an in vitro phenomenon not reflecting in vivo responses, which are apparently strongly circadian stage dependent. The second portion of the study demonstrated a dose-response effect on length of mitotic delay and, to a lesser extent, degree of recovery.

  19. Targeting Transmission Pathways for Emerging Zoonotic Disease Surveillance and Control.

    PubMed

    Loh, Elizabeth H; Zambrana-Torrelio, Carlos; Olival, Kevin J; Bogich, Tiffany L; Johnson, Christine K; Mazet, Jonna A K; Karesh, William; Daszak, Peter

    2015-07-01

    We used literature searches and a database of all reported emerging infectious diseases (EIDs) to analyze the most important transmission pathways (e.g., vector-borne, aerosol droplet transmitted) for emerging zoonoses. Our results suggest that at the broad scale, the likelihood of transmission occurring through any one pathway is approximately equal. However, the major transmission pathways for zoonoses differ widely according to the specific underlying drivers of EID events (e.g., land-use change, agricultural intensification). These results can be used to develop better targeting of surveillance for, and more effective control of newly emerged zoonoses in regions under different underlying pressures that drive disease emergence. PMID:26186515

  20. Robust mitotic entry is ensured by a latching switch

    PubMed Central

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

    2013-01-01

    Summary 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. PMID:24143279

  1. Maintaining Genome Stability in Defiance of Mitotic DNA Damage

    PubMed Central

    Ferrari, Stefano; Gentili, Christian

    2016-01-01

    The implementation of decisions affecting cell viability and proliferation is based on prompt detection of the issue to be addressed, formulation and transmission of a correct set of instructions and fidelity in the execution of orders. While the first and the last are purely mechanical processes relying on the faithful functioning of single proteins or macromolecular complexes (sensors and effectors), information is the real cue, with signal amplitude, duration, and frequency ultimately determining the type of response. The cellular response to DNA damage is no exception to the rule. In this review article we focus on DNA damage responses in G2 and Mitosis. First, we set the stage describing mitosis and the machineries in charge of assembling the apparatus responsible for chromosome alignment and segregation as well as the inputs that control its function (checkpoints). Next, we examine the type of issues that a cell approaching mitosis might face, presenting the impact of post-translational modifications (PTMs) on the correct and timely functioning of pathways correcting errors or damage before chromosome segregation. We conclude this essay with a perspective on the current status of mitotic signaling pathway inhibitors and their potential use in cancer therapy. PMID:27493659

  2. Control devices and steering strategies in pathway surgery.

    PubMed

    Fan, Chunman; Jelínek, Filip; Dodou, Dimitra; Breedveld, Paul

    2015-02-01

    For pathway surgery, that is, minimally invasive procedures carried out transluminally or through instrument-created pathways, handheld maneuverable instruments are being developed. As the accompanying control interfaces of such instruments have not been optimized for intuitive manipulation, we investigated the effect of control mode (1DoF or 2DoF), and control device (joystick or handgrip) on human performance in a navigation task. The experiments were conducted using the Endo-PaC (Endoscopic-Path Controller), a simulator that emulates the shaft and handle of a maneuverable instrument, combined with custom-developed software animating pathway surgical scenarios. Participants were asked to guide a virtual instrument without collisions toward a target located at the end of a virtual curved tunnel. The performance was assessed in terms of task completion time, path length traveled by the virtual instrument, motion smoothness, collision metrics, subjective workload, and personal preference. The results indicate that 2DoF control leads to faster task completion and fewer collisions with the tunnel wall combined with a strong subjective preference compared with 1DoF control. Handgrip control appeared to be more intuitive to master than joystick control. However, the participants experienced greater physical demand and had longer path lengths with handgrip than joystick control. PMID:25438958

  3. A controlled vocabulary for pathway entities and events.

    PubMed

    Jupe, Steve; Jassal, Bijay; Williams, Mark; Wu, Guanming

    2014-01-01

    Entities involved in pathways and the events they participate in require descriptive and unambiguous names that are often not available in the literature or elsewhere. Reactome is a manually curated open-source resource of human pathways. It is accessible via a website, available as downloads in standard reusable formats and via Representational State Transfer (REST)-ful and Simple Object Access Protocol (SOAP) application programming interfaces (APIs). We have devised a controlled vocabulary (CV) that creates concise, unambiguous and unique names for reactions (pathway events) and all the molecular entities they involve. The CV could be reapplied in any situation where names are used for pathway entities and events. Adoption of this CV would significantly improve naming consistency and readability, with consequent benefits for searching and data mining within and between databases. Database URL: http://www.reactome.org. PMID:24951798

  4. Asymmetrically dividing Drosophila neuroblasts utilize two spatially and temporally independent cytokinesis pathways

    PubMed Central

    Roth, Michaela; Roubinet, Chantal; Iffländer, Niklas; Ferrand, Alexia; Cabernard, Clemens

    2015-01-01

    Precise cleavage furrow positioning is required for faithful chromosome segregation and cell fate determinant distribution. In most metazoan cells, contractile ring placement is regulated by the mitotic spindle through the centralspindlin complex, and potentially also the chromosomal passenger complex (CPC). Drosophila neuroblasts, asymmetrically dividing neural stem cells, but also other cells utilize both spindle-dependent and spindle-independent cleavage furrow positioning pathways. However, the relative contribution of each pathway towards cytokinesis is currently unclear. Here we report that in Drosophila neuroblasts, the mitotic spindle, but not polarity cues, controls the localization of the CPC component Survivin. We also show that Survivin and the mitotic spindle are required to stabilize the position of the cleavage furrow in late anaphase and to complete furrow constriction. These results support the model that two spatially and temporally separate pathways control different key aspects during asymmetric cell division, ensuring correct cell fate determinant segregation and neuroblast self-renewal. PMID:25791062

  5. Atomic Force Microscopy to Study Mechanics of Living Mitotic Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Toyoda, Yusuke; Stewart, Martin P.; Hyman, Anthony A.; Müller, Daniel J.

    2011-08-01

    While biochemical pathways within mitotic cells have been intensively studied, the mechanics of dividing cells is only poorly understood. In our recent report, an experimental system combining fluorescence and atomic force microscopy was set up to study dynamics of mitotic rounding of mammalian cells. We show that cells have a rounding pressure that increases upon mitotic entry. Using specific inhibitors or perturbations, we revealed biological processes required for force generation that underpin the cell rounding shape change during mitosis. The significance of the finding and an outlook are discussed.

  6. 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

  7. CDK1 Phosphorylation of YAP Promotes Mitotic Defects and Cell Motility and Is Essential for Neoplastic Transformation

    PubMed Central

    Yang, Shuping; Zhang, Lin; Liu, Miao; Chong, Rong; Ding, Shi-Jian; Chen, Yuanhong; Dong, Jixin

    2013-01-01

    The Yes-associated protein YAP is a downstream effector of the Hippo pathway of cell cycle control which plays important roles in tumorigenesis. Hippo-mediated phosphorylation YAP, mainly at S127, inactivates YAP function. In this study, we define a mechanism for positive regulation of YAP activity that is critical for its oncogenic function. Specifically, we found that YAP is phosphorylated in vitro and in vivo by the cell cycle kinase CDK1 at T119, S289, and S367 during G2/M phase of the cell cycle. We also found that ectopic expression of a phosphomimetic YAP mutant (YAP3D, harboring T119D/S289D/S367D) was sufficient to induce mitotic defects in immortalized epithelial cells, including centrosome amplification, multipolar spindles and chromosome missegregation. Finally, we documented that mitotic phosphorylation of YAP was sufficient to promote cell migration and invasion in a manner essential for neoplastic cell transformation. In support of our findings, CDK1 inhibitors largely suppressed cell motility mediated by activated YAP-S127A but not the phosphomimetic mutant YAP3D. Collectively, our results reveal a previously unrecognized mechanism for controlling the activity of YAP that is crucial for its oncogenic function mediated by mitotic dysregulation. PMID:24101154

  8. Structure-biological function relationship extended to mitotic arrest-deficient 2-like protein Mad2 native and mutants-new opportunity for genetic disorder control.

    PubMed

    Avram, Speranta; Milac, Adina; Mernea, Maria; Mihailescu, Dan; Putz, Mihai V; Buiu, Catalin

    2014-01-01

    Overexpression of mitotic arrest-deficient proteins Mad1 and Mad2, two components of spindle assembly checkpoint, is a risk factor for chromosomal instability (CIN) and a trigger of many genetic disorders. Mad2 transition from inactive open (O-Mad2) to active closed (C-Mad2) conformations or Mad2 binding to specific partners (cell-division cycle protein 20 (Cdc20) or Mad1) were targets of previous pharmacogenomics studies. Here, Mad2 binding to Cdc20 and the interconversion rate from open to closed Mad2 were predicted and the molecular features with a critical contribution to these processes were determined by extending the quantitative structure-activity relationship (QSAR) method to large-size proteins such as Mad2. QSAR models were built based on available published data on 23 Mad2 mutants inducing CIN-related functional changes. The most relevant descriptors identified for predicting Mad2 native and mutants action mechanism and their involvement in genetic disorders are the steric (van der Waals area and solvent accessible area and their subdivided) and energetic van der Waals energy descriptors. The reliability of our QSAR models is indicated by significant values of statistical coefficients: Cross-validated correlation q2 (0.53-0.65) and fitted correlation r2 (0.82-0.90). Moreover, based on established QSAR equations, we rationally design and analyze nine de novo Mad2 mutants as possible promoters of CIN. PMID:25411801

  9. Control systems and coordination protocols of the secretory pathway.

    PubMed

    Luini, Alberto; Mavelli, Gabriella; Jung, Juan; Cancino, Jorge

    2014-01-01

    Like other cellular modules, the secretory pathway and the Golgi complex are likely to be supervised by control systems that support homeostasis and optimal functionality under all conditions, including external and internal perturbations. Moreover, the secretory apparatus must be functionally connected with other cellular modules, such as energy metabolism and protein degradation, via specific rules of interaction, or "coordination protocols". These regulatory devices are of fundamental importance for optimal function; however, they are generally "hidden" at steady state. The molecular components and the architecture of the control systems and coordination protocols of the secretory pathway are beginning to emerge through studies based on the use of controlled transport-specific perturbations aimed specifically at the detection and analysis of these internal regulatory devices. PMID:25374666

  10. Regulation of mitotic spindle orientation: an integrated view.

    PubMed

    di Pietro, Florencia; Echard, Arnaud; Morin, Xavier

    2016-08-01

    Mitotic spindle orientation is essential for cell fate decisions, epithelial maintenance, and tissue morphogenesis. In most animal cell types, the dynein motor complex is anchored at the cell cortex and exerts pulling forces on astral microtubules to position the spindle. Early studies identified the evolutionarily conserved Gαi/LGN/NuMA complex as a key regulator that polarizes cortical force generators. In recent years, a combination of genetics, biochemistry, modeling, and live imaging has contributed to decipher the mechanisms of spindle orientation. Here, we highlight the dynamic nature of the assembly of this complex and discuss the molecular regulation of its localization. Remarkably, a number of LGN-independent mechanisms were described recently, whereas NuMA remains central in most pathways involved in recruiting force generators at the cell cortex. We also describe the emerging role of the actin cortex in spindle orientation and discuss how dynamic astral microtubule formation is involved. We further give an overview on instructive external signals that control spindle orientation in tissues. Finally, we discuss the influence of cell geometry and mechanical forces on spindle orientation. PMID:27432284

  11. Engineering Heteromaterials to Control Lithium Ion Transport Pathways

    DOE PAGESBeta

    Liu, Yang; Vishniakou, Siarhei; Yoo, Jinkyoung; Dayeh, Shadi A.

    2015-12-21

    Safe and efficient operation of lithium ion batteries requires precisely directed flow of lithium ions and electrons to control the first directional volume changes in anode and cathode materials. Understanding and controlling the lithium ion transport in battery electrodes becomes crucial to the design of high performance and durable batteries. Recent work revealed that the chemical potential barriers encountered at the surfaces of heteromaterials play an important role in directing lithium ion transport at nanoscale. Here, we utilize in situ transmission electron microscopy to demonstrate that we can switch lithiation pathways from radial to axial to grain-by-grain lithiation through themore » systematic creation of heteromaterial combinations in the Si-Ge nanowire system. Lastly, our systematic studies show that engineered materials at nanoscale can overcome the intrinsic orientation-dependent lithiation, and open new pathways to aid in the development of compact, safe, and efficient batteries.« less

  12. Engineering Heteromaterials to Control Lithium Ion Transport Pathways

    SciTech Connect

    Liu, Yang; Vishniakou, Siarhei; Yoo, Jinkyoung; Dayeh, Shadi A.

    2015-12-21

    Safe and efficient operation of lithium ion batteries requires precisely directed flow of lithium ions and electrons to control the first directional volume changes in anode and cathode materials. Understanding and controlling the lithium ion transport in battery electrodes becomes crucial to the design of high performance and durable batteries. Recent work revealed that the chemical potential barriers encountered at the surfaces of heteromaterials play an important role in directing lithium ion transport at nanoscale. Here, we utilize in situ transmission electron microscopy to demonstrate that we can switch lithiation pathways from radial to axial to grain-by-grain lithiation through the systematic creation of heteromaterial combinations in the Si-Ge nanowire system. Lastly, our systematic studies show that engineered materials at nanoscale can overcome the intrinsic orientation-dependent lithiation, and open new pathways to aid in the development of compact, safe, and efficient batteries.

  13. Engineering Heteromaterials to Control Lithium Ion Transport Pathways

    PubMed Central

    Liu, Yang; Vishniakou, Siarhei; Yoo, Jinkyoung; Dayeh, Shadi A.

    2015-01-01

    Safe and efficient operation of lithium ion batteries requires precisely directed flow of lithium ions and electrons to control the first directional volume changes in anode and cathode materials. Understanding and controlling the lithium ion transport in battery electrodes becomes crucial to the design of high performance and durable batteries. Recent work revealed that the chemical potential barriers encountered at the surfaces of heteromaterials play an important role in directing lithium ion transport at nanoscale. Here, we utilize in situ transmission electron microscopy to demonstrate that we can switch lithiation pathways from radial to axial to grain-by-grain lithiation through the systematic creation of heteromaterial combinations in the Si-Ge nanowire system. Our systematic studies show that engineered materials at nanoscale can overcome the intrinsic orientation-dependent lithiation, and open new pathways to aid in the development of compact, safe, and efficient batteries. PMID:26686655

  14. Engineering Heteromaterials to Control Lithium Ion Transport Pathways

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Vishniakou, Siarhei; Yoo, Jinkyoung; Dayeh, Shadi A.

    2015-12-01

    Safe and efficient operation of lithium ion batteries requires precisely directed flow of lithium ions and electrons to control the first directional volume changes in anode and cathode materials. Understanding and controlling the lithium ion transport in battery electrodes becomes crucial to the design of high performance and durable batteries. Recent work revealed that the chemical potential barriers encountered at the surfaces of heteromaterials play an important role in directing lithium ion transport at nanoscale. Here, we utilize in situ transmission electron microscopy to demonstrate that we can switch lithiation pathways from radial to axial to grain-by-grain lithiation through the systematic creation of heteromaterial combinations in the Si-Ge nanowire system. Our systematic studies show that engineered materials at nanoscale can overcome the intrinsic orientation-dependent lithiation, and open new pathways to aid in the development of compact, safe, and efficient batteries.

  15. 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

  16. Closed MAD2 (C-MAD2) is selectively incorporated into the mitotic checkpoint complex (MCC)

    PubMed Central

    Tipton, Aaron R; Tipton, Michael; Yen, Tim

    2011-01-01

    The mitotic checkpoint is a specialized signal transduction pathway that monitors kinetochore-microtubule attachment to achieve faithful chromosome segregation. MAD2 is an evolutionarily conserved mitotic checkpoint protein that exists in open (O) and closed (C) conformations. The increase of intracellular C-MAD2 level during mitosis, through O→C-MAD2 conversion as catalyzed by unattached kinetochores, is a critical signaling event for the mitotic checkpoint. However, it remains controversial whether MAD2 is an integral component of the effector of the mitotic checkpoint—the mitotic checkpoint complex (MCC). We show here that endogenous human MCC is assembled by first forming a BUBR1:BUB3:CDC20 complex in G2 and then selectively incorporating C-MAD2 during mitosis. Nevertheless, MCC can be induced to form in G1/S cells by expressing a C-conformation locked MAD2 mutant, indicating intracellular level of C-MAD2 as a major limiting factor for MCC assembly. In addition, a recombinant MCC containing C-MAD2 exhibits effective inhibitory activity toward APC/C isolated from mitotic HeLa cells, while a recombinant BUBR1:BUB3:CDC20 ternary complex is ineffective at comparable concentrations despite association with APC/C. These results help establish a direct connection between a major signal transducer (C-MAD2) and the potent effector (MCC) of the mitotic checkpoint, and provide novel insights into protein-protein interactions during assembly of a functional MCC. PMID:22037211

  17. Axin localizes to mitotic spindles and centrosomes in mitotic cells

    SciTech Connect

    Kim, Shi-Mun; Choi, Eun-Jin; Song, Ki-Joon; Kim, Sewoon; Seo, Eunjeong; Jho, Eek-Hoon; Kee, Sun-Ho

    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 in 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.

  18. Single-walled carbon nanotube-induced mitotic disruption⋆

    PubMed Central

    Sargent, L.M.; Hubbs, A.F.; Young, S.-H.; Kashon, M.L.; Dinu, C.Z.; Salisbury, J.L.; Benkovic, S.A.; Lowry, D.T.; Murray, A.R.; Kisin, E.R.; Siegrist, K.J.; Battelli, L.; Mastovich, J.; Sturgeon, J.L.; Bunker, K.L.; Shvedova, A.A.; Reynolds, S.H.

    2015-01-01

    Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 μg/cm2 single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24–72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 μg/cm2 SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24 h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter

  19. Organ Size Control by Hippo and TOR Pathways

    PubMed Central

    Tumaneng, Karen; Russell, Ryan C.; Guan, Kun-Liang

    2013-01-01

    The determination of final organ size is a highly coordinated and complex process that relies on the precise regulation of cell number and/or cell size. Perturbation of organ size control contributes to many human diseases, including hypertrophy, degenerative diseases, and cancer. Hippo and TOR are among the key signaling pathways involved in the regulation of organ size through their respective functions in the regulation of cell number and cell size. Here, we review the general mechanisms that regulate organ growth, describe how Hippo and TOR control key aspects of growth, and discuss recent findings that highlight a possible coordination between Hippo and TOR in organ size regulation. PMID:22575479

  20. A comprehensive model to predict mitotic division in budding yeasts

    PubMed Central

    Sutradhar, Sabyasachi; Yadav, Vikas; Sridhar, Shreyas; Sreekumar, Lakshmi; Bhattacharyya, Dibyendu; Ghosh, Santanu Kumar; Paul, Raja; Sanyal, Kaustuv

    2015-01-01

    High-fidelity chromosome segregation during cell division depends on a series of concerted interdependent interactions. Using a systems biology approach, we built a robust minimal computational model to comprehend mitotic events in dividing budding yeasts of two major phyla: Ascomycota and Basidiomycota. This model accurately reproduces experimental observations related to spindle alignment, nuclear migration, and microtubule (MT) dynamics during cell division in these yeasts. The model converges to the conclusion that biased nucleation of cytoplasmic microtubules (cMTs) is essential for directional nuclear migration. Two distinct pathways, based on the population of cMTs and cortical dyneins, differentiate nuclear migration and spindle orientation in these two phyla. In addition, the model accurately predicts the contribution of specific classes of MTs in chromosome segregation. Thus we present a model that offers a wider applicability to simulate the effects of perturbation of an event on the concerted process of the mitotic cell division. PMID:26310442

  1. Microelasticity of Single Mitotic Chromosomes

    NASA Astrophysics Data System (ADS)

    Poirier, Michael; Eroglu, Sertac; Chatenay, Didier; Marko, John F.; Hirano, Tatsuya

    2000-03-01

    The force-extension behavior of mitotic chromosomes from the newt TVI tumor cell line was studied using micropipette manipulation and force measuring techniques. Reversible, linear elastic response was observed for extensions up to 5 times the native length; the force required to double chromosome length was 1 nanonewton (nN). For further elongations, the linear response teminates at a force plateau of 15 nN and at an extension of 20x. Beyond this extension, the chromosome breaks at elongations between 20x and 70x. These results will be compared to the similar behavior of mitotic chromosomes from explanted newt cells (Poirier, Eroglu, Chatenay and Marko, Mol. Biol. Cell, in press). Also, the effect of biochemical modifications on the elasticity was studied. Ethidium Bromide, which binds to DNA, induces up to a 10 times increase in the Young's modulus. Anti-XCAP-E, which binds to a putative chromosome folding protein, induces up to a 2 times increase in the Young's modulus. Preliminary results on the dynamical relaxation of chromosomes will also be presented. Support of this research through a Biomedical Engineering Research Grant from The Whitaker Foundation is gratefully acknowledged.

  2. Excess of miRNA-378a-5p perturbs mitotic fidelity and correlates with breast cancer tumourigenesis in vivo

    PubMed Central

    Winsel, S; Mäki-Jouppila, J; Tambe, M; Aure, M R; Pruikkonen, S; Salmela, A-L; Halonen, T; Leivonen, S-K; Kallio, L; Børresen-Dale, A-L; Kallio, M J

    2014-01-01

    Background: Optimal expression and proper function of key mitotic proteins facilitate control and repair processes that aim to prevent loss or gain of chromosomes, a hallmark of cancer. Altered expression of small regulatory microRNAs is associated with tumourigenesis and metastasis but the impact on mitotic signalling has remained unclear. Methods: Cell-based high-throughput screen identified miR-378a-5p as a mitosis perturbing microRNA. Transient transfections, immunofluorescence, western blotting, time-lapse microscopy, FISH and reporter assays were used to characterise the mitotic anomalies by excess miR-378a-5p. Analysis of microRNA profiles in breast tumours was performed. Results: Overexpression of miR-378a-5p induced numerical chromosome changes in cells and abrogated taxol-induced mitotic block via premature inactivation of the spindle assembly checkpoint. Moreover, excess miR-378a-5p triggered receptor tyrosine kinase–MAP kinase pathway signalling, and was associated with suppression of Aurora B kinase. In breast cancer in vivo, we found that high miR-378a-5p levels correlate with the most aggressive, poorly differentiated forms of cancer. Interpretation: Downregulation of Aurora B by excess miR-378a-5p can explain the observed microtubule drug resistance and increased chromosomal imbalance in the microRNA-overexpressing cells. The results suggest that breast tumours may deploy high miR-378a-5p levels to gain growth advantage and antagonise taxane therapy. PMID:25268374

  3. OTSSP167 Abrogates Mitotic Checkpoint through Inhibiting Multiple Mitotic Kinases.

    PubMed

    Ji, Wenbin; Arnst, Christopher; Tipton, Aaron R; Bekier, Michael E; Taylor, William R; Yen, Tim J; Liu, Song-Tao

    2016-01-01

    OTSSP167 was recently characterized as a potent inhibitor for maternal embryonic leucine zipper kinase (MELK) and is currently tested in Phase I clinical trials for solid tumors that have not responded to other treatment. Here we report that OTSSP167 abrogates the mitotic checkpoint at concentrations used to inhibit MELK. The abrogation is not recapitulated by RNAi mediated silencing of MELK in cells. Although OTSSP167 indeed inhibits MELK, it exhibits off-target activity against Aurora B kinase in vitro and in cells. Furthermore, OTSSP167 inhibits BUB1 and Haspin kinases, reducing phosphorylation at histones H2AT120 and H3T3 and causing mislocalization of Aurora B and associated chromosomal passenger complex from the centromere/kinetochore. The results suggest that OTSSP167 may have additional mechanisms of action for cancer cell killing and caution the use of OTSSP167 as a MELK specific kinase inhibitor in biochemical and cellular assays. PMID:27082996

  4. OTSSP167 Abrogates Mitotic Checkpoint through Inhibiting Multiple Mitotic Kinases

    PubMed Central

    Tipton, Aaron R.; Bekier, Michael E.; Taylor, William R.; Yen, Tim J.; Liu, Song-Tao

    2016-01-01

    OTSSP167 was recently characterized as a potent inhibitor for maternal embryonic leucine zipper kinase (MELK) and is currently tested in Phase I clinical trials for solid tumors that have not responded to other treatment. Here we report that OTSSP167 abrogates the mitotic checkpoint at concentrations used to inhibit MELK. The abrogation is not recapitulated by RNAi mediated silencing of MELK in cells. Although OTSSP167 indeed inhibits MELK, it exhibits off-target activity against Aurora B kinase in vitro and in cells. Furthermore, OTSSP167 inhibits BUB1 and Haspin kinases, reducing phosphorylation at histones H2AT120 and H3T3 and causing mislocalization of Aurora B and associated chromosomal passenger complex from the centromere/kinetochore. The results suggest that OTSSP167 may have additional mechanisms of action for cancer cell killing and caution the use of OTSSP167 as a MELK specific kinase inhibitor in biochemical and cellular assays. PMID:27082996

  5. Rab11-endosomes contribute to mitotic spindle orientation

    PubMed Central

    Hehnly, Heidi; Doxsey, Stephen

    2014-01-01

    During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is known about Rab11 and endosomes in mitosis. Here we show that Rab11 localizes to the mitotic spindle and regulates dynein-dependent endosome localization at poles. We found that mitotic recycling endosomes bind γ-TuRC components and associate with tubulin in vitro. Rab11-depletion or dominant-negative Rab11 expression disrupts astral microtubules, delays mitosis, and redistributes spindle pole proteins. Reciprocally, constitutively-active Rab11 increases astral microtubules, restores γ-tubulin spindle pole localization and generates robust spindles. This suggests a fundamental role for Rab11 activity in spindle pole maturation during mitosis. Rab11 depletion causes misorientation of the mitotic spindle and the plane of cell division. These findings suggest a molecular mechanism for the organization of astral microtubules and the mitotic spindle through Rab11-dependent control of spindle pole assembly and function. We propose that Rab11 and its associated endosomes co-contribute to these processes through retrograde transport to poles by dynein. PMID:24561039

  6. Rab11 endosomes contribute to mitotic spindle organization and orientation.

    PubMed

    Hehnly, Heidi; Doxsey, Stephen

    2014-03-10

    During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is known about Rab11 endosomes in mitosis. Here, we show that Rab11 localizes to the mitotic spindle and regulates dynein-dependent endosome localization at poles. We found that mitotic recycling endosomes bind γ-TuRC components and associate with tubulin in vitro. Rab11 depletion or dominant-negative Rab11 expression disrupts astral microtubules, delays mitosis, and redistributes spindle pole proteins. Reciprocally, constitutively active Rab11 increases astral microtubules, restores γ-tubulin spindle pole localization, and generates robust spindles. This suggests a role for Rab11 activity in spindle pole maturation during mitosis. Rab11 depletion causes misorientation of the mitotic spindle and the plane of cell division. These findings suggest a molecular mechanism for the organization of astral microtubules and the mitotic spindle through Rab11-dependent control of spindle pole assembly and function. We propose that Rab11 and its associated endosomes cocontribute to these processes through retrograde transport to poles by dynein. PMID:24561039

  7. 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

  8. 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. PMID:27114510

  9. Cell-cycle-regulated localization of tyrosine and threonine phosphoepitopes at the kinetochores of mitotic chromosomes.

    PubMed

    Taagepera, S; Campbell, M S; Gorbsky, G J

    1995-11-01

    We have detected novel phosphotyrosine epitopes at the kinetochores of mitotic chromosomes in rat kangaroo PtK1 and mouse P388D1 tissue culture cells. Immunofluorescence labeling of detergent-resistant cytoskeletons reveals that these phosphotyrosine epitopes are tightly bound at the centrosomes and kinetochores of mitotic cells. These phosphoepitopes are found at the kinetochores during only prophase and prometaphase. Inclusion of a mixture of phosphatase inhibitors in the cell extraction procedure was necessary to preserve these previously undetected phosphotyrosine epitopes. The use of the phosphatase inhibitor mixture also improved the detection of the centrosome and kinetochore antigens recognized by the monoclonal antibody MPM-2. The MPM-2 antibody labels a subset of phosphothreonine-containing antigens found primarily during M phase. Ultrastructural immunolabeling studies indicated that both the phosphotyrosine and the MPM-2 phosphoepitopes were contained in both the outer and the inner dense plaques of the kinetochore. We developed large-scale chromosome isolation procedures designed to maintain chromosome protein phosphorylation. Immunoblot analysis revealed that the phosphotyrosine and MPM-2 antibodies recognized a number of chromosomal proteins, some of which were concentrated in the chromosome scaffold fraction prepared by nuclease digestion and salt extraction of whole chromosomes. The strictly regulated appearance of the phosphotyrosine and MPM-2 epitopes at the kinetochores of chromosomes during various stages of mitosis suggests that these phosphoepitopes may be involved in signal transduction pathways controlling kinetochore assembly and function during mitosis. PMID:7589252

  10. Mitotic chromosome condensation in vertebrates

    SciTech Connect

    Vagnarelli, Paola

    2012-07-15

    Work from several laboratories over the past 10-15 years has revealed that, within the interphase nucleus, chromosomes are organized into spatially distinct territories [T. Cremer, C. Cremer, Chromosome territories, nuclear architecture and gene regulation in mammalian cells, Nat. Rev. Genet. 2 (2001) 292-301 and T. Cremer, M. Cremer, S. Dietzel, S. Muller, I. Solovei, S. Fakan, Chromosome territories-a functional nuclear landscape, Curr. Opin. Cell Biol. 18 (2006) 307-316]. The overall compaction level and intranuclear location varies as a function of gene density for both entire chromosomes [J.A. Croft, J.M. Bridger, S. Boyle, P. Perry, P. Teague,W.A. Bickmore, Differences in the localization and morphology of chromosomes in the human nucleus, J. Cell Biol. 145 (1999) 1119-1131] and specific chromosomal regions [N.L. Mahy, P.E. Perry, S. Gilchrist, R.A. Baldock, W.A. Bickmore, Spatial organization of active and inactive genes and noncoding DNA within chromosome territories, J. Cell Biol. 157 (2002) 579-589] (Fig. 1A, A'). In prophase, when cyclin B activity reaches a high threshold, chromosome condensation occurs followed by Nuclear Envelope Breakdown (NEB) [1]. At this point vertebrate chromosomes appear as compact structures harboring an attachment point for the spindle microtubules physically recognizable as a primary constriction where the two sister chromatids are held together. The transition from an unshaped interphase chromosome to the highly structured mitotic chromosome (compare Figs. 1A and B) has fascinated researchers for several decades now; however a definite picture of how this process is achieved and regulated is not yet in our hands and it will require more investigation to comprehend the complete process. From a biochemical point of view a vertebrate mitotic chromosomes is composed of DNA, histone proteins (60%) and non-histone proteins (40%) [6]. I will discuss below what is known to date on the contribution of these two different classes of

  11. Control the kinetics and pathway of insulin fibril formation

    NASA Astrophysics Data System (ADS)

    Zheng, Zhongli; Jing, Benxin; Zhu, Y. Elaine

    2012-02-01

    Protein fibrils have been proposed as possible toxic agents for many amyloid related diseases, such as Alzheimer's disease, however the reaction pathway toward the amyloid fibrillation remain inadequately understood. In this work, we examine the conformational transition of human insulin as the model amyloid protein by single-molecule fluorescence spectroscopy and imaging. By controlling the pH cycling, insulin monomer and oligomers are indentified at given pH variation condition. Furthermore, low frequency ac-electric fields are employed to control the insulin aggregation from its monomers in a microchannel. It is observed that lag time to induce insulin fibrillation can be significantly shortened, in compassion to the commonly used cooling and seeding methods, and exhibits a strong dependence on applied ac-field strength. Additionally, the structure of insulin aggregates under ac-electric fields is observed to be drastically different from that under the temperature control.

  12. Light-Mediated Remote Control of Signaling Pathways

    PubMed Central

    Priestman, Melanie A.; Lawrence, David S.

    2009-01-01

    Summary Cell signaling networks display an extraordinary range of temporal and spatial plasticity. Our programmatic approach focuses on the construction of intracellular probes, including sensors, inhibitors, and functionally unique proteins that can be temporally and spatially controlled by the investigator even after they have entered the cell. We have designed and evaluated protein kinase sensors that furnish a fluorescent readout upon phosphorylation. In addition, since the sensors are inert (i.e. cannot be phosphorylated) until activated by light, they can be carried through the various stages of any given cell-based behavior without being consumed. Using this strategy, we have shown that PKCβ is essential for nuclear envelope breakdown and thus the transition from prophase to metaphase in actively dividing cells. Photoactivatable proteins furnish the means to initiate cellular signaling pathways with a high degree of spatial and temporal control. We have used this approach to demonstrate that cofilin serves as a component of the steering apparatus of the cell. Finally, inhibitors are commonly used to assess the participation of specific enzymes in signaling pathways that control cellular behavior. We have constructed a photo-deactivatable inhibitor, an inhibitory species that can be switched off with light. In the absence of light, the target enzyme is inactive due to the presence of the potent inhibitory molecule. Upon photolysis, the inhibitory molecule is destroyed and enzymatic activity is released. PMID:19765679

  13. Metabolic control of YAP and TAZ by the mevalonate pathway.

    PubMed

    Sorrentino, Giovanni; Ruggeri, Naomi; Specchia, Valeria; Cordenonsi, Michelangelo; Mano, Miguel; Dupont, Sirio; Manfrin, Andrea; Ingallina, Eleonora; Sommaggio, Roberta; Piazza, Silvano; Rosato, Antonio; Piccolo, Stefano; Del Sal, Giannino

    2014-04-01

    The YAP and TAZ mediators of the Hippo pathway (hereafter called YAP/TAZ) promote tissue proliferation and organ growth. However, how their biological properties intersect with cellular metabolism remains unexplained. Here, we show that YAP/TAZ activity is controlled by the SREBP/mevalonate pathway. Inhibition of the rate-limiting enzyme of this pathway (HMG-CoA reductase) by statins opposes YAP/TAZ nuclear localization and transcriptional responses. Mechanistically, the geranylgeranyl pyrophosphate produced by the mevalonate cascade is required for activation of Rho GTPases that, in turn, activate YAP/TAZ by inhibiting their phosphorylation and promoting their nuclear accumulation. The mevalonate-YAP/TAZ axis is required for proliferation and self-renewal of breast cancer cells. In Drosophila melanogaster, inhibition of mevalonate biosynthesis and geranylgeranylation blunts the eye overgrowth induced by Yorkie, the YAP/TAZ orthologue. In tumour cells, YAP/TAZ activation is promoted by increased levels of mevalonic acid produced by SREBP transcriptional activity, which is induced by its oncogenic cofactor mutant p53. These findings reveal an additional layer of YAP/TAZ regulation by metabolic cues. PMID:24658687

  14. Nanoparticle hardness controls the internalization pathway for drug delivery

    NASA Astrophysics Data System (ADS)

    Li, Ye; Zhang, Xianren; Cao, Dapeng

    2015-01-01

    Nanoparticle (NP)-based drug delivery systems offer fundamental advantages over current therapeutic agents that commonly display a longer circulation time, lower toxicity, specific targeted release, and greater bioavailability. For successful NP-based drug delivery it is essential that the drug-carrying nanocarriers can be internalized by the target cells and transported to specific sites, and the inefficient internalization of nanocarriers is often one of the major sources for drug resistance. In this work, we use the dissipative particle dynamics simulation to investigate the effect of NP hardness on their internalization efficiency. Three simplified models of NP platforms for drug delivery, including polymeric NP, liposome and solid NP, are designed here to represent increasing nanocarrier hardness. Simulation results indicate that NP hardness controls the internalization pathway for drug delivery. Rigid NPs can enter the cell by a pathway of endocytosis, whereas for soft NPs the endocytosis process can be inhibited or frustrated due to wrapping-induced shape deformation and non-uniform ligand distribution. Instead, soft NPs tend to find one of three penetration pathways to enter the cell membrane via rearranging their hydrophobic and hydrophilic segments. Finally, we show that the interaction between nanocarriers and drug molecules is also essential for effective drug delivery.

  15. Mice produced by mitotic reprogramming of sperm injected into haploid parthenogenotes.

    PubMed

    Suzuki, Toru; Asami, Maki; Hoffmann, Martin; Lu, Xin; Gužvić, Miodrag; Klein, Christoph A; Perry, Anthony C F

    2016-01-01

    Sperm are highly differentiated and the activities that reprogram them for embryonic development during fertilization have historically been considered unique to the oocyte. We here challenge this view and demonstrate that mouse embryos in the mitotic cell cycle can also directly reprogram sperm for full-term development. Developmentally incompetent haploid embryos (parthenogenotes) injected with sperm developed to produce healthy offspring at up to 24% of control rates, depending when in the embryonic cell cycle injection took place. This implies that most of the first embryonic cell cycle can be bypassed in sperm genome reprogramming for full development. Remodelling of histones and genomic 5'-methylcytosine and 5'-hydroxymethylcytosine following embryo injection were distinct from remodelling in fertilization and the resulting 2-cell embryos consistently possessed abnormal transcriptomes. These studies demonstrate plasticity in the reprogramming of terminally differentiated sperm nuclei and suggest that different epigenetic pathways or kinetics can establish totipotency. PMID:27623537

  16. Viral Evasion and Manipulation of Host RNA Quality Control Pathways.

    PubMed

    Hogg, J Robert

    2016-08-15

    Viruses have evolved diverse strategies to maximize the functional and coding capacities of their genetic material. Individual viral RNAs are often used as substrates for both replication and translation and can contain multiple, sometimes overlapping open reading frames. Further, viral RNAs engage in a wide variety of interactions with both host and viral proteins to modify the activities of important cellular factors and direct their own trafficking, packaging, localization, stability, and translation. However, adaptations increasing the information density of small viral genomes can have unintended consequences. In particular, viral RNAs have developed features that mark them as potential targets of host RNA quality control pathways. This minireview focuses on ways in which viral RNAs run afoul of the cellular mRNA quality control and decay machinery, as well as on strategies developed by viruses to circumvent or exploit cellular mRNA surveillance. PMID:27226372

  17. Controlling a spillover pathway with the molecular cork effect

    NASA Astrophysics Data System (ADS)

    Marcinkowski, Matthew D.; Jewell, April D.; Stamatakis, Michail; Boucher, Matthew B.; Lewis, Emily A.; Murphy, Colin J.; Kyriakou, Georgios; Sykes, E. Charles H.

    2013-06-01

    Spillover of reactants from one active site to another is important in heterogeneous catalysis and has recently been shown to enhance hydrogen storage in a variety of materials. The spillover of hydrogen is notoriously hard to detect or control. We report herein that the hydrogen spillover pathway on a Pd/Cu alloy can be controlled by reversible adsorption of a spectator molecule. Pd atoms in the Cu surface serve as hydrogen dissociation sites from which H atoms can spillover onto surrounding Cu regions. Selective adsorption of CO at these atomic Pd sites is shown to either prevent the uptake of hydrogen on, or inhibit its desorption from, the surface. In this way, the hydrogen coverage on the whole surface can be controlled by molecular adsorption at a minority site, which we term a ‘molecular cork’ effect. We show that the molecular cork effect is present during a surface catalysed hydrogenation reaction and illustrate how it can be used as a method for controlling uptake and release of hydrogen in a model storage system.

  18. (Controls of the plant endomembrane-secretory pathway): Performance report

    SciTech Connect

    Not Available

    1987-01-01

    This project has been directed towards an understanding of the cellular and molecular mechanisms by which higher plants control the composition of the plasma membrane, through analysis of the biosynthesis, modification and targeting of plasma membrane proteins and glycoproteins. We have undertaken an identification of molecular markers both for the plasma membrane and for the biosynthetic processes, and the development of techniques for the isolation of conditional-lethal mutants defective at defined stages within the endomembrane-secretory pathway responsible for the biosynthesis, modification and targeting of plasma membrane proteins and glycoproteins. For the identification of molecular markers for the plasma membrane, monoclonal antibodies directed against epitopes present at the plant cell surface have been developed. Novel molecular markers for the plant plasma membrane and for the endomembrane-secretory pathway have been sought. Methods for the analysis of beta-glucuronidase in higher plants have been developed. These technologies have involved the use of flow cytometry and fluorescence-activated cell sorting. In addition, we have been investigating the feasibility of expression of animal plasma membrane marker proteins in plants, specifically the VSV G-protein. 5 refs., 6 figs.

  19. Neurophysiological Pathways to Obesity: Below Awareness and Beyond Individual Control

    PubMed Central

    Cohen, Deborah A.

    2008-01-01

    A global obesity epidemic is occurring simultaneously with ongoing increases in the availability and salience of food in the environment. Obesity is increasing across all socioeconomic groups and educational levels and occurs even among individuals with the highest levels of education and expertise in nutrition and related fields. Given these circumstances, it is plausible that excessive food consumption occurs in ways that defy personal insight or are below individual awareness. The current food environment stimulates automatic reflexive responses that enhance the desire to eat and increase caloric intake, making it exceedingly difficult for individuals to resist, especially because they may not be aware of these influences. This article identifies 10 neurophysiological pathways that can lead people to make food choices subconsciously or, in some cases, automatically. These pathways include reflexive and uncontrollable neurohormonal responses to food images, cues, and smells; mirror neurons that cause people to imitate the eating behavior of others without awareness; and limited cognitive capacity to make informed decisions about food. Given that people have limited ability to shape the food environment individually and no ability to control automatic responses to food-related cues that are unconsciously perceived, it is incumbent upon society as a whole to regulate the food environment, including the number and types of food-related cues, portion sizes, food availability, and food advertising. PMID:18586908

  20. Paradoxical control properties of enzymes within pathways: can activation cause an enzyme to have increased control?

    PubMed Central

    Kholodenko, B N; Brown, G C

    1996-01-01

    It is widely assumed that within a metabolic pathway inhibition of an enzyme causes the control exerted by that enzyme over the flux through its own reaction to increase, whereas activation causes its control to decrease. This assumption forms the basis of a number of experimental methods. For a pathway conceptually divided into two enzyme groups connected via a single metabolite we have derived a general condition under which this assumption is false, and thus the pathway shows paradoxical control behaviour, i.e. increased control with activation and decreased control with inhibition of an enzyme or group of enzymes. Paradoxical control behaviour occurs widely when enzyme activity is altered by changing Km (if an enzyme is already close to saturation by its substrate), but may also occur with changes in Vmax. when the elasticity to the linking metabolite increases with its concentration (as in some cases of sigmoidal and exponential kinetics or for reactions catalysed by isoenzymes). These findings suggest that enzymes with sigmoidal kinetics may have low control in the absence of activation, but may gain control with activation, and thus have beneficial regulatory properties. PMID:8615766

  1. A Central Neural Pathway Controlling Odor Tracking in Drosophila

    PubMed Central

    Slater, Gemma; Levy, Peter; Chan, K.L. Andrew

    2015-01-01

    Chemotaxis is important for the survival of most animals. How the brain translates sensory input into motor output beyond higher olfactory processing centers is largely unknown. We describe a group of excitatory neurons, termed Odd neurons, which are important for Drosophila larval chemotaxis. Odd neurons receive synaptic input from projection neurons in the calyx of the mushroom body and project axons to the central brain. Functional imaging shows that some of the Odd neurons respond to odor. Larvae in which Odd neurons are silenced are less efficient at odor tracking than controls and sample the odor space more frequently. Larvae in which the excitability of Odd neurons is increased are better at odor intensity discrimination and odor tracking. Thus, the Odd neurons represent a distinct pathway that regulates the sensitivity of the olfactory system to odor concentrations, demonstrating that efficient chemotaxis depends on processing of odor strength downstream of higher olfactory centers. PMID:25653345

  2. Fault-controlled hydrocarbon pathways in the Monterey Formation, California

    SciTech Connect

    Dholkakia, S.K.; Aydin, A.; Pollard, D.D.; Zoback, M.D.

    1998-08-01

    Field studies of low-permeability siliceous shale units of the Monterey Formation in the southern San Joaquin Valley and coastal California show evidence for fault control on hydrocarbon transport important for both migration and production. Shearing along preexisting discontinuities, such as bedding planes and joints, locally increases permeability in the sheared zone and surrounding fractured rock. As the rock is subjected to shear, it begins to systematically fragment and subsequently to brecciate, thereby creating interconnected voids for hydrocarbon transport. A outcrop-based conceptual model for the development of hydrocarbon pathways in the Monterey Formation is applied to the subsurface using formation microscanner (FMS) data and core. Bed-parallel breccia zones are identified in the Antelope Shale at Buena Vista Hills oil field.

  3. Metabolite Valves: Dynamic Control of Metabolic Flux for Pathway Engineering

    NASA Astrophysics Data System (ADS)

    Prather, Kristala

    2015-03-01

    Microbial strains have been successfully engineered to produce a wide variety of chemical compounds, several of which have been commercialized. As new products are targeted for biological synthesis, yield is frequently considered a primary driver towards determining feasibility. Theoretical yields can be calculated, establishing an upper limit on the potential conversion of starting substrates to target compounds. Such yields typically ignore loss of substrate to byproducts, with the assumption that competing reactions can be eliminated, usually by deleting the genes encoding the corresponding enzymes. However, when an enzyme encodes an essential gene, especially one involved in primary metabolism, deletion is not a viable option. Reducing gene expression in a static fashion is possible, but this solution ignores the metabolic demand needed for synthesis of the enzymes required for the desired pathway. We have developed Metabolite valves to address this challenge. The valves are designed to allow high flux through the essential enzyme during an initial period where growth is favored. Following an external perturbation, enzyme activity is then reduced, enabling a higher precursor pool to be diverted towards the pathway of interest. We have designed valves with control at both the transcriptional and post-translational levels. In both cases, key enzymes in glucose metabolism are regulated, and two different compounds are targeted for heterologous production. We have measured increased concentrations of intracellular metabolites once the valve is closed, and have demonstrated that these increased pools lead to increased product yields. These metabolite valves should prove broadly useful for dynamic control of metabolic flux, resulting in improvements in product yields.

  4. DNA profiles in mitotic cells from gastric adenomas.

    PubMed Central

    Rubio, C. A.; Kato, Y.

    1988-01-01

    Quantitative DNA measurements were done in mitotic figures from 17 gastric adenomas having slight (3 cases), moderate (8 cases), or severe dysplasia (3 cases) or foci of invasive adenocarcinoma (3 cases). Values higher than for normal diploid control cells (2c) or their estimated tetraploid values (4c) were found to increase gradually from slight dysplasia to invasive adenocarcinoma through moderate and severe dysplasia. While none of the adenomas having slight or moderate dysplasia demonstrated aneuploid mitoses (ie, values higher than 5c), 1% of the mitoses in severe dysplasia and 27% of those with invasive adenocarcinoma had values higher than 5c. The present results thus suggest that aneuploid mitotic figures may help to recognize those gastric adenomas having invasive growth. Images Figure 1 PMID:3348355

  5. 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.

  6. Toward a systems-level view of mitotic checkpoints.

    PubMed

    Ibrahim, Bashar

    2015-03-01

    Reproduction and natural selection are the key elements of life. In order to reproduce, the genetic material must be doubled, separated and placed into two new daughter cells, each containing a complete set of chromosomes and organelles. In mitosis, transition from one process to the next is guided by intricate surveillance mechanisms, known as the mitotic checkpoints. Dis-regulation of cell division through checkpoint malfunction can lead to developmental defects and contribute to the development or progression of tumors. This review approaches two important mitotic checkpoints, the spindle assembly checkpoint (SAC) and the spindle position checkpoint (SPOC). The highly conserved spindle assembly checkpoint (SAC) controls the onset of anaphase by preventing premature segregation of the sister chromatids of the duplicated genome, to the spindle poles. In contrast, the spindle position checkpoint (SPOC), in the budding yeast Saccharomyces cerevisiae, ensures that during asymmetric cell division mitotic exit does not occur until the spindle is properly aligned with the cell polarity axis. Although there are no known homologs, there is indication that functionally similar checkpoints exist also in animal cells. This review can be regarded as an "executable model", which could be easily translated into various quantitative concrete models like Petri nets, ODEs, PDEs, or stochastic particle simulations. It can also function as a base for developing quantitative models explaining the interplay of the various components and proteins controlling mitosis. PMID:25722206

  7. The NOXA-MCL1-BIM axis defines lifespan on extended mitotic arrest.

    PubMed

    Haschka, Manuel D; Soratroi, Claudia; Kirschnek, Susanne; Häcker, Georg; Hilbe, Richard; Geley, Stephan; Villunger, Andreas; Fava, Luca L

    2015-01-01

    Cell death on extended mitotic arrest is considered arguably most critical for the efficacy of microtubule-targeting agents (MTAs) in anticancer therapy. While the molecular machinery controlling mitotic arrest on MTA treatment, the spindle assembly checkpoint (SAC), appears well defined, the molecular components executing cell death, as well as factors connecting both networks remain poorly understood. Here we conduct a mini screen exploring systematically the contribution of individual BCL2 family proteins at single cell resolution to death on extended mitotic arrest, and demonstrate that the mitotic phosphorylation of BCL2 and BCLX represent a priming event for apoptosis that is ultimately triggered by NOXA-dependent MCL1 degradation, enabling BIM-dependent cell death. Our findings provide a comprehensive model for the initiation of apoptosis in cells stalled in mitosis and provide a molecular basis for the increased efficacy of combinatorial treatment of cancer cells using MTAs and BH3 mimetics. PMID:25922916

  8. The NOXA–MCL1–BIM axis defines lifespan on extended mitotic arrest

    PubMed Central

    Haschka, Manuel D.; Soratroi, Claudia; Kirschnek, Susanne; Häcker, Georg; Hilbe, Richard; Geley, Stephan; Villunger, Andreas; Fava, Luca L.

    2015-01-01

    Cell death on extended mitotic arrest is considered arguably most critical for the efficacy of microtubule-targeting agents (MTAs) in anticancer therapy. While the molecular machinery controlling mitotic arrest on MTA treatment, the spindle assembly checkpoint (SAC), appears well defined, the molecular components executing cell death, as well as factors connecting both networks remain poorly understood. Here we conduct a mini screen exploring systematically the contribution of individual BCL2 family proteins at single cell resolution to death on extended mitotic arrest, and demonstrate that the mitotic phosphorylation of BCL2 and BCLX represent a priming event for apoptosis that is ultimately triggered by NOXA-dependent MCL1 degradation, enabling BIM-dependent cell death. Our findings provide a comprehensive model for the initiation of apoptosis in cells stalled in mitosis and provide a molecular basis for the increased efficacy of combinatorial treatment of cancer cells using MTAs and BH3 mimetics. PMID:25922916

  9. Auxin and ethylene interactions control mitotic activity of the quiescent centre, root cap size, and pattern of cap cell differentiation in maize.

    PubMed

    Ponce, Georgina; Barlow, Peter W; Feldman, Lewis J; Cassab, Gladys I

    2005-06-01

    Root caps (RCs) are the terminal tissues of higher plant roots. In the present study the factors controlling RC size, shape and structure were examined. It was found that this control involves interactions between the RC and an adjacent population of slowly dividing cells, the quiescent centre, QC. Using the polar auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA), the effects of QC activation on RC gene expression and border cell release was characterized. Ethylene was found to regulate RC size and cell differentiation, since its addition, or the inhibition of its synthesis, affected RC development. The stimulation of cell division in the QC following NPA treatment was reversed by ethylene, and quiescence was re-established. Moreover, inhibition of both ethylene synthesis and auxin polar transport triggered a new pattern of cell division in the root epidermis and led to the appearance of supernumerary epidermal cell files with cap-like characteristics. The data suggest that the QC ensures an ordered internal distribution of auxin, and thereby regulates not only the planes of growth and division in both the root apex proper and the RC meristem, but also regulates cell fate in the RC. Ethylene appears to regulate the auxin redistribution system that resides in the RC. Experiments with Arabidopsis roots also reveal that ethylene plays an important role in regulating the auxin sink, and consequently cell fate in the RC. PMID:16010724

  10. Cyclic Dinucleotide-Controlled Regulatory Pathways in Streptomyces Species

    PubMed Central

    2015-01-01

    The cyclic dinucleotides cyclic 3′,5′-diguanylate (c-di-GMP) and cyclic 3′,5′-diadenylate (c-di-AMP) have emerged as key components of bacterial signal transduction networks. These closely related second messengers follow the classical general principles of nucleotide signaling by integrating diverse signals into regulatory pathways that control cellular responses to changing environments. They impact distinct cellular processes, with c-di-GMP having an established role in promoting bacterial adhesion and inhibiting motility and c-di-AMP being involved in cell wall metabolism, potassium homeostasis, and DNA repair. The involvement of c-dinucleotides in the physiology of the filamentous, nonmotile streptomycetes remained obscure until recent discoveries showed that c-di-GMP controls the activity of the developmental master regulator BldD and that c-di-AMP determines the level of the resuscitation-promoting factor A(RpfA) cell wall-remodelling enzyme. Here, I summarize our current knowledge of c-dinucleotide signaling in Streptomyces species and highlight the important roles of c-di-GMP and c-di-AMP in the biology of these antibiotic-producing, multicellular bacteria. PMID:26216850

  11. Cross-Talk between AURKA and Plk1 in Mitotic Entry and Spindle Assembly

    PubMed Central

    Asteriti, Italia Anna; De Mattia, Fabiola; Guarguaglini, Giulia

    2015-01-01

    The Aurora kinase A (AURKA) is involved in different aspects of mitotic control, from mitotic entry to cytokinesis. Consistent with its pleiotropic roles, several AURKA interactors are able to modulate its activity, the best characterized being the microtubule-binding protein TPX2, the centrosomal protein Cep192, and Bora. Bora has been described as an essential cofactor of AURKA for phosphorylation-mediated activation of the mitotic kinase polo-like kinase 1 (Plk1) at the G2/M transition. A complex AURKA/Plk1 signaling axis is emerging, with multiple involved actors; recent data suggest that this control network is not restricted to mitotic entry only, but operates throughout mitosis. Here, we integrate available data from the literature to depict the complex interplay between AURKA and Plk1 in G2 and mitosis and how it contributes to their mitotic functions. We will particularly focus on how the activity of specifically localized AURKA/Plk1 pools is modulated in time and space by their reciprocal regulation to ensure the timely and coordinated unfolding of downstream mitotic events. PMID:26779436

  12. The leukemogenic t(8;21) fusion protein AML1-ETO controls ribosomal RNA genes and associates with nucleolar organizing regions at mitotic chromosomes

    PubMed Central

    Bakshi, Rachit; Zaidi, Sayyed K.; Pande, Sandhya; Hassan, Mohammad Q.; Young, Daniel W.; Lian, Jane B.; van Wijnen, Andre J.; Stein, Janet L.; Stein, Gary S.

    2010-01-01

    SUMMARY RUNX1/AML1 is required for definitive hematopoiesis and is frequently targeted by chromosomal translocation in acute myeloid leukemias (AML). The t(8;21) related AML1-ETO fusion protein blocks differentiation of myeloid progenitors. Here, we show by immunofluorescence microscopy that during interphase, endogenous AML1-ETO localizes to nuclear microenvironments distinct from those containing native RUNX1/AML1 protein. At mitosis, we clearly detect binding of AML1-ETO to nucleolar organizing regions (NORs) in AML derived Kasumi-1 cells and binding of RUNX1/AML1 to NORs in Jurkat cells. Both RUNX1/AML1 and AML1-ETO occupy ribosomal DNA repeats during interphase, as well as interact with the endogenous RNA Pol I transcription factor UBF-1. Promoter cytosine methylation analysis indicates that RUNX1/AML1 binds to rDNA repeats that are more highly CpG methylated than those bound by AML1-ETO. Down-regulation by RNA interference reveals that RUNX1/AML1 negatively regulates rDNA transcription, while AML1-ETO is a positive regulator in Kasumi-1 cells. Taken together, our findings identify a novel role for the leukemia-related AML1-ETO protein in epigenetic control of cell growth through upregulation of RNA Pol I-mediated ribosomal gene transcription, consistent with the hyper-proliferative phenotype of myeloid cells in AML patients. PMID:19001502

  13. Mitotic Diversity in Homeostatic Human Interfollicular Epidermis

    PubMed Central

    Nöske, Katharina; Stark, Hans-Jürgen; Nevaril, Leonard; Berning, Manuel; Langbein, Lutz; Goyal, Ashish; Diederichs, Sven; Boukamp, Petra

    2016-01-01

    Despite decades of skin research, regulation of proliferation and homeostasis in human epidermis is still insufficiently understood. To address the role of mitoses in tissue regulation, we utilized human long-term skin equivalents and systematically assessed mitoses during early epidermal development and long-term epidermal regeneration. We now demonstrate four different orientations: (1) horizontal, i.e., parallel to the basement membrane (BM) and suggestive of symmetric divisions; (2) oblique with an angle of 45°–70°; or (3) perpendicular, suggestive of asymmetric division. In addition, we demonstrate a fourth substantial fraction of suprabasal mitoses, many of which are committed to differentiation (Keratin K10-positive). As verified also for normal human skin, this spatial mitotic organization is part of the regulatory program of human epidermal tissue homeostasis. As a potential marker for asymmetric division, we investigated for Numb and found that it was evenly spread in almost all undifferentiated keratinocytes, but indeed asymmetrically distributed in some mitoses and particularly frequent under differentiation-repressing low-calcium conditions. Numb deletion (stable knockdown by CRISPR/Cas9), however, did not affect proliferation, neither in a three-day follow up study by life cell imaging nor during a 14-day culture period, suggesting that Numb is not essential for the general control of keratinocyte division. PMID:26828486

  14. Pancreatic cancer cells retain the epithelial-related phenotype and modify mitotic spindle microtubules after the administration of ukrain in vitro.

    PubMed

    Gagliano, Nicoletta; Volpari, Tatiana; Clerici, Marco; Pettinari, Letizia; Barajon, Isabella; Portinaro, Nicola; Colombo, Graziano; Milzani, Aldo; Dalle-Donne, Isabella; Martinelli, Carla

    2012-10-01

    The aim of this study is to characterize the phenotype of pancreatic ductal adenocarcinoma (PDAC) cells in relation to the expression of epithelial-to-mesenchymal transition (EMT) markers and determine whether ukrain, an anticancer drug based on the alkaloids extracted from greater celandine, modulates in vitro the malignant behavior of PDAC cells in order to extend our understanding of its therapeutic potential. Three cell lines (HPAF-II, HPAC, and PL45) were treated with ukrain (5, 10, and 20 μmol/l) for 48 h or left untreated (control). Cell proliferation was assessed by growth curves. Apoptosis was determined by Hoechst nuclear staining and by cytochrome c and caspase-8 expressions. The EMT markers E-cadherin, β-catenin, and vimentin, as well as actin and tubulin cytoskeletons, were analyzed by immunofluorescence. Interphase and mitotic microtubules as well as abnormal mitotic figures were studied by fluorescence microscopy after tubulin immunolabeling. Ukrain strongly suppressed cell proliferation and induced apoptosis possibly through an extrinsic pathway as cytochrome c immunoreactivity suggested that the integrity of the mitochondria was not affected. Tubulin expression indicated an antiproliferative effect of ukrain on the basis of alterations in mitotic spindle microtubule dynamics, leading to abnormal mitosis. Membranous E-cadherin/β-catenin immunoreactivity was similarly expressed in control-treated and ukrain-treated cells, although the drug upregulated E-cadherin in cell lysates. Our results suggest that ukrain exerts its chemotherapeutic action on PDAC cells targeting mitotic spindle microtubules, leading to abnormal mitosis and apoptosis, and favoring cell cohesiveness. The differentiated epithelial phenotype of HPAF-II, HPAC, and PL45 cell lines concomitant with a highly invasive potential suggests that further experiments will be necessary to definitively clarify the role of EMT in PDAC progression. PMID:22700003

  15. Oncogenic KRAS triggers MAPK-dependent errors in mitosis and MYC-dependent sensitivity to anti-mitotic agents.

    PubMed

    Perera, David; Venkitaraman, Ashok R

    2016-01-01

    Oncogenic KRAS induces cell proliferation and transformation, but little is known about its effects on cell division. Functional genetic screens have recently revealed that cancer cell lines expressing oncogenic KRAS are sensitive to interference with mitosis, but neither the mechanism nor the uniformity of anti-mitotic drug sensitivity connected with mutant KRAS expression are yet clear. Here, we report that acute expression of oncogenic KRAS in HeLa cells induces mitotic delay and defects in chromosome segregation through mitogen-activated protein kinase (MAPK) pathway activation and de-regulated expression of several mitosis-related genes. These anomalies are accompanied by increased sensitivity to anti-mitotic agents, a phenotype dependent on the transcription factor MYC and its downstream target anti-apoptotic protein BCL-XL. Unexpectedly, we find no correlation between KRAS mutational status or MYC expression levels and anti-mitotic drug sensitivity when surveying a large database of anti-cancer drug responses. However, we report that the co-existence of KRAS mutations and high MYC expression predicts anti-mitotic drug sensitivity. Our findings reveal a novel function of oncogenic KRAS in regulating accurate mitotic progression and suggest new avenues to therapeutically target KRAS-mutant tumours and stratify patients in ongoing clinical trials of anti-mitotic drugs. PMID:27412232

  16. Oncogenic KRAS triggers MAPK-dependent errors in mitosis and MYC-dependent sensitivity to anti-mitotic agents

    PubMed Central

    Perera, David; Venkitaraman, Ashok R.

    2016-01-01

    Oncogenic KRAS induces cell proliferation and transformation, but little is known about its effects on cell division. Functional genetic screens have recently revealed that cancer cell lines expressing oncogenic KRAS are sensitive to interference with mitosis, but neither the mechanism nor the uniformity of anti-mitotic drug sensitivity connected with mutant KRAS expression are yet clear. Here, we report that acute expression of oncogenic KRAS in HeLa cells induces mitotic delay and defects in chromosome segregation through mitogen-activated protein kinase (MAPK) pathway activation and de-regulated expression of several mitosis-related genes. These anomalies are accompanied by increased sensitivity to anti-mitotic agents, a phenotype dependent on the transcription factor MYC and its downstream target anti-apoptotic protein BCL-XL. Unexpectedly, we find no correlation between KRAS mutational status or MYC expression levels and anti-mitotic drug sensitivity when surveying a large database of anti-cancer drug responses. However, we report that the co-existence of KRAS mutations and high MYC expression predicts anti-mitotic drug sensitivity. Our findings reveal a novel function of oncogenic KRAS in regulating accurate mitotic progression and suggest new avenues to therapeutically target KRAS-mutant tumours and stratify patients in ongoing clinical trials of anti-mitotic drugs. PMID:27412232

  17. Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

    SciTech Connect

    Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao

    2013-04-05

    Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

  18. The Mitotic Checkpoint Gene, SIL is Regulated by E2F1

    PubMed Central

    Erez, Ayelet; Chaussepied, Marie; Tina, Colaizzo-Anas; Aplan, Peter; Ginsberg, Doron; Izraeli, Shai

    2009-01-01

    The SIL gene expression is increased in multiple cancers and correlates with the expression of mitotic spindle checkpoint genes and with increased metastatic potential. SIL regulates mitotic entry, organization of the mitotic spindle and cell survival. The E2F transcription factors regulate cell cycle progression by controlling the expression of genes mediating the G1/S transition. More recently E2F has been shown to regulate mitotic spindle checkpoint genes as well. As SIL expression correlates with mitotic checkpoint genes we hypothesized that SIL is regulated by E2F. We mined raw data of published experiments and performed new experiments by modification of E2F expression in cell lines, reporter assays and chromatin immunoprecipitation. Ectopic expression or endogenous activation of E2F induced the expression of SIL, while knockdown of E2F by shRNA, downregulated SIL expression. E2F activated SIL promoter by reporter assay and bound to SIL promoter in-vivo. Taken together these data demonstrate that SIL is regulated by E2F. As SIL is essential for mitotic entry, E2F may regulate G2/M transition through the induction of SIL. Furthermore, as silencing of SIL cause apoptosis in cancer cells, these finding may have therapeutic relevance in tumors with constitutive activation of E2F. PMID:18649360

  19. Mitotic activation: a convergent mechanism for a cohort of neurodegenerative diseases.

    PubMed

    Husseman, J W; Nochlin, D; Vincent, I

    2000-01-01

    Previous evidence from our lab and others has implicated the mitotic cdc2/cyclin B1 kinase in the neurofibrillary degeneration of Alzheimer's disease. To examine the specificity of this relationship, and define conditions leading to atypical activation of mitotic kinase in postmitotic neurons, we have applied antibodies specific for the cdc2 kinase, its activator, cyclin B1, and three cdc2 produced phosphoepitopes: the TG-3 phosphoepitope in tau and nucleolin, the MPM-2 phosphoepitope in a variety of substrates, and the H5 phosphoepitope in RNA polymerase II, to affected brain regions from a spectrum of neurodegenerative disorders. Our results demonstrate that neurons containing characteristic lesions in a subset of diseases including Down Syndrome (DS), Frontotemporal Dementia linked to chromosome 17 (FTD-17), Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Parkinson-Amyotrophic Lateral Sclerosis of Guam (GP-ALS), Niemann Pick disease type C (NPDC), and Pick's disease, display mitotic indices, implicating diverse etiologies in mitotic activation. The convergence of various degenerative schemes into a unified mitotic kinase-driven pathway provides a common target for therapeutic treatment of these different disorders. PMID:11124425

  20. Mitotic Exit Function of Polo-like Kinase Cdc5 Is Dependent on Sequential Activation by Cdk1.

    PubMed

    Rodriguez-Rodriguez, Jose-Antonio; Moyano, Yolanda; Játiva, Soraya; Queralt, Ethel

    2016-05-31

    To complete mitosis, Saccharomyces cerevisiae needs to activate the mitotic phosphatase Cdc14. Two pathways contribute to Cdc14 regulation: FEAR (Cdc14 early anaphase release) and MEN (mitotic exit network). Cdc5 polo-like kinase was found to be an important mitotic exit component. However, its specific role in mitotic exit regulation and its involvement in Cdc14 release remain unclear. Here, we provide insight into the mechanism by which Cdc5 contributes to the timely release of Cdc14. Our genetic and biochemical data indicate that Cdc5 acts in parallel with MEN during anaphase. This MEN-independent Cdc5 function requires active separase and activation by Cdk1-dependent phosphorylation. Cdk1 first phosphorylates Cdc5 to activate it in early anaphase, and then, in late anaphase, further phosphorylation of Cdc5 by Cdk1 is needed to promote its MEN-related functions. PMID:27210759

  1. Sorting Nexin 9 Recruits Clathrin Heavy Chain to the Mitotic Spindle for Chromosome Alignment and Segregation

    PubMed Central

    Ma, Maggie P. C.; Robinson, Phillip J.; Chircop, Megan

    2013-01-01

    Sorting nexin 9 (SNX9) and clathrin heavy chain (CHC) each have roles in mitosis during metaphase. Since the two proteins directly interact for their other cellular function in endocytosis we investigated whether they also interact for metaphase and operate on the same pathway. We report that SNX9 and CHC functionally interact during metaphase in a specific molecular pathway that contributes to stabilization of mitotic spindle kinetochore (K)-fibres for chromosome alignment and segregation. This function is independent of their endocytic role. SNX9 residues in the clathrin-binding low complexity domain are required for CHC association and for targeting both CHC and transforming acidic coiled-coil protein 3 (TACC3) to the mitotic spindle. Mutation of these sites to serine increases the metaphase plate width, indicating inefficient chromosome congression. Therefore SNX9 and CHC function in the same molecular pathway for chromosome alignment and segregation, which is dependent on their direct association. PMID:23861900

  2. Characterization of TcCYC6 from Trypanosoma cruzi, a gene with homology to mitotic cyclins.

    PubMed

    Di Renzo, María Agostina; Laverrière, Marc; Schenkman, Sergio; Wehrendt, Diana Patricia; Tellez-Iñón, María Teresa; Potenza, Mariana

    2016-06-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is a protozoan parasite with a life cycle that alternates between replicative and non-replicative forms, but the components and mechanisms that regulate its cell cycle are poorly described. In higher eukaryotes, cyclins are proteins that activate cyclin-dependent kinases (CDKs), by associating with them along the different stages of the cell cycle. These cyclin-CDK complexes exert their role as major modulators of the cell cycle by phosphorylating specific substrates. For the correct progression of the cell cycle, the mechanisms that regulate the activity of cyclins and their associated CDKs are diverse and must be controlled precisely. Different types of cyclins are involved in specific phases of the eukaryotic cell cycle, preferentially activating certain CDKs. In this work, we characterized TcCYC6, a putative coding sequence of T. cruzi which encodes a protein with homology to mitotic cyclins. The overexpression of this sequence, fused to a tag of nine amino acids from influenza virus hemagglutinin (TcCYC6-HA), showed to be detrimental for the proliferation of epimastigotes in axenic culture and affected the cell cycle progression. In silico analysis revealed an N-terminal segment similar to the consensus sequence of the destruction box, a hallmark for the degradation of several mitotic cyclins. We experimentally determined that the TcCYC6-HA turnover decreased in the presence of proteasome inhibitors, suggesting that TcCYC6 degradation occurs via ubiquitin-proteasome pathway. The results obtained in this study provide first evidence that TcCYC6 expression and degradation are finely regulated in T. cruzi. PMID:26709077

  3. Advanced Sensors and Controls for Building Applications: Market Assessment and Potential R&D Pathways

    SciTech Connect

    Brambley, M. R.; Haves, P.; McDonald, S. C.; Torcellini, P.; Hansen, D.; Holmberg, D. R.; Roth, K. W.

    2005-04-01

    This document provides a market assessment of existing building sensors and controls and presents a range of technology pathways (R&D options) for pursuing advanced sensors and building control strategies.

  4. Control of Proliferation and Cancer Growth by the Hippo Signaling Pathway.

    PubMed

    Ehmer, Ursula; Sage, Julien

    2016-02-01

    The control of cell division is essential for normal development and the maintenance of cellular homeostasis. Abnormal cell proliferation is associated with multiple pathological states, including cancer. Although the Hippo/YAP signaling pathway was initially thought to control organ size and growth, increasing evidence indicates that this pathway also plays a major role in the control of proliferation independent of organ size control. In particular, accumulating evidence indicates that the Hippo/YAP signaling pathway functionally interacts with multiple other cellular pathways and serves as a central node in the regulation of cell division, especially in cancer cells. Here, recent observations are highlighted that connect Hippo/YAP signaling to transcription, the basic cell-cycle machinery, and the control of cell division. Furthermore, the oncogenic and tumor-suppressive attributes of YAP/TAZ are reviewed, which emphasizes the relevance of the Hippo pathway in cancer. Mol Cancer Res; 14(2); 127-40. ©2015 AACR. PMID:26432795

  5. Rapid measurement of mitotic spindle orientation in cultured mammalian cells.

    PubMed

    Decarreau, Justin; Driver, Jonathan; Asbury, Charles; Wordeman, Linda

    2014-01-01

    Factors that influence the orientation of the mitotic spindle are important for the maintenance of stem cell populations and in cancer development. However, screening for these factors requires rapid quantification of alterations of the angle of the mitotic spindle in cultured cell lines. Here we describe a method to image mitotic cells and rapidly score the angle of the mitotic spindle using a simple MATLAB application to analyze a stack of Z-images. PMID:24633791

  6. Mitotic Stress and Chromosomal Instability in Cancer

    PubMed Central

    Malumbres, Marcos

    2012-01-01

    Cell cycle deregulation is a common motif in human cancer, and multiple therapeutic strategies are aimed to prevent tumor cell proliferation. Whereas most current therapies are designed to arrest cell cycle progression either in G1/S or in mitosis, new proposals include targeting the intrinsic chromosomal instability (CIN, an increased rate of gain or losses of chromosomes during cell division) or aneuploidy (a genomic composition that differs from diploid) that many tumor cells display. Why tumors cells are chromosomally unstable or aneuploid and what are the consequences of these alterations are not completely clear at present. Several mitotic regulators are overexpressed as a consequence of oncogenic alterations, and they are likely to alter the proper regulation of chromosome segregation in cancer cells. In this review, we propose the relevance of TPX2, a mitotic regulator involved in the formation of the mitotic spindle, in oncogene-induced mitotic stress. This protein, as well as its partner Aurora-A, is frequently overexpressed in human cancer, and its deregulation may participate not only in chromosome numeric aberrations but also in other forms of genomic instability in cancer cells. PMID:23634259

  7. Molecular Evolution of Multiple-Level Control of Heme Biosynthesis Pathway in Animal Kingdom

    PubMed Central

    Tzou, Wen-Shyong; Chu, Ying; Lin, Tzung-Yi; Hu, Chin-Hwa; Pai, Tun-Wen; Liu, Hsin-Fu; Lin, Han-Jia; Cases, Ildeofonso; Rojas, Ana; Sanchez, Mayka; You, Zong-Ye; Hsu, Ming-Wei

    2014-01-01

    Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzyme-encoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 5′ untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway. PMID:24489775

  8. Transdominant genetic analysis of a growth control pathway

    PubMed Central

    Caponigro, Giordano; Abedi, Majid R.; Hurlburt, Anthony P.; Maxfield, Andrew; Judd, Weston; Kamb, Alexander

    1998-01-01

    Genetic selections that use proteinaceous transdominant inhibitors encoded by DNA libraries to cause mutant phenocopies may facilitate genetic analysis in traditionally nongenetic organisms. We performed a selection for random short peptides and larger protein fragments (collectively termed “perturbagens”) that inhibit the yeast pheromone response pathway. Peptide and protein fragment perturbagens that permit cell division in the presence of pheromone were recovered. Two perturbagens were derived from proteins required for pheromone response, and an additional two were derived from proteins that may negatively influence the pheromone response pathway. Furthermore, three known components of the pathway were identified as probable perturbagen targets based on physical interaction assays. Thus, by selection for transdominant inhibitors of pheromone response, multiple pathway components were identified either directly as gene fragments or indirectly as the likely targets of specific perturbagens. These results, combined with the results of previous work [Holzmayer, T. A., Pestov, D. G. & Roninson, I. B. (1992) Nucl. Acids. Res. 20, 711–717; Whiteway, M., Dignard, D. & Thomas, D. Y. (1992) Proc. Natl. Acad. Sci. USA 89, 9410–9414; and Gudkov, A. V., Kazarov, A. R., Thimmapaya, R., Axenovich, S. A., Mazo, I. A. & Roninson, I. B. (1994) Proc. Natl. Acad. Sci. USA 91, 3744–3748], suggest that transdominant genetic analysis of the type described here will be broadly applicable. PMID:9636180

  9. Beyond microarrays: Finding key transcription factors controlling signal transduction pathways

    PubMed Central

    Kel, Alexdander; Voss, Nico; Jauregui, Ruy; Kel-Margoulis, Olga; Wingender, Edgar

    2006-01-01

    Background Massive gene expression changes in different cellular states measured by microarrays, in fact, reflect just an "echo" of real molecular processes in the cells. Transcription factors constitute a class of the regulatory molecules that typically require posttranscriptional modifications or ligand binding in order to exert their function. Therefore, such important functional changes of transcription factors are not directly visible in the microarray experiments. Results We developed a novel approach to find key transcription factors that may explain concerted expression changes of specific components of the signal transduction network. The approach aims at revealing evidence of positive feedback loops in the signal transduction circuits through activation of pathway-specific transcription factors. We demonstrate that promoters of genes encoding components of many known signal transduction pathways are enriched by binding sites of those transcription factors that are endpoints of the considered pathways. Application of the approach to the microarray gene expression data on TNF-alpha stimulated primary human endothelial cells helped to reveal novel key transcription factors potentially involved in the regulation of the signal transduction pathways of the cells. Conclusion We developed a novel computational approach for revealing key transcription factors by knowledge-based analysis of gene expression data with the help of databases on gene regulatory networks (TRANSFAC® and TRANSPATH®). The corresponding software and databases are available at . PMID:17118134

  10. MEK1act/tubulin interaction is an important determinant of mitotic stability in cultured HT1080 human fibrosarcoma cells

    PubMed Central

    Cao, Jia-ning; Shafee, Norazizah; Vickery, Larry; Kaluz, Stefan; Ru, Ning; Stanbridge, Eric J.

    2010-01-01

    Activation of the MAPK pathway plays a major role in neoplastic cell transformation. Using a proteomics approach we identified α tubulin and β tubulin as proteins that interact with activated MEK1, a central MAPK regulatory kinase. Confocal analysis revealed spatio-temporal control of MEK1-tubulin co-localization that was most prominent in the mitotic spindle apparatus in variant HT1080 human fibrosarcoma cells. Peptide arrays identified the critical role of positively charged amino acids R108, R113, R160 and K157 on the surface of MEK1 for tubulin interaction. Overexpression of activated MEK1 caused defects in spindle arrangement, chromosome segregation and ploidy. In contrast, chromosome polyploidy was reduced in the presence of an activated MEK1 mutant (R108A, R113A) that disrupted interactions with tubulin. Our findings indicate the importance of signaling by activated MEK1-tubulin in spindle organization and chromosomal instability. PMID:20570892

  11. 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

  12. Phosphorylation of Plk1 at Ser326 regulates its functions during mitotic progression

    PubMed Central

    Tang, J; Yang, X; Liu, X

    2009-01-01

    Polo-like kinase 1 (Plk1), the best characterized member of the mammalian polo-like kinase family, is well regulated throughout the cell cycle at the protein expression level. Moreover, it is known that Plk1 kinase activity is also regulated at the post-translational level through phosphorylation. However, the upstream kinases of Plk1 have not been identified. Although the involvement of the p38 MAP kinase pathway in cellular responses to stress has been well documented, the role of this pathway in normal cell cycle progression is unclear. Here, we show that phosphorylated p38 and MAP kinase-activated protein kinase 2 (MK2) are colocalized with Plk1 to the spindle poles during prophase and metaphase. Specific depletion of various members of the p38 MAP kinase pathway by the use of RNA interference revealed that the pathway is required for mitotic progression under normal growth conditions. Furthermore, MK2 directly phosphorylates Ser326 of Plk1. Ectopic expression of Plk1-S326A completely blocked cells at mitosis, likely due to the defect of bipolar spindle formation and subsequent activation of the spindle checkpoint. Only Plk1-S326E, but not the Plk1-S326A, efficiently rescued the p38 or MK2-depletion-induced mitotic defects, further solidifying the requirement of S326 phosphorylation during mitotic progression. PMID:18695677

  13. Signaling Pathways That Control mRNA Turnover

    PubMed Central

    Thapar, Roopa; Denmon, Andria P.

    2013-01-01

    Cells regulate their genomes mainly at the level of transcription and at the level of mRNA decay. While regulation at the level of transcription is clearly important, the regulation of mRNA turnover by signaling networks is essential for a rapid response to external stimuli. Signaling pathways result in posttranslational modification of RNA binding proteins by phosphorylation, ubiquitination, methylation, acetylation etc. These modifications are important for rapid remodeling of dynamic ribonucleoprotein complexes and triggering mRNA decay. Understanding how these posttranslational modifications alter gene expression is therefore a fundamental question in biology. In this review we highlight recent findings on how signaling pathways and cell cycle checkpoints involving phosphorylation, ubiquitination, and arginine methylation affect mRNA turnover. PMID:23602935

  14. Trophic Status Controls Mercury Methylation Pathways in Northern Peats

    NASA Astrophysics Data System (ADS)

    Hines, M. E.; Zhang, L.; Barkay, T.; Krabbenhoft, D. P.; Schaefer, J.; Hu, H.; Sidelinger, W.; Liu, X.; Wang, Y.

    2015-12-01

    Methyl mercury (MeHg) can be produced by a variety of microbes including syntrophs, methanogens, acetogens, and fermenters, besides sulfate (SO42-, SRB) and iron- reducing bacteria. Many freshwater wetlands are deficient in electron acceptors that support the traditional respiratory pathways of methylation, yet they accumulate high levels of MeHg. To investigate methylation in these wetlands and to connect these pathways with vegetation and microbial communities, incubation experiments were conducted using peats from 26 sites in Alaska. The sites were clustered using multiple factor analysis based on pH, temp, CH4 and volatile fatty acids production rates, and surface vegetation composition. Three clusters were generated and corresponded to three trophic levels that were manifested by three pH levels (3.5, 4.5, and 5). Hg methylation activity in laboratory incubations was determined using the short-lived radioisotope 197Hg. In the low pH, Sphagnum-dominated cluster, methylation rates were less than 1% day-1 and likely conducted by primary fermenters. Conversely, the high pH trophic cluster dominated by Carex aquatilis and active syntrophy exhibited Hg methylation rates as high as 12% day-1. In intermediate sites, rich in Sphagnum magellanicum with less Carex, a gradient in syntrophy and Hg methylation paths was observed. Amendments with process-stimulators and inhibitors revealed no evidence of SO42- reduction, but suggested that SRB, metabolizing either syntrophically with methanogens and/or by fermentation, likely methylated Hg. While on going metatranscriptomics studies are required to verify the role of syntrophs, fermenters, and methanogens as methylators, these results revealed that Hg methylation pathways change greatly along trophic gradients with a dominance of respiratory pathways in mineral-rich sites, syntrophy dominance in intermediate sites, and fermentation dominance in nutrient-poor sites.

  15. The Aedes aegypti Toll Pathway Controls Dengue Virus Infection

    PubMed Central

    Xi, Zhiyong; Ramirez, Jose L.; Dimopoulos, George

    2008-01-01

    Aedes aegypti, the mosquito vector of dengue viruses, utilizes its innate immune system to ward off a variety of pathogens, some of which can cause disease in humans. To date, the features of insects' innate immune defenses against viruses have mainly been studied in the fruit fly Drosophila melanogaster, which appears to utilize different immune pathways against different types of viruses, in addition to an RNA interference–based defense system. We have used the recently released whole-genome sequence of the Ae. aegypti mosquito, in combination with high-throughput gene expression and RNA interference (RNAi)-based reverse genetic analyses, to characterize its response to dengue virus infection in different body compartments. We have further addressed the impact of the mosquito's endogenous microbial flora on virus infection. Our findings indicate a significant role for the Toll pathway in regulating resistance to dengue virus, as indicated by an infection-responsive regulation and functional assessment of several Toll pathway–associated genes. We have also shown that the mosquito's natural microbiota play a role in modulating the dengue virus infection, possibly through basal-level stimulation of the Toll immune pathway. PMID:18604274

  16. Sensitive cells: enabling tools for static and dynamic control of microbial metabolic pathways.

    PubMed

    Cress, Brady F; Trantas, Emmanouil A; Ververidis, Filippos; Linhardt, Robert J; Koffas, Mattheos Ag

    2015-12-01

    Natural metabolic pathways are dynamically regulated at the transcriptional, translational, and protein levels. Despite this, traditional pathway engineering has relied on static control strategies to engender changes in metabolism, most likely due to ease of implementation and perceived predictability of design outcome. Increasingly in recent years, however, metabolic engineers have drawn inspiration from natural systems and have begun to harness dynamically controlled regulatory machinery to improve design of engineered microorganisms for production of specialty and commodity chemicals. Here, we review recent enabling technologies for engineering static control over pathway expression levels, and we discuss state-of-the-art dynamic control strategies that have yielded improved outcomes in the field of microbial metabolic engineering. Furthermore, we emphasize design of a novel class of genetically encoded controllers that will facilitate automatic, transient tuning of synthetic and endogenous pathways. PMID:26453934

  17. PP1 initiates the dephosphorylation of MASTL, triggering mitotic exit and bistability in human cells

    PubMed Central

    Rogers, Samuel; Fey, Dirk; McCloy, Rachael A.; Parker, Benjamin L.; Mitchell, Nicholas J.; Payne, Richard J.; Daly, Roger J.; James, David E.; Caldon, C. Elizabeth; Watkins, D. Neil; Croucher, David R.; Burgess, Andrew

    2016-01-01

    ABSTRACT Entry into mitosis is driven by the phosphorylation of thousands of substrates, under the master control of Cdk1. During entry into mitosis, Cdk1, in collaboration with MASTL kinase, represses the activity of the major mitotic protein phosphatases, PP1 and PP2A, thereby ensuring mitotic substrates remain phosphorylated. For cells to complete and exit mitosis, these phosphorylation events must be removed, and hence, phosphatase activity must be reactivated. This reactivation of phosphatase activity presumably requires the inhibition of MASTL; however, it is not currently understood what deactivates MASTL and how this is achieved. In this study, we identified that PP1 is associated with, and capable of partially dephosphorylating and deactivating, MASTL during mitotic exit. Using mathematical modelling, we were able to confirm that deactivation of MASTL is essential for mitotic exit. Furthermore, small decreases in Cdk1 activity during metaphase are sufficient to initiate the reactivation of PP1, which in turn partially deactivates MASTL to release inhibition of PP2A and, hence, create a feedback loop. This feedback loop drives complete deactivation of MASTL, ensuring a strong switch-like activation of phosphatase activity during mitotic exit. PMID:26872783

  18. Inhibition of mitotic-specific histone phophorylation by sodium arsenite

    SciTech Connect

    Cobo, J.M.; Valdez, J.G.; Gurley, L.R.

    1994-10-01

    Synchronized cultures of Chinese hamster cells (line CHO) were used to measure the effects of 10{mu}M sodium arsenite on histone phosphorylation. This treatment caused cell proliferation to be temporarily arrested, after which the cells spontaneously resumed cell proliferation in a radiomimetric manner. Immediately following treatment, it was found that sodium arsenite affected only mitotic-specific HI and H3 phosphorylations. Neither interphase, nor mitotic, H2A and H4 phosphorylations were affected, nor was interphase HI Phosphorylation affected. The phosphorylation of HI was inhibited only in mitosis, reducing HI phosphorylation to 38.1% of control levels, which was the level of interphase HI phosphorylation. The phosphorylation of both H3 variants was inhibited in mitosis, the less hydrophobic H3 to 19% and the more hydrophobic H3 to 24% of control levels. These results suggest that sodium arsenite may inhibite cell proliferation by interfering with the cyclin B/p34{sup cdc2} histone kinase activity which is thought to play a key role in regulating the cell cycle. It has been proposed by our laboratory that HI and H3 phosphorylations play a role in restructuring interphase chromatin into metaphase chromosomes. Interference of this process by sodium arsenite may lead to structurally damaged chromosomes resulting in the increased cancer risks known to be produced by arsenic exposure from the environment.

  19. The evolution of control and distribution of adaptive mutations in a metabolic pathway.

    PubMed

    Wright, Kevin M; Rausher, Mark D

    2010-02-01

    In an attempt to understand whether it should be expected that some genes tend to be used disproportionately often by natural selection, we investigated two related phenomena: the evolution of flux control among enzymes in a metabolic pathway and properties of adaptive substitutions in pathway enzymes. These two phenomena are related by the principle that adaptive substitutions should occur more frequently in enzymes with greater flux control. Predicting which enzymes will be preferentially involved in adaptive evolution thus requires an evolutionary theory of flux control. We investigated the evolution of enzyme control in metabolic pathways with two models of enzyme kinetics: metabolic control theory (MCT) and Michaelis-Menten saturation kinetics (SK). Our models generate two main predictions for pathways in which reactions are moderately to highly irreversible: (1) flux control will evolve to be highly unequal among enzymes in a pathway and (2) upstream enzymes evolve a greater control coefficient then those downstream. This results in upstream enzymes fixing the majority of beneficial mutations during adaptive evolution. Once the population has reached high fitness, the trend is reversed, with the majority of neutral/slightly deleterious mutations occurring in downstream enzymes. These patterns are the result of three factors (the first of these is unique to the MCT simulations while the other two seem to be general properties of the metabolic pathways): (1) the majority of randomly selected, starting combinations of enzyme kinetic rates generate pathways that possess greater control for the upstream enzymes compared to downstream enzymes; (2) selection against large pools of intermediate substrates tends to prevent majority control by downstream enzymes; and (3) equivalent mutations in enzyme kinetic rates have the greatest effect on flux for enzymes with high levels of flux control, and these enzymes will accumulate adaptive substitutions, strengthening their

  20. Metabolic Control Analysis: A Tool for Designing Strategies to Manipulate Metabolic Pathways

    PubMed Central

    Moreno-Sánchez, Rafael; Saavedra, Emma; Rodríguez-Enríquez, Sara; Olín-Sandoval, Viridiana

    2008-01-01

    The traditional experimental approaches used for changing the flux or the concentration of a particular metabolite of a metabolic pathway have been mostly based on the inhibition or over-expression of the presumed rate-limiting step. However, the attempts to manipulate a metabolic pathway by following such approach have proved to be unsuccessful. Metabolic Control Analysis (MCA) establishes how to determine, quantitatively, the degree of control that a given enzyme exerts on flux and on the concentration of metabolites, thus substituting the intuitive, qualitative concept of rate limiting step. Moreover, MCA helps to understand (i) the underlying mechanisms by which a given enzyme exerts high or low control and (ii) why the control of the pathway is shared by several pathway enzymes and transporters. By applying MCA it is possible to identify the steps that should be modified to achieve a successful alteration of flux or metabolite concentration in pathways of biotechnological (e.g., large scale metabolite production) or clinical relevance (e.g., drug therapy). The different MCA experimental approaches developed for the determination of the flux-control distribution in several pathways are described. Full understanding of the pathway properties when is working under a variety of conditions can help to attain a successful manipulation of flux and metabolite concentration. PMID:18629230

  1. Measuring mitotic spindle dynamics in budding yeast

    NASA Astrophysics Data System (ADS)

    Plumb, Kemp

    In order to carry out its life cycle and produce viable progeny through cell division, a cell must successfully coordinate and execute a number of complex processes with high fidelity, in an environment dominated by thermal noise. One important example of such a process is the assembly and positioning of the mitotic spindle prior to chromosome segregation. The mitotic spindle is a modular structure composed of two spindle pole bodies, separated in space and spanned by filamentous proteins called microtubules, along which the genetic material of the cell is held. The spindle is responsible for alignment and subsequent segregation of chromosomes into two equal parts; proper spindle positioning and timing ensure that genetic material is appropriately divided amongst mother and daughter cells. In this thesis, I describe fluorescence confocal microscopy and automated image analysis algorithms, which I have used to observe and analyze the real space dynamics of the mitotic spindle in budding yeast. The software can locate structures in three spatial dimensions and track their movement in time. By selecting fluorescent proteins which specifically label the spindle poles and cell periphery, mitotic spindle dynamics have been measured in a coordinate system relevant to the cell division. I describe how I have characterised the accuracy and precision of the algorithms by simulating fluorescence data for both spindle poles and the budding yeast cell surface. In this thesis I also describe the construction of a microfluidic apparatus that allows for the measurement of long time-scale dynamics of individual cells and the development of a cell population. The tools developed in this thesis work will facilitate in-depth quantitative analysis of the non-equilibrium processes in living cells.

  2. Automatic microscopy for mitotic cell location.

    NASA Technical Reports Server (NTRS)

    Herron, J.; Ranshaw, R.; Castle, J.; Wald, N.

    1972-01-01

    Advances are reported in the development of an automatic microscope with which to locate hematologic or other cells in mitosis for subsequent chromosome analysis. The system under development is designed to perform the functions of: slide scanning to locate metaphase cells; conversion of images of selected cells into binary form; and on-line computer analysis of the digitized image for significant cytogenetic data. Cell detection criteria are evaluated using a test sample of 100 mitotic cells and 100 artifacts.

  3. RGS14 is a mitotic spindle protein essential from the first division of the mammalian zygote.

    PubMed

    Martin-McCaffrey, Luke; Willard, Francis S; Oliveira-dos-Santos, Antonio J; Natale, David R C; Snow, Bryan E; Kimple, Randall J; Pajak, Agnieszka; Watson, Andrew J; Dagnino, Lina; Penninger, Josef M; Siderovski, David P; D'Souza, Sudhir J A

    2004-11-01

    Heterotrimeric G protein alpha subunits, RGS proteins, and GoLoco motif proteins have been recently implicated in the control of mitotic spindle dynamics in C. elegans and D. melanogaster. Here we show that "regulator of G protein signaling-14" (RGS14) is expressed by the mouse embryonic genome immediately prior to the first mitosis, where it colocalizes with the anastral mitotic apparatus of the mouse zygote. Loss of Rgs14 expression in the mouse zygote results in cytofragmentation and failure to progress to the 2-cell stage. RGS14 is found in all tissues and segregates to the nucleus in interphase and to the mitotic spindle and centrioles during mitosis. Alteration of RGS14 levels in exponentially proliferating cells leads to cell growth arrest. Our results indicate that RGS14 is one of the earliest essential product of the mammalian embryonic genome yet described and has a general role in mitosis. PMID:15525537

  4. 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. PMID:21628425

  5. Mitotic spindle studied using picosecond laser scissors

    NASA Astrophysics Data System (ADS)

    Baker, N. M.; Botvinick, E. L.; Shi, Linda; Berns, M. B.; Wu, George

    2006-08-01

    In previous studies we have shown that the second harmonic 532 nm, from a picosecond frequency doubled Nd:YAG laser, can cleanly and selectively disrupt spindle fiber microtubules in live cells (Botvinick et al 2004, Biophys. J. 87:4303-4212). In the present study we have ablated different locations and amounts of the metaphase mitotic spindle, and followed the cells in order to observe the fate of the irradiated spindle and the ability of the cell to continue through mitosis. Cells of the rat kangaroo line (PTK2) were stably transfected by ECFP-tubulin and, using fluorescent microscopy and the automated RoboLase microscope, (Botvinick and Berns, 2005, Micros. Res. Tech. 68:65-74) brightly fluorescent individual cells in metaphase were irradiated with 0.2447 nJ/micropulse corresponding to an irradiance of 1.4496*10^7 J/(ps*cm^2) . Upon irradiation the exposed part of the mitotic spindle immediately lost fluorescence and the following events were observed in the cells over time: (1) immediate contraction of the spindle pole towards the cut, (2) recovery of connection between pole and cut microtubule, (3) completion of mitosis. This system should be very useful in studying internal cellular dynamics of the mitotic spindle.

  6. Direct corticospinal pathways contribute to neuromuscular control of perturbed stance.

    PubMed

    Taube, Wolfgang; Schubert, Martin; Gruber, Markus; Beck, Sandra; Faist, Michael; Gollhofer, Albert

    2006-08-01

    The antigravity soleus muscle (Sol) is crucial for compensation of stance perturbation. A corticospinal contribution to the compensatory response of the Sol is under debate. The present study assessed spinal, corticospinal, and cortical excitability at the peaks of short- (SLR), medium- (MLR), and long-latency responses (LLR) after posterior translation of the feet. Transcranial magnetic stimulation (TMS) and peripheral nerve stimulation were individually adjusted so that the peaks of either motor evoked potential (MEP) or H reflex coincided with peaks of SLR, MLR, and LLR, respectively. The influence of specific, presumably direct, corticospinal pathways was investigated by H-reflex conditioning. When TMS was triggered so that the MEP arrived in the Sol at the same time as the peaks of SLR and MLR, EMG remained unaffected. Enhanced EMG was observed when the MEP coincided with the LLR peak (P < 0.001). Similarly, conditioning of the H reflex by subthreshold TMS facilitated H reflexes only at LLR (P < 0.001). The earliest facilitation after perturbation occurred after 86 ms. The TMS-induced H-reflex facilitation at LLR suggests that increased cortical excitability contributes to the augmentation of the LLR peaks. This provides evidence that the LLR in the Sol muscle is at least partly transcortical, involving direct corticospinal pathways. Additionally, these results demonstrate that approximately 86 ms after perturbation, postural compensatory responses are cortically mediated. PMID:16601305

  7. Control of the innate immune response by the mevalonate pathway.

    PubMed

    Akula, Murali K; Shi, Man; Jiang, Zhaozhao; Foster, Celia E; Miao, David; Li, Annie S; Zhang, Xiaoman; Gavin, Ruth M; Forde, Sorcha D; Germain, Gail; Carpenter, Susan; Rosadini, Charles V; Gritsman, Kira; Chae, Jae Jin; Hampton, Randolph; Silverman, Neal; Gravallese, Ellen M; Kagan, Jonathan C; Fitzgerald, Katherine A; Kastner, Daniel L; Golenbock, Douglas T; Bergo, Martin O; Wang, Donghai

    2016-08-01

    Deficiency in mevalonate kinase (MVK) causes systemic inflammation. However, the molecular mechanisms linking the mevalonate pathway to inflammation remain obscure. Geranylgeranyl pyrophosphate, a non-sterol intermediate of the mevalonate pathway, is the substrate for protein geranylgeranylation, a protein post-translational modification that is catalyzed by protein geranylgeranyl transferase I (GGTase I). Pyrin is an innate immune sensor that forms an active inflammasome in response to bacterial toxins. Mutations in MEFV (encoding human PYRIN) result in autoinflammatory familial Mediterranean fever syndrome. We found that protein geranylgeranylation enabled Toll-like receptor (TLR)-induced activation of phosphatidylinositol-3-OH kinase (PI(3)K) by promoting the interaction between the small GTPase Kras and the PI(3)K catalytic subunit p110δ. Macrophages that were deficient in GGTase I or p110δ exhibited constitutive release of interleukin 1β that was dependent on MEFV but independent of the NLRP3, AIM2 and NLRC4 inflammasomes. In the absence of protein geranylgeranylation, compromised PI(3)K activity allows an unchecked TLR-induced inflammatory responses and constitutive activation of the Pyrin inflammasome. PMID:27270400

  8. Proteomic profiling revealed the functional networks associated with mitotic catastrophe of HepG2 hepatoma cells induced by 6-bromine-5-hydroxy-4-methoxybenzaldehyde

    SciTech Connect

    Zhang Bo; Huang Bo; Guan Hua; Zhang Shimeng; Xu Qinzhi; He Xingpeng; Liu Xiaodan; Wang Yu; Shang Zengfu; Zhou Pingkun

    2011-05-01

    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, 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.

  9. Pathways controlling dNTP pools to maintain genome stability.

    PubMed

    Rudd, Sean G; Valerie, Nicholas C K; Helleday, Thomas

    2016-08-01

    Artificially modified nucleotides, in the form of nucleoside analogues, are widely used in the treatment of cancers and various other diseases, and have become important tools in the laboratory to characterise DNA repair pathways. In contrast, the role of endogenously occurring nucleotide modifications in genome stability is little understood. This is despite the demonstration over three decades ago that the cellular DNA precursor pool is orders of magnitude more susceptible to modification than the DNA molecule itself. More recently, underscoring the importance of this topic, oxidation of the cellular nucleotide pool achieved through targeting the sanitation enzyme MTH1, appears to be a promising anti-cancer strategy. This article reviews our current understanding of modified DNA precursors in genome stability, with a particular focus upon oxidised nucleotides, and outlines some important outstanding questions. PMID:27311542

  10. Applications of Genetically-Encoded Biosensors for the Construction and Control of Biosynthetic Pathways

    PubMed Central

    Michener, Josh K.; Thodey, Kate; Liang, Joe C.; Smolke, Christina D.

    2011-01-01

    Cells are filled with biosensors, molecular systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chemical reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metabolism have focused on introducing new pathways and removing existing pathway regulation. Synthetic biology is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biological systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of molecular tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability. PMID:21946159

  11. Critical Roles of the Direct GABAergic Pallido-cortical Pathway in Controlling Absence Seizures.

    PubMed

    Chen, Mingming; Guo, Daqing; Li, Min; Ma, Tao; Wu, Shengdun; Ma, Jingling; Cui, Yan; Xia, Yang; Xu, Peng; Yao, Dezhong

    2015-10-01

    The basal ganglia (BG), serving as an intermediate bridge between the cerebral cortex and thalamus, are believed to play crucial roles in controlling absence seizure activities generated by the pathological corticothalamic system. Inspired by recent experiments, here we systematically investigate the contribution of a novel identified GABAergic pallido-cortical pathway, projecting from the globus pallidus externa (GPe) in the BG to the cerebral cortex, to the control of absence seizures. By computational modelling, we find that both increasing the activation of GPe neurons and enhancing the coupling strength of the inhibitory pallido-cortical pathway can suppress the bilaterally synchronous 2-4 Hz spike and wave discharges (SWDs) during absence seizures. Appropriate tuning of several GPe-related pathways may also trigger the SWD suppression, through modulating the activation level of GPe neurons. Furthermore, we show that the previously discovered bidirectional control of absence seizures due to the competition between other two BG output pathways also exists in our established model. Importantly, such bidirectional control is shaped by the coupling strength of this direct GABAergic pallido-cortical pathway. Our work suggests that the novel identified pallido-cortical pathway has a functional role in controlling absence seizures and the presented results might provide testable hypotheses for future experimental studies. PMID:26496656

  12. Critical Roles of the Direct GABAergic Pallido-cortical Pathway in Controlling Absence Seizures

    PubMed Central

    Li, Min; Ma, Tao; Wu, Shengdun; Ma, Jingling; Cui, Yan; Xia, Yang; Xu, Peng; Yao, Dezhong

    2015-01-01

    The basal ganglia (BG), serving as an intermediate bridge between the cerebral cortex and thalamus, are believed to play crucial roles in controlling absence seizure activities generated by the pathological corticothalamic system. Inspired by recent experiments, here we systematically investigate the contribution of a novel identified GABAergic pallido-cortical pathway, projecting from the globus pallidus externa (GPe) in the BG to the cerebral cortex, to the control of absence seizures. By computational modelling, we find that both increasing the activation of GPe neurons and enhancing the coupling strength of the inhibitory pallido-cortical pathway can suppress the bilaterally synchronous 2–4 Hz spike and wave discharges (SWDs) during absence seizures. Appropriate tuning of several GPe-related pathways may also trigger the SWD suppression, through modulating the activation level of GPe neurons. Furthermore, we show that the previously discovered bidirectional control of absence seizures due to the competition between other two BG output pathways also exists in our established model. Importantly, such bidirectional control is shaped by the coupling strength of this direct GABAergic pallido-cortical pathway. Our work suggests that the novel identified pallido-cortical pathway has a functional role in controlling absence seizures and the presented results might provide testable hypotheses for future experimental studies. PMID:26496656

  13. LKB1 and Notch Pathways Interact and Control Biliary Morphogenesis

    PubMed Central

    Just, Pierre-Alexandre; Poncy, Alexis; Charawi, Sara; Dahmani, Rajae; Traore, Massiré; Dumontet, Typhanie; Drouet, Valérie; Dumont, Florent; Gilgenkrantz, Hélène; Colnot, Sabine; Terris, Benoit; Coulouarn, Cédric; Lemaigre, Frédéric; Perret, Christine

    2015-01-01

    Background LKB1 is an evolutionary conserved kinase implicated in a wide range of cellular functions including inhibition of cell proliferation, regulation of cell polarity and metabolism. When Lkb1 is inactivated in the liver, glucose homeostasis is perturbed, cellular polarity is affected and cholestasis develops. Cholestasis occurs as a result from deficient bile duct development, yet how LKB1 impacts on biliary morphogenesis is unknown. Methodology/Principal Findings We characterized the phenotype of mice in which deletion of the Lkb1 gene has been specifically targeted to the hepatoblasts. Our results confirmed that lack of LKB1 in the liver results in bile duct paucity leading to cholestasis. Immunostaining analysis at a prenatal stage showed that LKB1 is not required for differentiation of hepatoblasts to cholangiocyte precursors but promotes maturation of the primitive ductal structures to mature bile ducts. This phenotype is similar to that obtained upon inactivation of Notch signaling in the liver. We tested the hypothesis of a functional overlap between the LKB1 and Notch pathways by gene expression profiling of livers deficient in Lkb1 or in the Notch mediator RbpJκ and identified a mutual cross-talk between LKB1 and Notch signaling. In vitro experiments confirmed that Notch activity was deficient upon LKB1 loss. Conclusion LKB1 and Notch share a common genetic program in the liver, and regulate bile duct morphogenesis. PMID:26689699

  14. Dual Genetic Pathways Controlling Nodule Number in Medicago truncatula1

    PubMed Central

    Penmetsa, R. Varma; Frugoli, Julia A.; Smith, Lucinda S.; Long, Sharon R.; Cook, Douglas R.

    2003-01-01

    We report the isolation and characterization of a new Medicago truncatula hyper-nodulation mutant, designated sunn (super numeric nodules). Similar to the previously described ethylene-insensitive mutant sickle, sunn exhibits a 10-fold increase in the number of nodules within the primary nodulation zone. Despite this general similarity, these two mutants are readily distinguished based on anatomical, genetic, physiological, and molecular criteria. In contrast to sickle, where insensitivity to ethylene is thought to be causal to the hyper-nodulation phenotype (R.V. Penmetsa, D.R. Cook [1997] Science 275: 527–530), nodulation in sunn is normally sensitive to ethylene. Nevertheless, sunn exhibits seedling root growth that is insensitive to ethylene, although other aspects of the ethylene triple response are normal; these observations suggest that hormonal responses might condition the sunn phenotype in a manner distinct from sickle. The two mutants also differ in the anatomy of the nodulation zone: Successful infection and nodule development in sunn occur predominantly opposite xylem poles, similar to wild type. In sickle, however, both infection and nodulation occur randomly throughout the circumference of the developing root. Genetic analysis indicates that sunn and sickle correspond to separate and unlinked loci, whereas the sunn/skl double mutant exhibits a novel and additive super-nodulation phenotype. Taken together, these results suggest a working hypothesis wherein sunn and sickle define distinct genetic pathways, with skl regulating the number and distribution of successful infection events, and sunn regulating nodule organogenesis. PMID:12644652

  15. Aryl hydrocarbon receptor control of a disease tolerance defense pathway

    PubMed Central

    Bessede, Alban; Gargaro, Marco; Pallotta, Maria T; Matino, Davide; Servillo, Giuseppe; Brunacci, Cinzia; Bicciato, Silvio; Mazza, Emilia MC; Macchiarulo, Antonio; Vacca, Carmine; Iannitti, Rossana; Tissi, Luciana; Volpi, Claudia; Belladonna, Maria L; Orabona, Ciriana; Bianchi, Roberta; Lanz, Tobias; Platten, Michael; Fazia, Maria A Della; Piobbico, Danilo; Zelante, Teresa; Funakoshi, Hiroshi; Nakamura, Toshikazu; Gilot, David; Denison, Michael S; Guillemin, Gilles J; DuHadaway, James B; Prendergast, George C; Metz, Richard; Geffard, Michel; Boon, Louis; Pirro, Matteo; Iorio, Alfonso; Veyret, Bernard; Romani, Luigina; Grohmann, Ursula; Fallarino, Francesca; Puccetti, Paolo

    2014-01-01

    Summary Disease tolerance is the ability of the host to reduce the impact of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (“endotoxin tolerance”). We found that a first exposure to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase 2, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR complex-associated Src kinase activity promoted IDO1 phosphorylation and signaling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in gram-negative and gram-positive infections, pointing to a role for AhR in contributing to host fitness. PMID:24930766

  16. Quantitative assessment of chromosome instability induced through chemical disruption of mitotic progression

    PubMed Central

    Markossian, Sarine; Arnaoutov, Alexei; Saba, Nakhle S.; Larionov, Vladimir; Dasso, Mary

    2016-01-01

    ABSTRACT Most solid tumors are aneuploid, carrying an abnormal number of chromosomes, and they frequently missegregate whole chromosomes in a phenomenon termed chromosome instability (CIN). While CIN can be provoked through disruption of numerous mitotic pathways, it is not clear which of these mechanisms are most critical, or whether alternative mechanisms could also contribute significantly in vivo. One difficulty in determining the relative importance of candidate CIN regulators has been the lack of a straightforward, quantitative assay for CIN in live human cells: While gross mitotic abnormalities can be detected visually, moderate levels of CIN may not be obvious, and are thus problematic to measure. To address this issue, we have developed the first Human Artificial Chromosome (HAC)-based quantitative live-cell assay for mitotic chromosome segregation in human cells. We have produced U2OS-Phoenix cells carrying the alphoidtetO-HAC encoding copies of eGFP fused to the destruction box (DB) of anaphase promoting complex/cyclosome (APC/C) substrate hSecurin and sequences encoding the tetracycline repressor fused to mCherry (TetR-mCherry). Upon HAC missegregation, daughter cells that do not obtain a copy of the HAC are GFP negative in the subsequent interphase. The HAC can also be monitored live following the TetR-mCherry signal. U2OS-Phoenix cells show low inherent levels of CIN, which can be enhanced by agents that target mitotic progression through distinct mechanisms. This assay allows direct detection of CIN induced by clinically important agents without conspicuous mitotic defects, allowing us to score increased levels of CIN that fall below the threshold required for discernable morphological disruption. PMID:27104376

  17. Quantitative assessment of chromosome instability induced through chemical disruption of mitotic progression.

    PubMed

    Markossian, Sarine; Arnaoutov, Alexei; Saba, Nakhle S; Larionov, Vladimir; Dasso, Mary

    2016-07-01

    Most solid tumors are aneuploid, carrying an abnormal number of chromosomes, and they frequently missegregate whole chromosomes in a phenomenon termed chromosome instability (CIN). While CIN can be provoked through disruption of numerous mitotic pathways, it is not clear which of these mechanisms are most critical, or whether alternative mechanisms could also contribute significantly in vivo. One difficulty in determining the relative importance of candidate CIN regulators has been the lack of a straightforward, quantitative assay for CIN in live human cells: While gross mitotic abnormalities can be detected visually, moderate levels of CIN may not be obvious, and are thus problematic to measure. To address this issue, we have developed the first Human Artificial Chromosome (HAC)-based quantitative live-cell assay for mitotic chromosome segregation in human cells. We have produced U2OS-Phoenix cells carrying the alphoid(tetO)-HAC encoding copies of eGFP fused to the destruction box (DB) of anaphase promoting complex/cyclosome (APC/C) substrate hSecurin and sequences encoding the tetracycline repressor fused to mCherry (TetR-mCherry). Upon HAC missegregation, daughter cells that do not obtain a copy of the HAC are GFP negative in the subsequent interphase. The HAC can also be monitored live following the TetR-mCherry signal. U2OS-Phoenix cells show low inherent levels of CIN, which can be enhanced by agents that target mitotic progression through distinct mechanisms. This assay allows direct detection of CIN induced by clinically important agents without conspicuous mitotic defects, allowing us to score increased levels of CIN that fall below the threshold required for discernable morphological disruption. PMID:27104376

  18. Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses

    PubMed Central

    Holm, Christian K.; Rahbek, Stine H.; Gad, Hans Henrik; Bak, Rasmus O.; Jakobsen, Martin R.; Jiang, Zhaozaho; Hansen, Anne Louise; Jensen, Simon K.; Sun, Chenglong; Thomsen, Martin K.; Laustsen, Anders; Nielsen, Camilla G.; Severinsen, Kasper; Xiong, Yingluo; Burdette, Dara L.; Hornung, Veit; Lebbink, Robert Jan; Duch, Mogens; Fitzgerald, Katherine A.; Bahrami, Shervin; Mikkelsen, Jakob Giehm; Hartmann, Rune; Paludan, Søren R.

    2016-01-01

    Stimulator of interferon genes (STING) is known be involved in control of DNA viruses but has an unexplored role in control of RNA viruses. During infection with DNA viruses STING is activated downstream of cGAMP synthase (cGAS) to induce type I interferon. Here we identify a STING-dependent, cGAS-independent pathway important for full interferon production and antiviral control of enveloped RNA viruses, including influenza A virus (IAV). Further, IAV interacts with STING through its conserved hemagglutinin fusion peptide (FP). Interestingly, FP antagonizes interferon production induced by membrane fusion or IAV but not by cGAMP or DNA. Similar to the enveloped RNA viruses, membrane fusion stimulates interferon production in a STING-dependent but cGAS-independent manner. Abolishment of this pathway led to reduced interferon production and impaired control of enveloped RNA viruses. Thus, enveloped RNA viruses stimulate a cGAS-independent STING pathway, which is targeted by IAV. PMID:26893169

  19. Novel insights into mitotic chromosome condensation

    PubMed Central

    Piskadlo, Ewa; Oliveira, Raquel A.

    2016-01-01

    The fidelity of mitosis is essential for life, and successful completion of this process relies on drastic changes in chromosome organization at the onset of nuclear division. The mechanisms that govern chromosome compaction at every cell division cycle are still far from full comprehension, yet recent studies provide novel insights into this problem, challenging classical views on mitotic chromosome assembly. Here, we briefly introduce various models for chromosome assembly and known factors involved in the condensation process (e.g. condensin complexes and topoisomerase II). We will then focus on a few selected studies that have recently brought novel insights into the mysterious way chromosomes are condensed during nuclear division. PMID:27508072

  20. The homologous chromosome is an effective template for the repair of mitotic DNA double-strand breaks in Drosophila.

    PubMed Central

    Rong, Yikang S; Golic, Kent G

    2003-01-01

    In recombinational DNA double-strand break repair a homologous template for gene conversion may be located at several different genomic positions: on the homologous chromosome in diploid organisms, on the sister chromatid after DNA replication, or at an ectopic position. The use of the homologous chromosome in mitotic gene conversion is thought to be limited in the yeast Saccharomyces cerevisiae and mammalian cells. In contrast, by studying the repair of double-strand breaks generated by the I-SceI rare-cutting endonuclease, we find that the homologous chromosome is frequently used in Drosophila melanogaster, which we suggest is attributable to somatic pairing of homologous chromosomes in mitotic cells of Drosophila. We also find that Drosophila mitotic cells of the germ line, like yeast, employ the homologous recombinational repair pathway more often than imperfect nonhomologous end joining. PMID:14704169

  1. 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

  2. Disruption of IFT Complex A Causes Cystic Kidneys without Mitotic Spindle Misorientation

    PubMed Central

    Jonassen, Julie A.; SanAgustin, Jovenal; Baker, Stephen P.

    2012-01-01

    Intraflagellar transport (IFT) complexes A and B build and maintain primary cilia. In the mouse, kidney-specific or hypomorphic mutant alleles of IFT complex B genes cause polycystic kidneys, but the influence of IFT complex A proteins on renal development is not well understood. In the present study, we found that HoxB7-Cre–driven deletion of the complex A gene Ift140 from collecting ducts disrupted, but did not completely prevent, cilia assembly. Mutant kidneys developed collecting duct cysts by postnatal day 5, with rapid cystic expansion and renal dysfunction by day 15 and little remaining parenchymal tissue by day 20. In contrast to many models of polycystic kidney disease, precystic Ift140-deleted collecting ducts showed normal centrosomal positioning and no misorientation of the mitotic spindle axis, suggesting that disruption of oriented cell division is not a prerequisite to cyst formation in these kidneys. Precystic collecting ducts had an increased mitotic index, suggesting that cell proliferation may drive cyst expansion even with normal orientation of the mitotic spindle. In addition, we observed significant increases in expression of canonical Wnt pathway genes and mediators of Hedgehog and tissue fibrosis in highly cystic, but not precystic, kidneys. Taken together, these studies indicate that loss of Ift140 causes pronounced renal cystic disease and suggest that abnormalities in several different pathways may influence cyst progression. PMID:22282595

  3. (Controls of the plant endomembrane-secretory pathway)

    SciTech Connect

    Not Available

    1991-01-01

    These studies are focused on elucidating the molecular structure of plant cell membranes with special reference to cell surface glycoproteins. The studies reported herein include use of monoclonal antibodies to characterize cell surface epitopes, construction of cDNA libraries of cell surface proteins, isolation of plant cell mutants by flow cytometry, detection of beta-glucouronidase marker enzyme systems in plants, expression go VSVG (the major envelope glycoprotein of Vesicular Stomatis Virus) in plant cells, and control of gene expression of cell membrane glycoproteins.(DT)

  4. [Controls of the plant endomembrane-secretory pathway]. Final report

    SciTech Connect

    Not Available

    1991-12-31

    These studies are focused on elucidating the molecular structure of plant cell membranes with special reference to cell surface glycoproteins. The studies reported herein include use of monoclonal antibodies to characterize cell surface epitopes, construction of cDNA libraries of cell surface proteins, isolation of plant cell mutants by flow cytometry, detection of beta-glucouronidase marker enzyme systems in plants, expression go VSVG (the major envelope glycoprotein of Vesicular Stomatis Virus) in plant cells, and control of gene expression of cell membrane glycoproteins.(DT)

  5. Mitotic genes are transcriptionally upregulated in the fibroblast irradiated with very low doses of UV-C

    PubMed Central

    Takeuchi, Seiji; Matsuda, Toshiro; Ono, Ryusuke; Tsujimoto, Mariko; Nishigori, Chikako

    2016-01-01

    Ultraviolet (UV) radiation induces a variety of biological effects, including DNA damage response and cell signaling pathways. We performed transcriptome analysis using microarray in human primary cultured fibroblasts irradiated with UV-C (0.5 or 5 J/m2) and harvested at 4 or 12 h following UV exposure. All transcript data were analyzed by comparison with the corresponding results in non-irradiated (control) cells. The number of genes with significantly altered expression (≥2-fold difference relative to the control) is higher in the sample irradiated with high dose of UV, suggesting that gene expression was UV dose-dependent. Pathway analysis on the upregulated genes at 12 h indicates that the expression of some cell cycle-related genes was predominantly induced irrespective of UV-dose. Interestingly, almost all the genes with significant altered expression were cell cycle-related genes designated as ‘Mitotic Genes’, which function in the spindle assembly checkpoint. Therefore, even a low dose of UV could affect the transcriptional profile. PMID:27378355

  6. Mitotic genes are transcriptionally upregulated in the fibroblast irradiated with very low doses of UV-C.

    PubMed

    Takeuchi, Seiji; Matsuda, Toshiro; Ono, Ryusuke; Tsujimoto, Mariko; Nishigori, Chikako

    2016-01-01

    Ultraviolet (UV) radiation induces a variety of biological effects, including DNA damage response and cell signaling pathways. We performed transcriptome analysis using microarray in human primary cultured fibroblasts irradiated with UV-C (0.5 or 5 J/m(2)) and harvested at 4 or 12 h following UV exposure. All transcript data were analyzed by comparison with the corresponding results in non-irradiated (control) cells. The number of genes with significantly altered expression (≥2-fold difference relative to the control) is higher in the sample irradiated with high dose of UV, suggesting that gene expression was UV dose-dependent. Pathway analysis on the upregulated genes at 12 h indicates that the expression of some cell cycle-related genes was predominantly induced irrespective of UV-dose. Interestingly, almost all the genes with significant altered expression were cell cycle-related genes designated as 'Mitotic Genes', which function in the spindle assembly checkpoint. Therefore, even a low dose of UV could affect the transcriptional profile. PMID:27378355

  7. Chelidonine induces mitotic slippage and apoptotic-like death in SGC-7901 human gastric carcinoma cells.

    PubMed

    Qu, Zhongyuan; Zou, Xiang; Zhang, Xiujuan; Sheng, Jiejing; Wang, Yumeng; Wang, Jiaqi; Wang, Chao; Ji, Yubin

    2016-02-01

    mitotic slippage. In addition, apoptotic morphological changes in multinucleated cells were observed, the apoptosis rates increased gradually with administration of chelidonine in a time-dependent manner and the protein levels of caspase-3 increased significantly between 24 and 72 h. Thus, chelidonine induces mitotic slippage, and apoptotic-like death occurs in SGC-7901 cells undergoing mitotic catastrophe. Gastric cancer is a common malignancy, and ranks second in overall cancer-associated mortalities worldwide. The present study demonstrated that chelidonine induces M phase arrest and mitotic slippage of SGC-7901 human gastric carcinoma cells via downregulating the expression of BubR1, Cdk1 and cyclin B1 proteins. With the prolongation of chelidonine treatment, the giant cells with multiple micronuclei underwent mitotic slippage and were maintained in the G1 phase and did not survive. A number of multinucleated cells underwent apoptosis via a caspase-dependent signaling pathway. The current study proposes that chelidonine induces mitotic slippage and apoptotic-like death of SGC-7901 cells. PMID:26677104

  8. Induction of mitotic aneuploidy in lower eukaryotes

    SciTech Connect

    Kappas, A.

    1993-12-31

    Genetic tests for induction of mitotic aneuploidy in lower eukarotes used mainly the fungal systems of Aspergillus nidulans and Saccharomyces cerevisiae. There are several differences between the two systems such as the greater tolerance for aneuploidy and the fertility of triploids in S. cerevisiae, the stability of diploids and the selective advantage of haploids over diploids in Aspergillus and the mycelial growth of Aspergillus. On the other hand several similarities also exist between the two systems such as the general instability and varying growth rate of disomics and the random loss of extra chromosomes which produces more competitive types or the most frequent recovery of certain specific aneuploids. In using lower eukaryotes as test systems for the identification of aneugens several points should be considered which concern the relevance of such systems to higher organisms, the ability to identify primary aneuploidy and distinguish this from events, such as chromosomal breaks, which lead to secondary aneuploidy and the ability to obtain repeatable results. Within the framework of an EEC comparative study for evaluating assays for aneuploidy, a number of chemicals were assayed in A. nidulans for mitotic instability due to malsegregation of chromosomes at cell division.

  9. A cotranslational ubiquitination pathway for quality control of misfolded proteins.

    PubMed

    Wang, Feng; Durfee, Larissa A; Huibregtse, Jon M

    2013-05-01

    Previous studies have indicated that 6%-30% of newly synthesized proteins are rapidly degraded by the ubiquitin-proteasome system; however, the relationship of ubiquitination to translation for these proteins has been unclear. We report that cotranslational ubiquitination (CTU) is a robust process, with 12%-15% of nascent polypeptides being ubiquitinated in human cells. CTU products contained primarily K48-linked polyubiquitin chains, consistent with a proteasomal targeting function. While nascent chains have been shown previously to be ubiquitinated within stalled complexes (CTU(S)), the majority of nascent chain ubiquitination occurred within active translation complexes (CTU(A)). CTU(A) was increased in response to agents that induce protein misfolding, while CTU(S) was increased in response to agents that lead to translational errors or stalling. These results indicate that ubiquitination of nascent polypeptides occurs in two contexts and define CTU(A) as a component of a quality control system that marks proteins for destruction while they are being synthesized. PMID:23583076

  10. Directional Regulation of Enzyme Pathways through the Control of Substrate Channeling on a DNA Origami Scaffold.

    PubMed

    Ke, Guoliang; Liu, Minghui; Jiang, Shuoxing; Qi, Xiaodong; Yang, Yuhe Renee; Wootten, Shaun; Zhang, Fei; Zhu, Zhi; Liu, Yan; Yang, Chaoyong James; Yan, Hao

    2016-06-20

    Artificial multi-enzyme systems with precise and dynamic control over the enzyme pathway activity are of great significance in bionanotechnology and synthetic biology. Herein, we exploit a spatially addressable DNA nanoplatform for the directional regulation of two enzyme pathways (G6pDH-MDH and G6pDH-LDH) through the control of NAD(+) substrate channeling by specifically shifting NAD(+) between the two enzyme pairs. We believe that this concept will be useful for the design of regulatory biological circuits for synthetic biology and biomedicine. PMID:27159899

  11. Controlling pathway dynamics of a four-level quantum system with pulse shaping

    NASA Astrophysics Data System (ADS)

    Cao, Dewen; Yang, Ling; Wang, Yaoxiong; Shuang, Feng; Gao, Fang

    2016-07-01

    The dynamics of two two-photon absorption (TPA) pathways in a four-level quantum system driven by a laser pulse is investigated in this work. An analytical solution for pulse shaping is proposed to be globally optimal for constructive interference between the two pathways, and accurate spectral boundaries for phase modulation are obtained. The TPA rate can be enhanced by a factor of 8.33 with the optimal pulse instead of the transform limited pulse (TL pulse). Simple control strategies modulating both amplitudes and phases are also designed to increase the TPA amplitude along one pathway while decreasing that along the other simultaneously. The strategies are intuitive and the two pathway amplitudes can differ by two orders of magnitude.

  12. Redox Controls and Reaction Pathways during Serpentinization of Abyssal Peridotite

    NASA Astrophysics Data System (ADS)

    Klein, Frieder; Bach, Wolfgang; Kahl, Wolf-Achim; Humphris, Susan

    2013-04-01

    The aqueous alteration of ultramafic rocks liberates substantial amounts of hydrogen via the oxidation of ferrous iron in primary minerals to ferric iron in secondary minerals; however, the underlying mechanisms of this process remain poorly understood. We examined partly to completely serpentinized peridotites from continental rifted margins, mid-ocean ridges, and fore-arc settings of subduction zones recovered during DSDP/ODP Legs 82, 107, 125, 147, 149, 153, 173, 195, 209, 210, and 304/305 using a variety of microscopic and spectroscopic methods. We merged petrographic observations and chemical analyses with thermodynamic reaction path models and hydrothermal experiments to establish basic conceptual mechanisms of peridotite-seawater interactions. In principle, the complex variations in temperature, host-rock composition, water-to-rock ratio, and reaction kinetics are recorded by the secondary mineralogy. Our results suggest that high temperature (>250°C) serpentinization of olivine at low water-to-rock ratios leads to the formation of magnetite and serpentine together with relatively Fe-poor brucite in mesh texture, and thus to strongly reducing conditions (e.g., at Leg 153). If serpentinization of peridotite proceeds at low temperatures, production of magnetite is limited and brucite generally Fe-richer (e.g., Leg 173), which results in somewhat less reducing conditions. Serpentinization of orthopyroxene-rich lithologies does not generate significant amounts of magnetite (unless it undergoes post-serpentinization oxidation), while serpentine in bastite texture is Fe-rich. In addition to protolith composition, the relative dissolution kinetics of olivine and orthopyroxene can influence the silica activity during serpentinization of peridotite and thus control whether the assemblage brucite-serpentine-clinopyroxene-magnetite or serpentine-talc-tremolite-chlorite is stable. The former assemblage is associated with strongly reducing, high pH, silica poor and Ca

  13. Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)

    SciTech Connect

    Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick; Bang, Jiyoung; Kim, Eun-A; Sung, Ki Sa; Yoon, Hyun-Joo; Yoo, Hae Yong; Choi, Cheol Yong

    2014-01-03

    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 conjugation 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.

  14. Random mitotic activities across human embryonic stem cell colonies.

    SciTech Connect

    Jin, Q.; Duggan, R.; Dasa, S.; Li, F.; Chen, L.

    2010-08-01

    A systemic and quantitative study was performed to examine whether different levels of mitotic activities, assessed by the percentage of S-phase cells at any given time point, existed at different physical regions of human embryonic stem (hES) cell colonies at 2, 4, 6 days after cell passaging. Mitotically active cells were identified by the positive incorporation of 5-bromo-2-deoxyuridine (BrdU) within their newly synthesized DNA. Our data indicated that mitotically active cells were often distributed as clusters randomly across the colonies within the examined growth period, presumably resulting from local deposition of newly divided cells. This latter notion was further demonstrated by the confined growth of enhanced green florescence protein (EGFP) expressing cells amongst non-GFP expressing cells. Furthermore, the overall percentage of mitotically active cells remained constantly at about 50% throughout the 6-day culture period, indicating mitotic activities of hES cell cultures were time-independent under current growth conditions.

  15. Identification of a mitotic death signature in cancer cell lines.

    PubMed

    Sakurikar, Nandini; Eichhorn, Joshua M; Alford, Sarah E; Chambers, Timothy C

    2014-02-28

    This study examined the molecular mechanism of action of anti-mitotic drugs. The hypothesis was tested that death in mitosis occurs through sustained mitotic arrest with robust Cdk1 signaling causing complete phosphorylation of Mcl-1 and Bcl-xL, and conversely, that mitotic slippage is associated with incomplete phosphorylation of Mcl-1/Bcl-xL. The results, obtained from studying six different cancer cell lines, strongly support the hypothesis and identify for the first time a unique molecular signature for mitotic death. The findings represent an important advance in understanding anti-mitotic drug action and provide insight into cancer cell susceptibility to such drugs which has important clinical implications. PMID:24099917

  16. 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. PMID:26916504

  17. Adaptive Control Model Reveals Systematic Feedback and Key Molecules in Metabolic Pathway Regulation

    PubMed Central

    Moffitt, Richard A.; Merrill, Alfred H.; Wang, May D.

    2011-01-01

    Abstract Robust behavior in metabolic pathways resembles stabilized performance in systems under autonomous control. This suggests we can apply control theory to study existing regulation in these cellular networks. Here, we use model-reference adaptive control (MRAC) to investigate the dynamics of de novo sphingolipid synthesis regulation in a combined theoretical and experimental case study. The effects of serine palmitoyltransferase over-expression on this pathway are studied in vitro using human embryonic kidney cells. We report two key results from comparing numerical simulations with observed data. First, MRAC simulations of pathway dynamics are comparable to simulations from a standard model using mass action kinetics. The root-sum-square (RSS) between data and simulations in both cases differ by less than 5%. Second, MRAC simulations suggest systematic pathway regulation in terms of adaptive feedback from individual molecules. In response to increased metabolite levels available for de novo sphingolipid synthesis, feedback from molecules along the main artery of the pathway is regulated more frequently and with greater amplitude than from other molecules along the branches. These biological insights are consistent with current knowledge while being new that they may guide future research in sphingolipid biology. In summary, we report a novel approach to study regulation in cellular networks by applying control theory in the context of robust metabolic pathways. We do this to uncover potential insight into the dynamics of regulation and the reverse engineering of cellular networks for systems biology. This new modeling approach and the implementation routines designed for this case study may be extended to other systems. Supplementary Material is available at www.liebertonline.com/cmb. PMID:21314456

  18. Force and Length in the Mitotic Spindle

    PubMed Central

    Dumont, Sophie; Mitchison, Timothy J.

    2009-01-01

    The mitotic spindle assembles to a steady-state length at metaphase through the integrated action of molecular mechanisms that generate and respond to mechanical forces. While molecular mechanisms that produce force have been described, our understanding of how they integrate with each other, and with the assembly-disassembly mechanisms that regulate length, is poor. We review current understanding of the basic architecture and dynamics of the metaphase spindle, and some of the elementary force producing mechanisms. We then discuss models for force integration, and spindle length determination. We also emphasize key missing data that notably includes absolute values of forces, and how they vary as a function of position, within the spindle. PMID:19906577

  19. Prostaglandin D2 pathway upregulation: Relation to asthma severity, control, and TH2 inflammation

    PubMed Central

    Fajt, Merritt L.; Gelhaus, Stacy L.; Freeman, Bruce; Uvalle, Crystal E.; Trudeau, John B.; Holguin, Fernando; Wenzel, Sally E.

    2013-01-01

    Background Bronchoalveolar lavage (BAL) fluid prostaglandin D2 (PGD2) levels are increased in patients with severe, poorly controlled asthma in association with epithelial mast cells (MCs). PGD2, which is generated by hematopoietic prostaglandin D synthase (HPGDS), acts on 3 G protein–coupled receptors, including chemoattractant receptor–homologous molecule expressed on TH2 lymphocytes (CRTH2) and PGD2 receptor 1 (DP1). However, much remains to be understood regarding the presence and activation of these pathway elements in asthmatic patients. Objective We sought to compare the expression and activation of PGD2 pathway elements in bronchoscopically obtained samples from healthy control subjects and asthmatic patients across a range of disease severity and control, as well as in relation to TH2 pathway elements. Methods Epithelial cells and BAL fluid were evaluated for HPGDS (quantitative real-time PCR/immunohistochemistry [IHC]) and PGD2 (ELISA/liquid chromatography mass spectrometry) in relation to levels of MC proteases. Expression of the 2 inflammatory cell receptors DP1 and CRTH2 was evaluated on luminal cells. These PGD2 pathway markers were then compared with asthma severity, level of control, and markers of TH2 inflammation (blood eosinophils and fraction of exhaled nitric oxide). Results Confirming previous results, BAL fluid PGD2 levels were highest in patients with severe asthma (overall P = .0001). Epithelial cell compartment HPGDS mRNA and IHC values differed among groups (P = .008 and P < .0001, respectively) and correlated with MC protease mRNA. CRTH2 mRNA and IHC values were highest in patients with severe asthma (P = .001 and P = .0001, respectively). Asthma exacerbations, poor asthma control, and TH2 inflammatory markers were associated with higher PGD2, HPGDS, and CRTH2 levels. Conclusion The current study identifies coordinated upregulation of the PGD2 pathway in patients with severe, poorly controlled, TH2-high asthma despite corticosteroid

  20. Major Autonomic Neuroregulatory Pathways Underlying Short- and Long-Term Control of Cardiovascular Function.

    PubMed

    Salman, Ibrahim M

    2016-03-01

    Short-term and long-term blood pressure (BP) regulation and its maintenance at levels adequate to perfuse tissue organs involve an integrated action of multiple neural, cardiovascular, renal, endocrine and local tissue control systems. In the recent year, there has been a growing interest in the understanding of neural pathways key to BP control. For instance, through major advances in studies using both anesthetized and conscious animals, our knowledge of the essential neural mechanisms that subserve the baroreceptor, cardiopulmonary and chemoreceptor reflexes, and those evoked by the activation of stress pathways has dramatically increased. While the importance of these neural pathways in the maintenance of cardiovascular homeostasis is well established, the recognition of the central processing nuclei that integrate various afferent inputs to produce synchronous adjustments of autonomic outflows is still progressively expanding. Based on the literature provided thus far, the present review provides an overview in relation to the important neural determinants of BP control and later offers a concise description of major neuronal pathways that control autonomic outflows to the cardiovascular system in the short and long term. PMID:26838031

  1. Noncanonical Wnt signaling pathways in C. elegans converge on POP-1/TCF and control cell polarity.

    PubMed

    Herman, Michael A; Wu, Mingfu

    2004-05-01

    In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway controls a cell migration whereas noncanonical Wnt pathways control the polarities of individual cells. Despite the differences in the identities and interactions among canonical and noncanonical Wnt pathway components, as well as the processes they regulate, almost all C. elegans Wnt pathways involve the sole Tcf homolog, POP-1. Intriguingly, POP-1 is asymmetrically distributed between the daughters of an asymmetric cell division, with the anterior sister cell usually having a higher level of nuclear POP-1 than its posterior sister. At some divisions, asymmetric distribution of POP-1 is controlled by noncanonical Wnt signaling, but at others the asymmetry is generated independently. Recent experiments suggest that despite this elaborate anterior-posterior POP-1 asymmetry, the quantity of POP-1 protein may have less to do with the subsequent determination of fate than does the quality of the POP-1 protein in the cell. In this review, we will embark on a quest to understand Quality (1), at least from the standpoint of the effect POP/Tcf quality has on the control of cell polarity in C. elegans. PMID:14977564

  2. Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

    SciTech Connect

    Yang, Feng; Camp, David G.; Gritsenko, Marina A.; Luo, Quanzhou; Kelly, Ryan T.; Clauss, Therese RW; Brinkley, William R.; Smith, Richard D.; Stenoien, David L.

    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 including 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.

  3. Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging

    PubMed Central

    Sapir, Amir; Tsur, Assaf; Koorman, Thijs; Ching, Kaitlin; Mishra, Prashant; Bardenheier, Annabelle; Podolsky, Lisa; Bening-Abu-Shach, Ulrike; Boxem, Mike; Chou, Tsui-Fen; Broday, Limor; Sternberg, Paul W.

    2014-01-01

    Many metabolic pathways are critically regulated during development and aging but little is known about the molecular mechanisms underlying this regulation. One key metabolic cascade in eukaryotes is the mevalonate pathway. It catalyzes the synthesis of sterol and nonsterol isoprenoids, such as cholesterol and ubiquinone, as well as other metabolites. In humans, an age-dependent decrease in ubiquinone levels and changes in cholesterol homeostasis suggest that mevalonate pathway activity changes with age. However, our knowledge of the mechanistic basis of these changes remains rudimentary. We have identified a regulatory circuit controlling the sumoylation state of Caenorhabditis elegans HMG-CoA synthase (HMGS-1). This protein is the ortholog of human HMGCS1 enzyme, which mediates the first committed step of the mevalonate pathway. In vivo, HMGS-1 undergoes an age-dependent sumoylation that is balanced by the activity of ULP-4 small ubiquitin-like modifier protease. ULP-4 exhibits an age-regulated expression pattern and a dynamic cytoplasm-to-mitochondria translocation. Thus, spatiotemporal ULP-4 activity controls the HMGS-1 sumoylation state in a mechanism that orchestrates mevalonate pathway activity with the age of the organism. To expand the HMGS-1 regulatory network, we combined proteomic analyses with knockout studies and found that the HMGS-1 level is also governed by the ubiquitin–proteasome pathway. We propose that these conserved molecular circuits have evolved to govern the level of mevalonate pathway flux during aging, a flux whose dysregulation is associated with numerous age-dependent cardiovascular and cancer pathologies. PMID:25187565

  4. Transportin acts to regulate mitotic assembly events by target binding rather than Ran sequestration

    PubMed Central

    Bernis, Cyril; Swift-Taylor, Beth; Nord, Matthew; Carmona, Sarah; Chook, Yuh Min; Forbes, Douglass J.

    2014-01-01

    The nuclear import receptors importin β and transportin play a different role in mitosis: both act phenotypically as spatial regulators to ensure that mitotic spindle, nuclear membrane, and nuclear pore assembly occur exclusively around chromatin. Importin β is known to act by repressing assembly factors in regions distant from chromatin, whereas RanGTP produced on chromatin frees factors from importin β for localized assembly. The mechanism of transportin regulation was unknown. Diametrically opposed models for transportin action are as follows: 1) indirect action by RanGTP sequestration, thus down-regulating release of assembly factors from importin β, and 2) direct action by transportin binding and inhibiting assembly factors. Experiments in Xenopus assembly extracts with M9M, a superaffinity nuclear localization sequence that displaces cargoes bound by transportin, or TLB, a mutant transportin that can bind cargo and RanGTP simultaneously, support direct inhibition. Consistently, simple addition of M9M to mitotic cytosol induces microtubule aster assembly. ELYS and the nucleoporin 107–160 complex, components of mitotic kinetochores and nuclear pores, are blocked from binding to kinetochores in vitro by transportin, a block reversible by M9M. In vivo, 30% of M9M-transfected cells have spindle/cytokinesis defects. We conclude that the cell contains importin β and transportin “global positioning system”or “GPS” pathways that are mechanistically parallel. PMID:24478460

  5. Nercc1, a mammalian NIMA-family kinase, binds the Ran GTPase and regulates mitotic progression

    PubMed Central

    Roig, Joan; Mikhailov, Alexei; Belham, Christopher; Avruch, Joseph

    2002-01-01

    The protein kinase NIMA is an indispensable pleiotropic regulator of mitotic progression in Aspergillus. Although several mammalian NIMA-like kinases (Neks) are known, none appears to have the broad importance for mitotic regulation attributed to NIMA. Nercc1 is a new NIMA-like kinase that regulates chromosome alignment and segregation in mitosis. Its NIMA-like catalytic domain is followed by a noncatalytic tail containing seven repeats homologous to those of the Ran GEF, RCC1, a Ser/Thr/Pro-rich segment, and a coiled-coil domain. Nercc1 binds to another NIMA-like kinase, Nek6, and also binds specifically to the Ran GTPase through both its catalytic and its RCC1-like domains, preferring RanGDP in vivo. Nercc1 exists as a homooligomer and can autoactivate in vitro by autophosphorylation. Nercc1 is a cytoplasmic protein that is activated during mitosis and is avidly phosphorylated by active p34Cdc2. Microinjection of anti-Nercc1 antibodies in prophase results in spindle abnormalities and/or chromosomal misalignment. In Ptk2 cells the outcome is prometaphase arrest or aberrant chromosome segregation and aneuploidy, whereas in CFPAC-1 cells prolonged arrest in prometaphase is the usual response. Nercc1 and its partner Nek6 represent a new signaling pathway that regulates mitotic progression. PMID:12101123

  6. PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator

    PubMed Central

    Kim, Yoosik; Lee, Jung Hyun; Park, Jong-Eun; Cho, Jun; Yi, Hyerim; Kim, V. Narry

    2014-01-01

    dsRNA-dependent protein kinase R (PKR) is a ubiquitously expressed enzyme well known for its roles in immune response. Upon binding to viral dsRNA, PKR undergoes autophosphorylation, and the phosphorylated PKR (pPKR) regulates translation and multiple signaling pathways in infected cells. Here, we found that PKR is activated in uninfected cells, specifically during mitosis, by binding to dsRNAs formed by inverted Alu repeats (IRAlus). While PKR and IRAlu-containing RNAs are segregated in the cytosol and nucleus of interphase cells, respectively, they interact during mitosis when nuclear structure is disrupted. Once phosphorylated, PKR suppresses global translation by phosphorylating the α subunit of eukaryotic initiation factor 2 (eIF2α). In addition, pPKR acts as an upstream kinase for c-Jun N-terminal kinase and regulates the levels of multiple mitotic factors such as CYCLINS A and B and POLO-LIKE KINASE 1 and phosphorylation of HISTONE H3. Disruption of PKR activation via RNAi or expression of a transdominant-negative mutant leads to misregulation of the mitotic factors, delay in mitotic progression, and defects in cytokinesis. Our study unveils a novel function of PKR and endogenous dsRNAs as signaling molecules during the mitosis of uninfected cells. PMID:24939934

  7. Population dynamics of a meiotic/mitotic expansion model for the fragile X syndrome

    SciTech Connect

    Ashley, A.E.; Sherman, S.L.

    1995-12-01

    A model to explain the mutational process and population dynamics of the fragile X syndrome is presented. The mutational mechanism was assumed to be a multi-pathway, multistep process. Expansion of CGG repeats was based on an underlying biological process and was assumed to occur at two time points: meiosis and early embryonic development (mitosis). Meiotic expansion was assumed to occur equally in oogenesis and spermatogenesis, while mitotic expansion was restricted to somatic, or constitutional, alleles of maternal origin. Testable hypotheses were predicted by this meiotic/mitotic model. First, parental origin of mutation is predicted to be associated with the risk of a woman to have a full-mutation child. Second, {open_quotes}contractions{close_quotes} seen in premutation male transmissions are predicted not to be true contractions in repeat size, but a consequence of the lack of mitotic expansion in paternally derived alleles. Third, a portion of full-mutation males should have full-mutation alleles in their sperm, due to the lack of complete selection against the full-mutation female. Fourth, a specific premutation-allele frequency distribution is predicted and differs from that based on models assuming only meiotic expansion. Last, it is predicted that {approximately}65 generations are required to achieve equilibrium, but this depends greatly on the expansion probabilities. 42 refs., 4 figs., 4 tabs.

  8. Histone deacetylase inhibitors promote glioma cell death by G2 checkpoint abrogation leading to mitotic catastrophe.

    PubMed

    Cornago, M; Garcia-Alberich, C; Blasco-Angulo, N; Vall-Llaura, N; Nager, M; Herreros, J; Comella, J X; Sanchis, D; Llovera, M

    2014-01-01

    Glioblastoma multiforme is resistant to conventional anti-tumoral treatments due to its infiltrative nature and capability of relapse; therefore, research efforts focus on characterizing gliomagenesis and identifying molecular targets useful on therapy. New therapeutic strategies are being tested in patients, such as Histone deacetylase inhibitors (HDACi) either alone or in combination with other therapies. Here two HDACi included in clinical trials have been tested, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), to characterize their effects on glioma cell growth in vitro and to determine the molecular changes that promote cancer cell death. We found that both HDACi reduce glioma cell viability, proliferation and clonogenicity. They have multiple effects, such as inducing the production of reactive oxygen species (ROS) and activating the mitochondrial apoptotic pathway, nevertheless cell death is not prevented by the pan-caspase inhibitor Q-VD-OPh. Importantly, we found that HDACi alter cell cycle progression by decreasing the expression of G2 checkpoint kinases Wee1 and checkpoint kinase 1 (Chk1). In addition, HDACi reduce the expression of proteins involved in DNA repair (Rad51), mitotic spindle formation (TPX2) and chromosome segregation (Survivin) in glioma cells and in human glioblastoma multiforme primary cultures. Therefore, HDACi treatment causes glioma cell entry into mitosis before DNA damage could be repaired and to the formation of an aberrant mitotic spindle that results in glioma cell death through mitotic catastrophe-induced apoptosis. PMID:25275596

  9. Advanced Sensors and Controls for Building Applications: Market Assessment and Potential R&D Pathways

    SciTech Connect

    Brambley, Michael R.; Haves, Philip; McDonald, Sean C.; Torcellini, Paul; Hansen, David G.; Holmberg, David; Roth, Kurt

    2005-04-13

    Significant energy savings can be achieved in commercial building operation, along with increased comfort and control for occupants, through the implementation of advanced technologies. This document provides a market assessment of existing building sensors and controls and presents a range of technology pathways (R&D options) for pursuing advanced sensors and building control strategies. This paper is actually a synthesis of five other white papers: the first describes the market assessment including estimates of market potential and energy savings for sensors and control strategies currently on the market as well as a discussion of market barriers to these technologies. The other four cover technology pathways: (1) current applications and strategies for new applications, (2) sensors and controls, (3) networking, security, and protocols and standards, and (4) automated diagnostics, performance monitoring, commissioning, optimal control and tools. Each technology pathway chapter gives an overview of the technology or application. This is followed by a discussion of needs and the current status of the technology. Finally, a series of research topics is proposed.

  10. Nutritional Control of Cell Size by the Greatwall-Endosulfine-PP2A·B55 Pathway.

    PubMed

    Chica, Nathalia; Rozalén, Ana Elisa; Pérez-Hidalgo, Livia; Rubio, Angela; Novak, Bela; Moreno, Sergio

    2016-02-01

    Proliferating cells adjust their cell size depending on the nutritional environment. Cells are large in rich media and small in poor media. This physiological response has been demonstrated in both unicellular and multicellular organisms. Here we show that the greatwall-endosulfine (Ppk18-Igo1 in fission yeast) pathway couples the nutritional environment to the cell-cycle machinery by regulating the activity of PP2A·B55. In the presence of nutrients, greatwall (Ppk18) protein kinase is inhibited by TORC1 and PP2A·B55 is active. High levels of PP2A·B55 prevent the activation of mitotic Cdk1·Cyclin B, and cells increase in size in G2 before they undergo mitosis. When nutrients are limiting, TORC1 activity falls off, and the activation of greatwall (Ppk18) leads to the phosphorylation of endosulfine (Igo1) and inhibition of PP2A·B55, which in turn allows full activation of Cdk1·CyclinB and entry into mitosis with a smaller cell size. Given the conservation of this pathway, it is reasonable to assume that this mechanism operates in higher eukaryotes, as well. PMID:26776736

  11. Suicide by people in a community justice pathway: population-based nested case-control study.

    PubMed

    King, Carlene; Senior, Jane; Webb, Roger T; Millar, Tim; Piper, Mary; Pearsall, Alison; Humber, Naomi; Appleby, Louis; Shaw, Jenny

    2015-08-01

    The elevated risk of suicide in prison and after release is a well-recognised and serious problem. Despite this, evidence concerning community-based offenders' suicide risk is sparse. We conducted a population-based nested case-control study of all people in a community justice pathway in England and Wales. Our data show 13% of general population suicides were in community justice pathways before death. Suicide risks were highest among individuals receiving police cautions, and those having recent, or impending prosecution for sexual offences. Findings have implications for the training and practice of clinicians identifying and assessing suicidality, and offering support to those at elevated risk. PMID:26159602

  12. The LKB1-AMPK pathway: metabolism and growth control in tumor suppression

    PubMed Central

    Shackelford, David B.; Shaw, Reuben J.

    2009-01-01

    In the past decade, studies of the human tumor suppressor LKB1 have uncovered a novel signaling pathway that links cell metabolism to growth control and cell polarity. LKB1 encodes a serine/threonine kinase that directly phosphorylates and activates AMPK, a central metabolic sensor. AMPK regulates lipid, cholesterol and glucose metabolism in specialized metabolic tissues such as liver, muscle, and adipose, a function that has made it a key therapeutic target in patients with diabetes. The connection of AMPK with several tumor suppressors suggests that therapeutic manipulation of this pathway with established diabetes drugs warrants further investigation in patients with cancer. PMID:19629071

  13. Cardiomyocytes undergo apoptosis in human immunodeficiency virus cardiomyopathy through mitochondrion- and death receptor-controlled pathways

    PubMed Central

    Twu, Cheryl; Liu, Nancy Q.; Popik, Waldemar; Bukrinsky, Michael; Sayre, James; Roberts, Jaclyn; Rania, Shammas; Bramhandam, Vishnu; Roos, Kenneth P.; MacLellan, W. Robb; Fiala, Milan

    2002-01-01

    We investigated 18 AIDS hearts (5 with and 13 without cardiomyopathy) by using immunocytochemistry and computerized image analysis regarding the roles of HIV-1 proteins and tumor necrosis factor ligands in HIV cardiomyopathy (HIVCM). HIVCM and cardiomyocyte apoptosis were significantly related to each other and to the expression by inflammatory cells of gp120 and tumor necrosis factor-α. In HIVCM heart, active caspase 9, a component of the mitochondrion-controlled apoptotic pathway, and the elements of the death receptor-mediated pathway, tumor necrosis factor-α and Fas ligand, were expressed strongly on macrophages and weakly on cardiomyocytes. HIVCM showed significantly greater macrophage infiltration and cardiomyocyte apoptosis rate compared with non-HIVCM. HIV-1 entered cultured neonatal rat ventricular myocytes by macropinocytosis but did not replicate. HIV-1- or gp120-induced apoptosis of rat myocytes through a mitochondrion-controlled pathway, which was inhibited by heparin, AOP-RANTES, or pertussis toxin, suggesting that cardiomyocyte apoptosis is induced by signaling through chemokine receptors. In conclusion, in patients with HIVCM, cardiomyocytes die through both mitochondrion- and death receptor-controlled apoptotic pathways. PMID:12379743

  14. Improving fatty acids production by engineering dynamic pathway regulation and metabolic control

    PubMed Central

    Xu, Peng; Li, Lingyun; Zhang, Fuming; Stephanopoulos, Gregory; Koffas, Mattheos

    2014-01-01

    Global energy demand and environmental concerns have stimulated increasing efforts to produce carbon-neutral fuels directly from renewable resources. Microbially derived aliphatic hydrocarbons, the petroleum-replica fuels, have emerged as promising alternatives to meet this goal. However, engineering metabolic pathways with high productivity and yield requires dynamic redistribution of cellular resources and optimal control of pathway expression. Here we report a genetically encoded metabolic switch that enables dynamic regulation of fatty acids (FA) biosynthesis in Escherichia coli. The engineered strains were able to dynamically compensate the critical enzymes involved in the supply and consumption of malonyl-CoA and efficiently redirect carbon flux toward FA biosynthesis. Implementation of this metabolic control resulted in an oscillatory malonyl-CoA pattern and a balanced metabolism between cell growth and product formation, yielding 15.7- and 2.1-fold improvement in FA titer compared with the wild-type strain and the strain carrying the uncontrolled metabolic pathway. This study provides a new paradigm in metabolic engineering to control and optimize metabolic pathways facilitating the high-yield production of other malonyl-CoA–derived compounds. PMID:25049420

  15. p21-activated kinase 4 regulates mitotic spindle positioning and orientation.

    PubMed

    Bompard, Guillaume; Morin, Nathalie

    2012-01-01

    During mitosis, microtubules (MTs) are massively rearranged into three sets of highly dynamic MTs that are nucleated from the centrosomes to form the mitotic spindle. Tight regulation of spindle positioning in the dividing cell and chromosome alignment at the center of the metaphase spindle are required to ensure perfect chromosome segregation and to position the cytokinetic furrow that will specify the two daughter cells. Spindle positioning requires regulation of MT dynamics, involving depolymerase activities together with cortical and kinetochore-mediated pushing and pulling forces acting on astral MTs and kinetochore fibres. These forces rely on MT motor activities. Cortical pulling forces exerted on astral MTs depend upon dynein/dynactin complexes and are essential in both symmetric and asymmetric cell division. A well-established spindle positioning pathway regulating the cortical targeting of dynein/dynactin involves the conserved LGN (Leu-Gly-Asn repeat-enriched-protein) and NuMA (microtubule binding nuclear mitotic apparatus protein) complex. Spindle orientation is also regulated by integrin-mediated cell adhesion and actin retraction fibres that respond to mechanical stress and are influenced by the microenvironment of the dividing cell. Altering the capture of astral MTs or modulating pulling forces affects spindle position, which can impair cell division, differentiation and embryogenesis. In this general scheme, the activity of mitotic kinases such as Auroras and Plk1 (Polo-like kinase 1) is crucial. Recently, the p21-activated kinases (PAKs) emerged as novel important players in mitotic progression. In our recent article, we demonstrated that PAK4 regulates spindle positioning in symmetric cell division. In this commentary, and in light of recent published studies, we discuss how PAK4 could participate in the regulation of mechanisms involved in spindle positioning and orientation. PMID:22960742

  16. Cell-cycle control as a target for calcium, hormonal and developmental signals: the role of phosphorylation in the retinoblastoma-centred pathway

    PubMed Central

    Dudits, Dénes; Ábrahám, Edit; Miskolczi, Pál; Ayaydin, Ferhan; Bilgin, Metin; Horváth, Gábor V.

    2011-01-01

    Background During the life cycle of plants, both embryogenic and post-embryogenic growth are essentially based on cell division and cell expansion that are under the control of inherited developmental programmes modified by hormonal and environmental stimuli. Considering either stimulation or inhibition of plant growth, the key role of plant hormones in the modification of cell division activities or in the initiation of differentiation is well supported by experimental data. At the same time there is only limited insight into the molecular events that provide linkage between the regulation of cell-cycle progression and hormonal and developmental control. Studies indicate that there are several alternative ways by which hormonal signalling networks can influence cell division parameters and establish functional links between regulatory pathways of cell-cycle progression and genes and protein complexes involved in organ development. Scope An overview is given here of key components in plant cell division control as acceptors of hormonal and developmental signals during organ formation and growth. Selected examples are presented to highlight the potential role of Ca2+-signalling, the complex actions of auxin and cytokinins, regulation by transcription factors and alteration of retinoblastoma-related proteins by phosphorylation. Conclusions Auxins and abscisic acid can directly influence expression of cyclin, cyclin-dependent kinase (CDK) genes and activities of CDK complexes. D-type cyclins are primary targets for cytokinins and over-expression of CyclinD3;1 can enhance auxin responses in roots. A set of auxin-activated genes (AXR1–ARGOS–ANT) controls cell number and organ size through modification of CyclinD3;1 gene expression. The SHORT ROOT (SHR) and SCARECROW (SCR) transcriptional factors determine root patterning by activation of the CYCD6;1 gene. Over-expression of the EBP1 gene (plant homologue of the ErbB-3 epidermal growth factor receptor-binding protein

  17. [Aging and the control of the insulin-FOXO signaling pathway].

    PubMed

    Brunet, Anne

    2012-03-01

    Aging is a complex process that is accompanied by the onset of a series of age-related diseases, including Alzheimer's disease. Aging is controlled by a combination of genetic and environmental factors. Among the genes that regulate aging, the insulin-FOXO signaling pathway plays a central role, as this pathway regulates lifespan in multiple species, such as worms, flies, and mice. In humans, exceptional longevity - being a centenarian - is also associated with genetic variation in this insulin-FOXO pathway. Recent evidence indicates that the FOXO family of transcription factors plays a key role in the self-renewal of adult and embryonic stem cells, which could contribute to tissue regeneration. Understanding the mechanisms underlying aging should help better prevent and treat age-dependent diseases. PMID:22480657

  18. Coupling spindle position with mitotic exit in budding yeast: The multifaceted role of the small GTPase Tem1

    PubMed Central

    Scarfone, Ilaria; Piatti, Simonetta

    2015-01-01

    The budding yeast S. cerevisiae divides asymmetrically and is an excellent model system for asymmetric cell division. As for other asymmetrically dividing cells, proper spindle positioning along the mother-daughter polarity axis is crucial for balanced chromosome segregation. Thus, a surveillance mechanism named Spindle Position Checkpoint (SPOC) inhibits mitotic exit and cytokinesis until the mitotic spindle is properly oriented, thereby preventing the generation of cells with aberrant ploidies. The small GTPase Tem1 is required to trigger a Hippo-like protein kinase cascade, named Mitotic Exit Network (MEN), that is essential for mitotic exit and cytokinesis but also contributes to correct spindle alignment in metaphase. Importantly, Tem1 is the target of the SPOC, which relies on the activity of the GTPase-activating complex (GAP) Bub2-Bfa1 to keep Tem1 in the GDP-bound inactive form. Tem1 forms a hetero-trimeric complex with Bub2-Bfa1 at spindle poles (SPBs) that accumulates asymmetrically on the bud-directed spindle pole during mitosis when the spindle is properly positioned. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. We have recently shown that Tem1 residence at SPBs depends on its nucleotide state and, importantly, asymmetry of the Bub2-Bfa1-Tem1 complex does not promote mitotic exit but rather controls spindle positioning. PMID:26507466

  19. Regulation of the Action of Early Mitotic Inhibitor 1 on the Anaphase-promoting Complex/Cyclosome by Cyclin-dependent Kinases*

    PubMed Central

    Moshe, Yakir; Bar-On, Ortal; Ganoth, Dvora; Hershko, Avram

    2011-01-01

    Cell cycle regulation is characterized by alternating activities of cyclin-dependent kinases (CDKs) and of the ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). During S-phase APC/C is inhibited by early mitotic inhibitor 1 (Emi1) to allow the accumulation of cyclins A and B and to prevent re-replication. Emi1 is degraded at prophase by a Plk1-dependent pathway. Recent studies in which the degradation pathway of Emi1 was disrupted have shown that APC/C is activated at mitotic entry despite stabilization of Emi1. These results suggested the possibility of additional mechanisms other than degradation of Emi1, which release APC/C from inhibition by Emi1 upon entry into mitosis. In this study we report one such mechanism, by which the ability of Emi1 to inhibit APC/C is negatively regulated by CDKs. We show that in Plk1-inhibited cells Emi1 is stabilized and phosphorylated, that Emi1 is phosphorylated by CDKs in mitotic but not S-phase cell extracts, and that Emi1 phosphorylation by mitotic cell extracts or purified CDKs markedly reduces the ability of Emi1 to bind and to inhibit APC/C. Finally, we show that the addition of extracts from S-phase cells to extracts from mitotic cells protects Emi1 from CDK-mediated inactivation. PMID:21454540

  20. 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.

  1. Paravascular pathways contribute to vasculitis and neuroinflammation after subarachnoid hemorrhage independently of glymphatic control

    PubMed Central

    Luo, C; Yao, X; Li, J; He, B; Liu, Q; Ren, H; Liang, F; Li, M; Lin, H; Peng, J; Yuan, T F; Pei, Z; Su, H

    2016-01-01

    Subarachnoid hemorrhage (SAH) is a devastating disease with high mortality. The mechanisms underlying its pathological complications have not been fully identified. Here, we investigate the potential involvement of the glymphatic system in the neuropathology of SAH. We demonstrate that blood components rapidly enter the paravascular space following SAH and penetrate into the perivascular parenchyma throughout the brain, causing disastrous events such as cerebral vasospasm, delayed cerebral ischemia, microcirculation dysfunction and widespread perivascular neuroinflammation. Clearance of the paravascular pathway with tissue-type plasminogen activator ameliorates the behavioral deficits and alleviates histological injury of SAH. Interestingly, AQP4−/− mice showed no improvements in neurological deficits and neuroinflammation at day 7 after SAH compared with WT control mice. In conclusion, our study proves that the paravascular pathway dynamically mediates the pathological complications following acute SAH independently of glymphatic control. PMID:27031957

  2. Curcumin suppresses the dynamic instability of microtubules, activates the mitotic checkpoint and induces apoptosis in MCF-7 cells.

    PubMed

    Banerjee, Mithu; Singh, Parminder; Panda, Dulal

    2010-08-01

    In this study, curcumin, a potential anticancer agent, was found to dampen the dynamic instability of individual microtubules in living MCF-7 cells. It strongly reduced the rate and extent of shortening states, and modestly reduced the rate and extent of growing states. In addition, curcumin decreased the fraction of time microtubules spent in the growing state and strongly increased the time microtubules spent in the pause state. Brief treatment with curcumin depolymerized mitotic microtubules, perturbed microtubule-kinetochore attachment and disturbed the mitotic spindle structure. Curcumin also perturbed the localization of the kinesin protein Eg5 and induced monopolar spindle formation. Further, curcumin increased the accumulation of Mad2 and BubR1 at the kinetochores, indicating that it activated the mitotic checkpoint. In addition, curcumin treatment increased the metaphase/anaphase ratio, indicating that it can delay mitotic progression from the metaphase to anaphase. We provide evidence suggesting that the affected cells underwent apoptosis via the p53-dependent apoptotic pathway. The results support the idea that kinetic stabilization of microtubule dynamics assists in the nuclear translocation of p53. Curcumin exerted additive effects when combined with vinblastine, a microtubule depolymerizing drug, whereas the combination of curcumin with paclitaxel, a microtubule-stabilizing drug, produced an antagonistic effect on the inhibition of MCF-7 cell proliferation. The results together suggested that curcumin inhibited MCF-7 cell proliferation by inhibiting the assembly dynamics of microtubules. PMID:20646066

  3. 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

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

    PubMed

    Platani, Melpomeni; Trinkle-Mulcahy, Laura; Porter, Michael; Jeyaprakash, A Arockia; Earnshaw, William C

    2015-07-01

    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

  5. Metabolic Pathway Signatures Associated with Urinary Metabolite Biomarkers Differentiate Bladder Cancer Patients from Healthy Controls

    PubMed Central

    Kim, Won Tae; Yun, Seok Joong; Yan, Chunri; Jeong, Pildu; Kim, Ye Hwan; Lee, Il-Seok; Kang, Ho-Won; Park, Sunghyouk; Moon, Sung-Kwon; Choi, Yung-Hyun; Choi, Young Deuk; Kim, Isaac Yi

    2016-01-01

    Purpose Our previous high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry study identified bladder cancer (BCA)-specific urine metabolites, including carnitine, acylcarnitines, and melatonin. The objective of the current study was to determine which metabolic pathways are perturbed in BCA, based on our previously identified urinary metabolome. Materials and Methods A total of 135 primary BCA samples and 26 control tissue samples from healthy volunteers were analyzed. The association between specific urinary metabolites and their related encoding genes was analyzed. Results Significant alterations in the carnitine-acylcarnitine and tryptophan metabolic pathways were detected in urine specimens from BCA patients compared to those of healthy controls. The expression of eight genes involved in the carnitine-acylcarnitine metabolic pathway (CPT1A, CPT1B, CPT1C, CPT2, SLC25A20, and CRAT) or tryptophan metabolism (TPH1 and IDO1) was assessed by RT-PCR in our BCA cohort (n=135). CPT1B, CPT1C, SLC25A20, CRAT, TPH1, and IOD1 were significantly downregulated in tumor tissues compared to normal bladder tissues (p<0.05 all) of patients with non-muscle invasive BCA, whereas CPT1B, CPT1C, CRAT, and TPH1 were downregulated in those with muscle invasive BCA (p<0.05), with no changes in IDO1 expression. Conclusion Alterations in the expression of genes associated with the carnitine-acylcarnitine and tryptophan metabolic pathways, which were the most perturbed pathways in BCA, were determined. PMID:27189278

  6. 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.

  7. Sharing of mitotic pre-ribosomal particles between daughter cells.

    PubMed

    Sirri, Valentina; Jourdan, Nathalie; Hernandez-Verdun, Danièle; Roussel, Pascal

    2016-04-15

    Ribosome biogenesis is a fundamental multistep process initiated by the synthesis of 90S pre-ribosomal particles in the nucleoli of higher eukaryotes. Even though synthesis of ribosomes stops during mitosis while nucleoli disappear, mitotic pre-ribosomal particles persist as observed in pre-nucleolar bodies (PNBs) during telophase. To further understand the relationship between the nucleolus and the PNBs, the presence and the fate of the mitotic pre-ribosomal particles during cell division were investigated. We demonstrate that the recently synthesized 45S precursor ribosomal RNAs (pre-rRNAs) as well as the 32S and 30S pre-rRNAs are maintained during mitosis and associated with the chromosome periphery together with pre-rRNA processing factors. Maturation of the mitotic pre-ribosomal particles, as assessed by the stability of the mitotic pre-rRNAs, is transiently arrested during mitosis by a cyclin-dependent kinase (CDK)1-cyclin-B-dependent mechanism and can be restored by CDK inhibitor treatments. At the M-G1 transition, the resumption of mitotic pre-rRNA processing in PNBs does not induce the disappearance of PNBs; this only occurs when functional nucleoli reform. Strikingly, during their maturation process, mitotic pre-rRNAs localize in reforming nucleoli. PMID:26929073

  8. Timeless links replication termination to mitotic kinase activation.

    PubMed

    Dheekollu, Jayaraju; Wiedmer, Andreas; Hayden, James; Speicher, David; Gotter, Anthony L; Yen, Tim; Lieberman, Paul M

    2011-01-01

    The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication. PMID:21573113

  9. Gradual implementation of the meiotic recombination program via checkpoint pathways controlled by global DSB levels.

    PubMed

    Joshi, Neeraj; Brown, M Scott; Bishop, Douglas K; Börner, G Valentin

    2015-03-01

    During meiosis, Spo11-induced double-strand breaks (DSBs) are processed into crossovers, ensuring segregation of homologous chromosomes (homologs). Meiotic DSB processing entails 5' end resection and preferred strand exchange with the homolog rather than the sister chromatid (homolog bias). In many organisms, DSBs appear gradually along the genome. Here we report unexpected effects of global DSB levels on local recombination events. Early-occurring, low-abundance "scout" DSBs lack homolog bias. Their resection and interhomolog processing are controlled by the conserved checkpoint proteins Tel1(ATM) kinase and Pch2(TRIP13) ATPase. Processing pathways controlled by Mec1(ATR) kinase take over these functions only above a distinct DSB threshold, resulting in progressive strengthening of the homolog bias. We conclude that Tel1(ATM)/Pch2 and Mec1(ATR) DNA damage response pathways are sequentially activated during wild-type meiosis because of their distinct sensitivities to global DSB levels. Moreover, relative DSB order controls the DSB repair pathway choice and, ultimately, recombination outcome. PMID:25661491

  10. Phosphorylation of EB2 by Aurora B and CDK1 ensures mitotic progression and genome stability

    PubMed Central

    Iimori, Makoto; Watanabe, Sugiko; Kiyonari, Shinichi; Matsuoka, Kazuaki; Sakasai, Ryo; Saeki, Hiroshi; Oki, Eiji; Kitao, Hiroyuki; Maehara, Yoshihiko

    2016-01-01

    Temporal regulation of microtubule dynamics is essential for proper progression of mitosis and control of microtubule plus-end tracking proteins by phosphorylation is an essential component of this regulation. Here we show that Aurora B and CDK1 phosphorylate microtubule end-binding protein 2 (EB2) at multiple sites within the amino terminus and a cluster of serine/threonine residues in the linker connecting the calponin homology and end-binding homology domains. EB2 phosphorylation, which is strictly associated with mitotic entry and progression, reduces the binding affinity of EB2 for microtubules. Expression of non-phosphorylatable EB2 induces stable kinetochore microtubule dynamics and delays formation of bipolar metaphase plates in a microtubule binding-dependent manner, and leads to aneuploidy even in unperturbed mitosis. We propose that Aurora B and CDK1 temporally regulate the binding affinity of EB2 for microtubules, thereby ensuring kinetochore microtubule dynamics, proper mitotic progression and genome stability. PMID:27030108

  11. Mitotic stopwatch for the blast fungus Magnaporthe oryzae during invasion of rice cells.

    PubMed

    Jones, Kiersun; Jenkinson, Cory B; Borges Araújo, Maíra; Zhu, Jie; Kim, Rebecca Y; Kim, Dong Won; Khang, Chang Hyun

    2016-08-01

    To study nuclear dynamics of Magnaporthe oryzae, we developed a novel mitotic reporter strain with GFP-NLS (localized in nuclei during interphase but in the cytoplasm during mitosis) and H1-tdTomato (localized in nuclei throughout the cell cycle). Time-lapse confocal microscopy of the reporter strain during host cell invasion provided several new insights into nuclear division and migration in M. oryzae: (i) mitosis lasts about 5min; (ii) mitosis is semi-closed; (iii) septal pores are closed during mitosis; and (iv) a nucleus exhibits extreme constriction (approximately from 2μm to 0.5μm), elongation (over 5μm), and long migration (over 16μm). Our observations raise new questions about mechanisms controlling the mitotic dynamics, and the answers to these questions may result in new means to prevent fungal proliferation without negatively affecting the host cell cycle. PMID:27321562

  12. Kizuna is a novel mitotic substrate for CDC25B phosphatase.

    PubMed

    Thomas, Yann; Peter, Marion; Mechali, Francisca; Blanchard, Jean-Marie; Coux, Olivier; Baldin, Véronique

    2014-01-01

    CDC25 dual-specificity phosphatases play a central role in cell cycle control through the activation of Cyclin-Dependent Kinases (CDKs). Expression during mitosis of a stabilized CDC25B mutant (CDC25B-DDA), which cannot interact with the F-box protein βTrCP for proteasome-dependent degradation, causes mitotic defects and chromosome segregation errors in mammalian cells. We found, using the same CDC25B mutant, that stabilization and failure to degrade CDC25B during mitosis lead to the appearance of multipolar spindle cells resulting from a fragmentation of pericentriolar material (PCM) and abolish mitotic Plk1-dependent phosphorylation of Kizuna (Kiz), which is essential for the function of Kiz in maintaining spindle pole integrity. Thus, in mitosis Kiz is a new substrate of CDC25B whose dephosphorylation following CDC25B stabilization leads to the formation of multipolar spindles. Furthermore, endogenous Kiz and CDC25B interact only in mitosis, suggesting that Kiz phosphorylation depends on a balance between CDC25B and Plk1 activities. Our data identify a novel mitotic substrate of CDC25B phosphatase that plays a key role in mitosis control. PMID:25558830

  13. Observing and Controlling the Folding Pathway of DNA Origami at the Nanoscale.

    PubMed

    Lee Tin Wah, Jonathan; David, Christophe; Rudiuk, Sergii; Baigl, Damien; Estevez-Torres, André

    2016-02-23

    DNA origami is a powerful method to fold DNA into rationally designed nanostructures that holds great promise for bionanotechnology. However, the folding mechanism has yet to be fully resolved, principally due to a lack of data with single molecule resolution. To address this issue, we have investigated in detail, using atomic force microscopy, the morphological evolution of hundreds of individual rectangular origamis in solution as a function of temperature. Significant structural changes were observed between 65 and 55 °C both for folding and melting, and six structural intermediates were identified. Under standard conditions, folding was initiated at the edges of the rectangle and progressed toward the center. Melting occurred through the reverse pathway until the structures were significantly disrupted but ended through a different pathway involving out-of-equilibrium chainlike structures. Increasing the relative concentration of center to edge staples dramatically modified the folding pathway to a mechanism progressing from the center toward the edges. These results indicate that the folding pathway is determined by thermodynamics and suggest a way of controlling it. PMID:26795025

  14. Reduced O-GlcNAcase expression promotes mitotic errors and spindle defects.

    PubMed

    Lanza, Chris; Tan, Ee Phie; Zhang, Zhen; Machacek, Miranda; Brinker, Amanda E; Azuma, Mizuki; Slawson, Chad

    2016-05-18

    Alterations in O-GlcNAc cycling, the addition and removal of O-GlcNAc, lead to mitotic defects and increased aneuploidy. Herein, we generated stable O-GlcNAcase (OGA, the enzyme that removes O-GlcNAc) knockdown HeLa cell lines and characterized the effect of the reduction in OGA activity on cell cycle progression. After release from G1/S, the OGA knockdown cells progressed normally through S phase but demonstrated mitotic exit defects. Cyclin A was increased in the knockdown cells while Cyclin B and D expression was reduced. Retinoblastoma protein (RB) phosphorylation was also increased in the knockdown compared to control. At M phase, the knockdown cells showed more compact spindle chromatids than control cells and had a greater percentage of cells with multipolar spindles. Furthermore, the timing of the inhibitory tyrosine phosphorylation of Cyclin Dependent Kinase 1 (CDK1) was altered in the OGA knockdown cells. Although expression and localization of the chromosomal passenger protein complex (CPC) was unchanged, histone H3 threonine 3 phosphorylation was decreased in one of the OGA knockdown cell lines. The Ewing Sarcoma Breakpoint Region 1 Protein (EWS) participates in organizing the CPC at the spindle and is a known substrate for O-GlcNAc transferase (OGT, the enzyme that adds O-GlcNAc). EWS O-GlcNAcylation was significantly increased in the OGA knockdown cells promoting uneven localization of the mitotic midzone. Our data suggests that O-GlcNAc cycling is an essential mechanism for proper mitotic signaling and spindle formation, and alterations in the rate of O-GlcNAc cycling produces aberrant spindles and promotes aneuploidy. PMID:27070276

  15. Physical limits on kinesin-5–mediated chromosome congression in the smallest mitotic spindles

    PubMed Central

    McCoy, Kelsey M.; Tubman, Emily S.; Claas, Allison; Tank, Damien; Clancy, Shelly Applen; O’Toole, Eileen T.; Berman, Judith; Odde, David J.

    2015-01-01

    A characteristic feature of mitotic spindles is the congression of chromosomes near the spindle equator, a process mediated by dynamic kinetochore microtubules. A major challenge is to understand how precise, submicrometer-scale control of kinetochore micro­tubule dynamics is achieved in the smallest mitotic spindles, where the noisiness of microtubule assembly/disassembly will potentially act to overwhelm the spatial information that controls microtubule plus end–tip positioning to mediate congression. To better understand this fundamental limit, we conducted an integrated live fluorescence, electron microscopy, and modeling analysis of the polymorphic fungal pathogen Candida albicans, which contains one of the smallest known mitotic spindles (<1 μm). Previously, ScCin8p (kinesin-5 in Saccharomyces cerevisiae) was shown to mediate chromosome congression by promoting catastrophe of long kinetochore microtubules (kMTs). Using C. albicans yeast and hyphal kinesin-5 (Kip1p) heterozygotes (KIP1/kip1∆), we found that mutant spindles have longer kMTs than wild-type spindles, consistent with a less-organized spindle. By contrast, kinesin-8 heterozygous mutant (KIP3/kip3∆) spindles exhibited the same spindle organization as wild type. Of interest, spindle organization in the yeast and hyphal states was indistinguishable, even though yeast and hyphal cell lengths differ by two- to fivefold, demonstrating that spindle length regulation and chromosome congression are intrinsic to the spindle and largely independent of cell size. Together these results are consistent with a kinesin-5–mediated, length-dependent depolymerase activity that organizes chromosomes at the spindle equator in C. albicans to overcome fundamental noisiness in microtubule self-assembly. More generally, we define a dimensionless number that sets a fundamental physical limit for maintaining congression in small spindles in the face of assembly noise and find that C. albicans operates very close to

  16. Physical limits on kinesin-5-mediated chromosome congression in the smallest mitotic spindles.

    PubMed

    McCoy, Kelsey M; Tubman, Emily S; Claas, Allison; Tank, Damien; Clancy, Shelly Applen; O'Toole, Eileen T; Berman, Judith; Odde, David J

    2015-11-01

    A characteristic feature of mitotic spindles is the congression of chromosomes near the spindle equator, a process mediated by dynamic kinetochore microtubules. A major challenge is to understand how precise, submicrometer-scale control of kinetochore micro-tubule dynamics is achieved in the smallest mitotic spindles, where the noisiness of microtubule assembly/disassembly will potentially act to overwhelm the spatial information that controls microtubule plus end-tip positioning to mediate congression. To better understand this fundamental limit, we conducted an integrated live fluorescence, electron microscopy, and modeling analysis of the polymorphic fungal pathogen Candida albicans, which contains one of the smallest known mitotic spindles (<1 μm). Previously, ScCin8p (kinesin-5 in Saccharomyces cerevisiae) was shown to mediate chromosome congression by promoting catastrophe of long kinetochore microtubules (kMTs). Using C. albicans yeast and hyphal kinesin-5 (Kip1p) heterozygotes (KIP1/kip1∆), we found that mutant spindles have longer kMTs than wild-type spindles, consistent with a less-organized spindle. By contrast, kinesin-8 heterozygous mutant (KIP3/kip3∆) spindles exhibited the same spindle organization as wild type. Of interest, spindle organization in the yeast and hyphal states was indistinguishable, even though yeast and hyphal cell lengths differ by two- to fivefold, demonstrating that spindle length regulation and chromosome congression are intrinsic to the spindle and largely independent of cell size. Together these results are consistent with a kinesin-5-mediated, length-dependent depolymerase activity that organizes chromosomes at the spindle equator in C. albicans to overcome fundamental noisiness in microtubule self-assembly. More generally, we define a dimensionless number that sets a fundamental physical limit for maintaining congression in small spindles in the face of assembly noise and find that C. albicans operates very close to this

  17. Primary Care Pathway for Childhood Asthma: Protocol for a Randomized Cluster-Controlled Trial

    PubMed Central

    Sharpe, Heather; Anselmo, Mark; Befus, A Dean; Currie, Gillian; Davey, Christina; Drummond, Neil; Graham, Jim; Green, Lee A; Grimshaw, Jeremy; Kam, Karen; Manca, Donna P; Nettel-Aguirre, Alberto; Potestio, Melissa L; Rowe, Brian H; Scott, Shannon D; Williamson, Tyler; Johnson, David W

    2016-01-01

    Background Asthma is the most common chronic condition in children. For many, the disease is inadequately controlled, which can burden the lives of children and their families as well as the health care system. Improved use of the best available scientific evidence by primary care practitioners could reduce the need for hospital care and improve quality of life and asthma control, thereby reducing overall costs to society and families. Objective The Primary Care Pathway for Childhood Asthma aims to improve the management of children with asthma by (1) providing primary care practitioners with an electronic guide (a clinical pathway) incorporated into the patient’s electronic medical record, and (2) providing train-the-trainer education to chronic disease management health professionals to promote the provision of asthma education in primary care. Methods The research will utilize a pragmatic cluster-controlled design, quantitative and qualitative research methodologies, and economic evaluation to assess the implementation of a pathway and education intervention in primary care. The intervention will be analyzed for effectiveness, and if the results are positive, a strategy will be developed to implement delivery to all primary care practices in Alberta. Results The research has been successfully funded and ethics approvals have been obtained. Practice recruitment began fall 2015, and we expect all study-related activities to be concluded by March 2018. Conclusions The proposed pathway and education intervention has the potential to improve pediatric asthma management in Alberta. The intervention is anticipated to result in better quality of care for equal or lesser cost. ClinicalTrial ClinicalTrials.gov NCT02481037; https://clinicaltrials.gov/ct2/show/NCT02481037 (Archived by WebCite at http://www.webcitation.org/6fPIQ02Ma). PMID:26955763

  18. Roles of Hippo signaling pathway in size control of organ regeneration.

    PubMed

    Hayashi, Shinichi; Yokoyama, Hitoshi; Tamura, Koji

    2015-05-01

    Animals have an intrinsic regeneration ability for injured tissues and organs. Species that have high regeneration ability such as newts can regenerate an organ with exactly the same size and shape as those of the original one. It has been unclear how a regenerating organ grows and ceases growth at an appropriate size. Organ size control in regeneration is seen in various organs of various species that have high regeneration ability. In animal species that do not have sufficient regeneration ability, a wound heals (the injury is closed, but lost parts are not regenerated), but an organ cannot be restored to its original size. On the other hand, perturbation of regeneration sometimes results in oversized or extra structures. In this sense, organ size control plays essential roles in proper regeneration. In this article, we introduce the concept of size control in organ regeneration regulated by the Hippo signaling pathway. We focused on the transcriptional regulator Yap, which shuttles between the nuclei and cytoplasm to exert a regulatory function in a context-dependent manner. The Yap-mediated Hippo pathway is thought to sense cell density, extracellular matrix (ECM) contact and cell position and to regulate gene expression for control of organ size. This mechanism can reasonably explain size control of organ regeneration. PMID:25867864

  19. Drosophila ATM and ATR checkpoint kinases control partially redundant pathways for telomere maintenance

    PubMed Central

    Bi, Xiaolin; Srikanta, Deepa; Fanti, Laura; Pimpinelli, Sergio; Badugu, RamaKrishna; Kellum, Rebecca; Rong, Yikang S.

    2005-01-01

    In higher eukaryotes, the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) checkpoint kinases play distinct, but partially overlapping, roles in DNA damage response. Yet their interrelated function has not been defined for telomere maintenance. We discover in Drosophila that the two proteins control partially redundant pathways for telomere protection: the loss of ATM leads to the fusion of some telomeres, whereas the loss of both ATM and ATR renders all telomeres susceptible to fusion. The ATM-controlled pathway includes the Mre11 and Nijmegen breakage syndrome complex but not the Chk2 kinase, whereas the ATR-regulated pathway includes its partner ATR-interacting protein but not the Chk1 kinase. This finding suggests that ATM and ATR regulate different molecular events at the telomeres compared with the sites of DNA damage. This compensatory relationship between ATM and ATR is remarkably similar to that observed in yeast despite the fact that the biochemistry of telomere elongation is completely different in the two model systems. We provide evidence suggesting that both the loading of telomere capping proteins and normal telomeric silencing requires ATM and ATR in Drosophila and propose that ATM and ATR protect telomere integrity by safeguarding chromatin architecture that favors the loading of telomere-elongating, capping, and silencing proteins. PMID:16203987

  20. Coordinate Control of Muscle Cell Survival by Distinct Insulin-like Growth Factor Activated Signaling Pathways

    PubMed Central

    Lawlor, Margaret A.; Rotwein, Peter

    2000-01-01

    Peptide growth factors control diverse cellular functions by regulating distinct signal transduction pathways. In cultured myoblasts, insulin-like growth factors (IGFs) stimulate differentiation and promote hypertrophy. IGFs also maintain muscle cell viability. We previously described C2 skeletal muscle lines lacking expression of IGF-II. These cells did not differentiate, but underwent progressive apoptotic death when incubated in differentiation medium. Viability could be sustained and differentiation enabled by IGF analogues that activated the IGF-I receptor; survival was dependent on stimulation of phosphatidylinositol 3-kinase (PI3-kinase). We now find that IGF action promotes myoblast survival through two distinguishable PI3-kinase–regulated pathways that culminate in expression of the cyclin-dependent kinase inhibitor, p21. Incubation with IGF-I or transfection with active PI3-kinase led to rapid induction of MyoD and p21, and forced expression of either protein maintained viability in the absence of growth factors. Ectopic expression of MyoD induced p21, and inhibition of p21 blocked MyoD-mediated survival, thus defining one PI3-kinase–dependent pathway as leading first to MyoD, and then to p21 and survival. Unexpectedly, loss of MyoD expression did not impede IGF-mediated survival, revealing a second pathway involving activation by PI3-kinase of Akt, and subsequent induction of p21. Since inhibition of p21 caused death even in the presence of IGF-I, these results establish a central role for p21 as a survival factor for muscle cells. Our observations also define a MyoD-independent pathway for regulating p21 in muscle, and demonstrate that distinct mechanisms help ensure appropriate expression of this key protein during differentiation. PMID:11121430

  1. Optical switching of electric charge transfer pathways in porphyrin: a light-controlled nanoscale current router.

    PubMed

    Thanopulos, Ioannis; Paspalakis, Emmanuel; Yannopapas, Vassilios

    2008-11-01

    We introduce a novel molecular junction based on a thiol-functionalized porphyrin derivative with two almost energetically degenerate equilibrium configurations. We show that each equilibrium structure defines a pathway of maximal electric charge transfer through the molecular junction and that these two conduction pathways are spatially orthogonal. We further demonstrate computationally how to switch between the two equilibrium structures of the compound by coherent light. The optical switching mechanism is presented in the relevant configuration subspace of the compound, and the corresponding potential and electric dipole surfaces are obtained by ab initio methods. The laser-induced isomerization takes place in two steps in tandem, while each step is induced by a two-photon process. The effect of metallic electrodes on the electromagnetic irradiation driving the optical switching is also investigated. Our study demonstrates the potential for using thiol-functionalized porphyrin derivatives for the development of a light-controlled nanoscale current router. PMID:21832723

  2. Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control

    PubMed Central

    McLelland, Gian-Luca; Soubannier, Vincent; Chen, Carol X; McBride, Heidi M; Fon, Edward A

    2014-01-01

    Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild-type but not PD-linked mutant parkin supports the biogenesis of a population of mitochondria-derived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin- and PINK1-dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD. PMID:24446486

  3. PKCι depletion initiates mitotic slippage-induced senescence in glioblastoma.

    PubMed

    Restall, Ian J; Parolin, Doris A E; Daneshmand, Manijeh; Hanson, Jennifer E L; Simard, Manon A; Fitzpatrick, Megan E; Kumar, Ritesh; Lavictoire, Sylvie J; Lorimer, Ian A J

    2015-01-01

    Cellular senescence is a tumor suppressor mechanism where cells enter a permanent growth arrest following cellular stress. Oncogene-induced senescence (OIS) is induced in non-malignant cells following the expression of an oncogene or inactivation of a tumor suppressor. Previously, we have shown that protein kinase C iota (PKCι) depletion induces cellular senescence in glioblastoma cells in the absence of a detectable DNA damage response. Here we demonstrate that senescent glioblastoma cells exhibit an aberrant centrosome morphology. This was observed in basal levels of senescence, in p21-induced senescence, and in PKCι depletion-induced senescence. In addition, senescent glioblastoma cells are polyploid, Ki-67 negative and arrest at the G1/S checkpoint, as determined by expression of cell cycle regulatory proteins. These markers are all consistent with cells that have undergone mitotic slippage. Failure of the spindle assembly checkpoint to function properly can lead to mitotic slippage, resulting in the premature exit of mitotic cells into the G1 phase of the cell cycle. Although in G1, these cells have the replicated DNA and centrosomal phenotype of a cell that has entered mitosis and failed to divide. Overall, we demonstrate that PKCι depletion initiates mitotic slippage-induced senescence in glioblastoma cells. To our knowledge, this is the first evidence of markers of mitotic slippage directly in senescent cells by co-staining for senescence-associated β-galactosidase and immunofluorescence markers in the same cell population. We suggest that markers of mitotic slippage be assessed in future studies of senescence to determine the extent of mitotic slippage in the induction of cellular senescence. PMID:26208522

  4. Transcriptional regulators in the Hippo signaling pathway control organ growth in Xenopus tadpole tail regeneration.

    PubMed

    Hayashi, Shinichi; Ochi, Haruki; Ogino, Hajime; Kawasumi, Aiko; Kamei, Yasuhiro; Tamura, Koji; Yokoyama, Hitoshi

    2014-12-01

    The size and shape of tissues are tightly controlled by synchronized processes among cells and tissues to produce an integrated organ. The Hippo signaling pathway controls both cell proliferation and apoptosis by dual signal-transduction states regulated through a repressive kinase cascade. Yap1 and Tead, transcriptional regulators that act downstream of the Hippo signaling kinase cascade, have essential roles in regulating cell proliferation. In amphibian limb or tail regeneration, the local tissue outgrowth terminates when the correct size is reached, suggesting that organ size is strictly controlled during epimorphic organ-level regeneration. We recently demonstrated that Yap1 is required for the regeneration of Xenopus tadpole limb buds (Hayashi et al., 2014, Dev. Biol. 388, 57-67), but the molecular link between the Hippo pathway and organ size control in vertebrate epimorphic regeneration is not fully understood. To examine the requirement of Hippo pathway transcriptional regulators in epimorphic regeneration, including organ size control, we inhibited these regulators during Xenopus tadpole tail regeneration by overexpressing a dominant-negative form of Yap (dnYap) or Tead4 (dnTead4) under a heat-shock promoter in transgenic animal lines. Each inhibition resulted in regeneration defects accompanied by reduced cell mitosis and increased apoptosis. Single-cell gene manipulation experiments indicated that Tead4 cell-autonomously regulates the survival of neural progenitor cells in the regenerating tail. In amphibians, amputation at the proximal level of the tail (deep amputation) results in faster regeneration than that at the distal level (shallow amputation), to restore the original-sized tail with similar timing. However, dnTead4 overexpression abolished the position-dependent differential growth rate of tail regeneration. These results suggest that the transcriptional regulators in the Hippo pathway, Tead4 and Yap1, are required for general vertebrate

  5. Physical Description of Mitotic Spindle Orientation During Cell Division

    NASA Astrophysics Data System (ADS)

    Jiménez-Dalmaroni, Andrea; Théry, Manuel; Racine, Victor; Bornens, Michel; Jülicher, Frank

    2009-03-01

    During cell division, the duplicated chromosomes are physically separated by the action of the mitotic spindle. The spindle is a dynamic structure of the cytoskeleton, which consists of two microtubule asters. Its orientation defines the axis along which the cell divides. Recent experiments show that the spindle orientation depends on the spatial distribution of cell adhesion sites. Here we show that the experimentally observed spindle orientation can be understood as the result of the action of cortical force generators acting on the spindle. We assume that the local activity of force generators is controlled by the spatial distribution of cell adhesion sites determined by the particular geometry of the adhesive substrate. We develop a simple physical description of the spindle mechanics, which allows us to calculate the torque acting on the spindle, as well as the energy profile and the angular distribution of spindle orientation. Our model accounts for the preferred spindle orientation, as well as the full shape of the angular distributions of spindle orientation observed in a large variety of pattern geometries. M. Th'ery, A. Jim'enez-Dalmaroni, et al., Nature 447, 493 (2007).

  6. PP2ARts1 is a master regulator of pathways that control cell size

    PubMed Central

    Zapata, Jessica; Dephoure, Noah; MacDonough, Tracy; Yu, Yaxin; Parnell, Emily J.; Mooring, Meghan; Gygi, Steven P.; Stillman, David J.

    2014-01-01

    Cell size checkpoints ensure that passage through G1 and mitosis occurs only when sufficient growth has occurred. The mechanisms by which these checkpoints work are largely unknown. PP2A associated with the Rts1 regulatory subunit (PP2ARts1) is required for cell size control in budding yeast, but the relevant targets are unknown. In this paper, we used quantitative proteome-wide mass spectrometry to identify proteins controlled by PP2ARts1. This revealed that PP2ARts1 controls the two key checkpoint pathways thought to regulate the cell cycle in response to cell growth. To investigate the role of PP2ARts1 in these pathways, we focused on the Ace2 transcription factor, which is thought to delay cell cycle entry by repressing transcription of the G1 cyclin CLN3. Diverse experiments suggest that PP2ARts1 promotes cell cycle entry by inhibiting the repressor functions of Ace2. We hypothesize that control of Ace2 by PP2ARts1 plays a role in mechanisms that link G1 cyclin accumulation to cell growth. PMID:24493588

  7. Dual Control of Muscle Cell Survival by Distinct Growth Factor-Regulated Signaling Pathways

    PubMed Central

    Lawlor, Margaret A.; Feng, Xiuhong; Everding, Daniel R.; Sieger, Kerry; Stewart, Claire E. H.; Rotwein, Peter

    2000-01-01

    In addition to their ability to stimulate cell proliferation, polypeptide growth factors are able to maintain cell survival under conditions that otherwise lead to apoptotic death. Growth factors control cell viability through regulation of critical intracellular signal transduction pathways. We previously characterized C2 muscle cell lines that lacked endogenous expression of insulin-like growth factor II (IGF-II). These cells did not differentiate but underwent apoptotic death in low-serum differentiation medium. Death could be prevented by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as platelet-derived growth factor BB (PDGF-BB). Here we analyze the signaling pathways involved in growth factor-mediated myoblast survival. PDGF treatment caused sustained activation of extracellular-regulated kinases 1 and 2 (ERK1 and -2), while IGF-I only transiently induced these enzymes. Transient transfection of a constitutively active Mek1, a specific upstream activator of ERKs, maintained myoblast viability in the absence of growth factors, while inhibition of Mek1 by the drug UO126 blocked PDGF-mediated but not IGF-stimulated survival. Although both growth factors activated phosphatidylinositol 3-kinase (PI3-kinase) to similar extents, only IGF-I treatment led to sustained stimulation of its downstream kinase, Akt. Transient transfection of a constitutively active PI3-kinase or an inducible Akt promoted myoblast viability in the absence of growth factors, while inhibition of PI3-kinase activity by the drug LY294002 selectively blocked IGF- but not PDGF-mediated muscle cell survival. In aggregate, these observations demonstrate that distinct growth factor-regulated signaling pathways independently control myoblast survival. Since IGF action also stimulates muscle differentiation, these results suggest a means to regulate myogenesis through selective manipulation of different signal transduction pathways. PMID:10757809

  8. Pharmacological inactivation of CHK1 and WEE1 induces mitotic catastrophe in nasopharyngeal carcinoma cells.

    PubMed

    Mak, Joyce P Y; Man, Wing Yu; Chow, Jeremy P H; Ma, Hoi Tang; Poon, Randy Y C

    2015-08-28

    Nasopharyngeal carcinoma (NPC) is a rare but highly invasive cancer. As radiotherapy is the primary treatment for NPC, this offers a rationale to investigate if uncoupling the DNA damage responses can sensitize this cancer type. The G2 DNA damage checkpoint is controlled by a cascade of protein kinases: ATM/ATR, which phosphorylates CHK1/CHK2, which in turn phosphorylates WEE1. A number of small molecule inhibitors have been developed against these kinases as potential therapeutic agents. Here we demonstrated that compare to that in immortalized nasopharyngeal epithelial cells, ATR, CHK1, and WEE1 were overexpressed in NPC cell lines. Inhibitors of these kinases were unable to promote extensive mitotic catastrophe in ionizing radiation-treated NPC cells, indicating that they are not very effective radiosensitizer for this cancer. In the absence of prior irradiation, however, mitotic catastrophe could be induced with inhibitors against CHK1 (AZD7762) or WEE1 (MK-1775). NPC cells were more sensitive to WEE1 inactivation than nasopharyngeal epithelial cells. Targeting CHK1 and WEE1 together induced more extensive mitotic catastrophe than the individual components alone. Taken together, our results show that NPC cells depend on CHK1 and WEE1 activity for growth and that inhibitors of these kinases may serve as potential therapeutics for NPC. PMID:26025928

  9. Pharmacological inactivation of CHK1 and WEE1 induces mitotic catastrophe in nasopharyngeal carcinoma cells

    PubMed Central

    Mak, Joyce P.Y.; Man, Wing Yu; Chow, Jeremy P.H.; Ma, Hoi Tang; Poon, Randy Y.C.

    2015-01-01

    Nasopharyngeal carcinoma (NPC) is a rare but highly invasive cancer. As radiotherapy is the primary treatment for NPC, this offers a rationale to investigate if uncoupling the DNA damage responses can sensitize this cancer type. The G2 DNA damage checkpoint is controlled by a cascade of protein kinases: ATM/ATR, which phosphorylates CHK1/CHK2, which in turn phosphorylates WEE1. A number of small molecule inhibitors have been developed against these kinases as potential therapeutic agents. Here we demonstrated that compare to that in immortalized nasopharyngeal epithelial cells, ATR, CHK1, and WEE1 were overexpressed in NPC cell lines. Inhibitors of these kinases were unable to promote extensive mitotic catastrophe in ionizing radiation-treated NPC cells, indicating that they are not very effective radiosensitizer for this cancer. In the absence of prior irradiation, however, mitotic catastrophe could be induced with inhibitors against CHK1 (AZD7762) or WEE1 (MK-1775). NPC cells were more sensitive to WEE1 inactivation than nasopharyngeal epithelial cells. Targeting CHK1 and WEE1 together induced more extensive mitotic catastrophe than the individual components alone. Taken together, our results show that NPC cells depend on CHK1 and WEE1 activity for growth and that inhibitors of these kinases may serve as potential therapeutics for NPC. PMID:26025928

  10. The mevalonate pathway as a metabolic requirement for autophagy-implications for growth control, proteostasis, and disease.

    PubMed

    Miettinen, Teemu P; Björklund, Mikael

    2016-05-01

    Autophagy is responsible for the degradation and recycling of cellular proteins and organelles. Our recent work shows that the mevalonate pathway influences cell size, growth, and proteostasis by regulating basal autophagic flux through geranylgeranylation of the small GTPase RAB11. The control of autophagy by the mevalonate/cholesterol pathway has potential implications for statin toxicity, inflammation, cancer, and neurodegenerative diseases. PMID:27314093

  11. Focal adhesions control cleavage furrow shape and spindle tilt during mitosis

    PubMed Central

    Taneja, Nilay; Fenix, Aidan M.; Rathbun, Lindsay; Millis, Bryan A.; Tyska, Matthew J.; Hehnly, Heidi; Burnette, Dylan T.

    2016-01-01

    The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critical role in stem cell differentiation and the relative positioning of daughter cells during development. Early observations of adhesive cell lines revealed asymmetry in the shape of the cleavage furrow, where the bottom (i.e., substrate attached side) of the cleavage furrow ingressed less than the top (i.e., unattached side). This data suggested substrate attachment could be regulating furrow ingression. Here we report a population of mitotic focal adhesions (FAs) controls the symmetry of the cleavage furrow. In single HeLa cells, stronger adhesion to the substrate directed less ingression from the bottom of the cell through a pathway including paxillin, focal adhesion kinase (FAK) and vinculin. Cell-cell contacts also direct ingression of the cleavage furrow in coordination with FAs in epithelial cells—MDCK—within monolayers and polarized cysts. In addition, mitotic FAs established 3D orientation of the mitotic spindle and the relative positioning of mother and daughter centrosomes. Therefore, our data reveals mitotic FAs as a key link between mitotic cell shape and spindle orientation, and may have important implications in our understanding stem cell homeostasis and tumorigenesis. PMID:27432211

  12. Controlling the intracellular fate of nano-bioconjugates: pathways for realizing nanoparticle-mediated theranostics

    NASA Astrophysics Data System (ADS)

    Delehanty, James B.; Blanco-Canosa, Juan B.; Bradburne, Christopher E.; Susumu, Kimihiro; Stewart, Michael H.; Prasuhn, Duane E.; Dawson, Philip E.; Medintz, Igor L.

    2014-08-01

    For nanomaterials to realize their full potential in disease diagnosis and drug delivery applications, one must be able to exert fine control over their cellular delivery, localization and long-term fate in biological systems. Our laboratory has been active in developing methodologies for the controlled and site-specific delivery of a range of nanomaterials (e.g., quantum dots, colloidal gold, nematic liquid crystals) for cellular labeling, imaging and sensing. This talk will highlight several examples from these efforts and will demonstrate the use of peptide- and protein-mediated facilitated delivery of nanomaterials to discrete cellular locations including the endocytic pathway, the plasma membrane and the cellular cytosol. The implications of the ability to exert fine control over nanomaterial constructs in biological settings will be discussed with a particular focus on their use in nanoparticle-based theranostics.

  13. A mitotic function for the high-mobility group protein HMG20b regulated by its interaction with the BRC repeats of the BRCA2 tumor suppressor.

    PubMed

    Lee, M; Daniels, M J; Garnett, M J; Venkitaraman, A R

    2011-07-28

    The inactivation of BRCA2, a suppressor of breast, ovarian and other epithelial cancers, triggers instability in chromosome structure and number, which are thought to arise from defects in DNA recombination and mitotic cell division, respectively. Human BRCA2 controls DNA recombination via eight BRC repeats, evolutionarily conserved motifs of ∼35 residues, that interact directly with the recombinase RAD51. How BRCA2 controls mitotic cell division is debated. Several studies by different groups report that BRCA2 deficiency affects cytokinesis. Moreover, its interaction with HMG20b, a protein of uncertain function containing a promiscuous DNA-binding domain and kinesin-like coiled coils, has been implicated in the G2-M transition. We show here that HMG20b depletion by RNA interference disturbs the completion of cell division, suggesting a novel function for HMG20b. In vitro, HMG20b binds directly to the BRC repeats of BRCA2, and exhibits the highest affinity for BRC5, a motif that binds poorly to RAD51. Conversely, the BRC4 repeat binds strongly to RAD51, but not to HMG20b. In vivo, BRC5 overexpression inhibits the BRCA2-HMG20b interaction, recapitulating defects in the completion of cell division provoked by HMG20b depletion. In contrast, BRC4 inhibits the BRCA2-RAD51 interaction and the assembly of RAD51 at sites of DNA damage, but not the completion of cell division. Our findings suggest that a novel function for HMG20b in cytokinesis is regulated by its interaction with the BRC repeats of BRCA2, and separate this unexpected function for the BRC repeats from their known activity in DNA recombination. We propose that divergent tumor-suppressive pathways regulating chromosome segregation as well as chromosome structure may be governed by the conserved BRC motifs in BRCA2. PMID:21399666

  14. 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).

  15. Mitotic Chromosome Loss in a Disomic Haploid of SACCHAROMYCES CEREVISIAE

    PubMed Central

    Campbell, D. A.; Fogel, S.; Lusnak, K.

    1975-01-01

    Experiments designed to characterize the incidence of mitotic chromosome loss in a yeast disomic haploid were performed. The selective methods employed utilize the non-mating property of strains disomic for linkage group III and heterozygous at the mating type locus. The principal findings are: (1) The frequency of spontaneous chromosome loss in the disome is of the order 10-4 per cell; this value approximates the frequency in the same population of spontaneous mitotic exchange resulting in homozygosity at the mating type locus. (2) The recovered diploids are pure clones, and thus represent unique events in the disomic haploid. (3) Of the euploid chromosomes recovered after events leading to chromosome loss, approximately 90% retain the parental marker configuration expected from segregation alone; however, the remainder are recombinant for marker genes, and are the result of mitotic exchanges in the disome, especially in regions near the centromere. The recombinant proportion significantly exceeds that expected if chromosome loss and mitotic exchange in the disome were independent events. The data are consistent with a model proposing mitotic nondisjunction as the event responsible for chromosome loss in the disomic haploid. PMID:1092597

  16. Isolation of Human Mitotic Protein Phosphatase Complexes: Identification of a Complex between Protein Phosphatase 1 and the RNA Helicase Ddx21

    PubMed Central

    De Wever, Veerle; Lloyd, David C.; Nasa, Isha; Nimick, Mhairi; Trinkle-Mulcahy, Laura; Gourlay, Robert; Morrice, Nick; Moorhead, Greg B. G.

    2012-01-01

    Metazoan mitosis requires remodelling of sub-cellular structures to ensure proper division of cellular and genetic material. Faults often lead to genomic instability, cell cycle arrests and disease onset. These key structural changes are under tight spatial-temporal and post-translational control, with crucial roles for reversible protein phosphorylation. The phosphoprotein phosphatases PP1 and PP2A are paramount for the timely execution of mitotic entry and exit but their interaction partners and substrates are still largely unresolved. High throughput, mass-spectrometry based studies have limited sensitivity for the detection of low-abundance and transient complexes, a typical feature of many protein phosphatase complexes. Moreover, the limited timeframe during which mitosis takes place reduces the likelihood of identifying mitotic phosphatase complexes in asynchronous cells. Hence, numerous mitotic protein phosphatase complexes still await identification. Here we present a strategy to enrich and identify serine/threonine protein phosphatase complexes at the mitotic spindle. We thus identified a nucleolar RNA helicase, Ddx21/Gu, as a novel, direct PP1 interactor. Furthermore, our results place PP1 within the toposome, a Topoisomerase II alpha (TOPOIIα) containing complex with a key role in mitotic chromatin regulation and cell cycle progression, possibly via regulated protein phosphorylation. This study provides a strategy for the identification of further mitotic PP1 partners and the unravelling of PP1 functions during mitosis. PMID:22761809

  17. Design, implementation, and quality control in the Pathways American-Indian multicenter trial

    PubMed Central

    Stone, Elaine J.; Norman, James E.; Davis, Sally M.; Stewart, Dawn; Clay, Theresa E.; Caballero, Ben; Lohman, Timothy G.; Murray, David M.

    2016-01-01

    Background Pathways was the first multicenter American-Indian school-based study to test the effectiveness of an obesity prevention program promoting healthy eating and physical activity. Methods Pathways employed a nested cohort design in which 41 schools were randomized to intervention or control conditions and students within these schools were followed as a cohort (1,704 third graders at baseline). The study’s primary endpoint was percent body fat. Secondary endpoints were levels of fat in school lunches; time spent in physical activity; and knowledge, attitudes, and behaviors regarding diet and exercise. Quality control (QC) included design of data management systems which provided standardization and quality assurance of data collection and processing. Data QC procedures at study centers included manuals of operation, training and certification, and monitoring of performance. Process evaluation was conducted to monitor dose and fidelity of the interventions. Registration and tracking systems were used for students and schools. Results No difference in mean percent body fat at fifth grade was found between the intervention and control schools. Percent of calories from fat and saturated fat in school lunches was significantly reduced in the intervention schools as was total energy intake from 24-hour recalls. Significant increases in self-reported physical activity levels and knowledge of healthy behaviors were found for the intervention school students. Conclusions The Pathways study results provide evidence demonstrating the role schools can play in public health promotion. Its study design and QC systems and procedures provide useful models for other similar school based multi- or single-site studies. PMID:14636805

  18. The ribosome quality control pathway can access nascent polypeptides stalled at the Sec61 translocon.

    PubMed

    von der Malsburg, Karina; Shao, Sichen; Hegde, Ramanujan S

    2015-06-15

    Cytosolic ribosomes that stall during translation are split into subunits, and nascent polypeptides trapped in the 60S subunit are ubiquitinated by the ribosome quality control (RQC) pathway. Whether the RQC pathway can also target stalls during cotranslational translocation into the ER is not known. Here we report that listerin and NEMF, core RQC components, are bound to translocon-engaged 60S subunits on native ER membranes. RQC recruitment to the ER in cultured cells is stimulated by translation stalling. Biochemical analyses demonstrated that translocon-targeted nascent polypeptides that subsequently stall are polyubiquitinated in 60S complexes. Ubiquitination at the translocon requires cytosolic exposure of the polypeptide at the ribosome-Sec61 junction. This exposure can result from either failed insertion into the Sec61 channel or partial backsliding of translocating nascent chains. Only Sec61-engaged nascent chains early in their biogenesis were relatively refractory to ubiquitination. Modeling based on recent 60S-RQC and 80S-Sec61 structures suggests that the E3 ligase listerin accesses nascent polypeptides via a gap in the ribosome-translocon junction near the Sec61 lateral gate. Thus the RQC pathway can target stalled translocation intermediates for degradation from the Sec61 channel. PMID:25877867

  19. Impact of Alkyl Spacer Length on Aggregation Pathways in Kinetically Controlled Supramolecular Polymerization.

    PubMed

    Ogi, Soichiro; Stepanenko, Vladimir; Thein, Johannes; Würthner, Frank

    2016-01-20

    We have investigated the kinetic and thermodynamic supramolecular polymerizations of a series of amide-functionalized perylene bisimide (PBI) organogelator molecules bearing alkyl spacers of varied lengths (ethylene to pentylene chains, PBI-1-C2 to PBI-1-C5) between the amide and PBI imide groups. These amide-functionalized PBIs form one-dimensional fibrous nanostructures as the thermodynamically favored states in solvents of low polarity. Our in-depth studies revealed, however, that the kinetic behavior of their supramolecular polymerization is dependent on the spacer length. Propylene- and pentylene-tethered PBIs follow a similar polymerization process as previously observed for the ethylene-tethered PBI. Thus, the monomers of these PBIs are kinetically trapped in conformationally restricted states through intramolecular hydrogen bonding between the amide and imide groups. In contrast, the intramolecularly hydrogen-bonded monomers of butylene-tethered PBI spontaneously self-assemble into nanoparticles, which constitute an off-pathway aggregate state with regard to the thermodynamically stable fibrous supramolecular polymers obtained. Thus, for this class of π-conjugated system, an unprecedented off-pathway aggregate with high kinetic stability could be realized for the first time by introducing an alkyl linker of optimum length (C4 chain) between the amide and imide groups. Our current system with an energy landscape of two competing nucleated aggregation pathways is applicable to the kinetic control over the supramolecular polymerization by the seeding approach. PMID:26699283

  20. A pharmaco-epistasis strategy reveals a new cell size controlling pathway in yeast

    PubMed Central

    Moretto, Fabien; Sagot, Isabelle; Daignan-Fornier, Bertrand; Pinson, Benoît

    2013-01-01

    Cell size is a complex quantitative trait resulting from interactions between intricate genetic networks and environmental conditions. Here, taking advantage of previous studies that uncovered hundreds of genes affecting budding yeast cell size homeostasis, we performed a wide pharmaco-epistasis analysis using drugs mimicking cell size mutations. Simple epistasis relationship emerging from this approach allowed us to characterize a new cell size homeostasis pathway comprising the sirtuin Sir2, downstream effectors including the large ribosomal subunit (60S) and the transcriptional regulators Swi4 and Swi6. We showed that this Sir2/60S signaling route acts independently of other previously described cell size controlling pathways and may integrate the metabolic status of the cell through NAD+ intracellular concentration. Finally, although Sir2 and the 60S subunits regulate both cell size and replicative aging, we found that there is no clear causal relationship between these two complex traits. This study sheds light on a pathway of >50 genes and illustrates how pharmaco-epistasis applied to yeast offers a potent experimental framework to explore complex genotype/phenotype relationships. PMID:24217298

  1. Dse1 may control cross talk between the pheromone and filamentation pathways in yeast.

    PubMed

    Draper, Edward; Dubrovskyi, Oleksii; Bar, Eli E; Stone, David E

    2009-12-01

    The filamentous/invasive growth pathway is activated by nutrient limitation in the haploid form of the yeast Saccharomyces cerevisiae, whereas exposure to mating-pheromone causes cells to differentiate into gametes. Although these two pathways respond to very different stimuli and generate very different responses, they utilize many of the same signaling components. This implies the need for robust mechanisms to maintain signal fidelity. Dse1 was identified in an allele-specific suppressor screen for proteins that interact with the pheromone-responsive Gbetagamma, and found to bind both to a Gbetagamma-affinity column, and to the shared MEKK, Ste11. Although overexpression of Dse1 stimulated invasive growth and transcription of both filamentation and mating-specific transcriptional reporters, deletion of DSE1 had no effect on these outputs. In contrast, pheromone hyper-induced transcription of the filamentation reporter in cells lacking Dse1 and in cells expressing a mutant form of Gbeta that exhibits diminished interaction with Dse1. Thus, the interaction of Dse1 with both Gbeta and Ste11 may be designed to control cross talk between the pheromone and filamentation pathways. PMID:19820940

  2. Conserved genetic pathways controlling the development of the diffuse endocrine system in vertebrates and Drosophila.

    PubMed

    Hartenstein, Volker; Takashima, Shigeo; Adams, Katrina L

    2010-05-01

    The midgut epithelium is formed by absorptive enterocytes, secretory cells and endocrine cells. Each of these lineages is derived from the pluripotent progenitors that constitute the embryonic endoderm; the mature midgut retains pools of self-renewing stem cells that continue to produce all lineages. Recent findings in vertebrates and Drosophila shed light on the genetic mechanism that specifies the fate of the different lineages. A pivotal role is played by the Notch signaling pathway that, in a manner that appears to be very similar to the way in which Notch signaling selects neural progenitors within the neurectoderm, distinguishes the fate of secretory/endocrine cells and enterocytes. Proneural genes encoding bHLH transcription factors are expressed and required in prospective endocrine cells; activation of the Notch pathways restricts the number of these cells and promotes enterocyte development. In this review we compare the development of the intestinal endocrine cells in vertebrates and insects and summarize recent findings dealing with genetic pathways controlling this cell type. PMID:20005229

  3. Conserved Genetic Pathways Controlling the Development of the Diffuse Endocrine System in Vertebrates and Drosophila

    PubMed Central

    Hartenstein, Volker; Takashima, Shigeo; Adams, Katrina

    2014-01-01

    The midgut epithelium is formed by absorptive enterocytes, secretory cells and endocrine cells. Each of these lineages is derived from the pluripotent progenitors that constitute the embryonic endoderm; the mature midgut retains pools of self-renewing stem cells that continue to produce all lineages. Recent findings in vertebrates and Drosophila shed light on the genetic mechanism that specifies the fate of the different lineages. A pivotal role is played by the Notch signaling pathway that, in a manner that appears to be very similar to the way in which Notch signaling selects neural progenitors within the neurectoderm, distinguishes the fate of secretory/endocrine cells and enterocytes. Proneural genes encoding bHLH transcription factors are expressed and required in prospective endocrine cells; activation of the Notch pathways restricts the number of these cells and promotes enterocyte development. In this review we compare the development of the intestinal endocrine cells in vertebrates and insects and summarize recent findings dealing with genetic pathways controlling this cell type. PMID:20005229

  4. Kinetics of nucleation-controlled polymerization. A perturbation treatment for use with a secondary pathway.

    PubMed Central

    Bishop, M F; Ferrone, F A

    1984-01-01

    We present a perturbation method for analyzing nucleation-controlled polymerization augmented by a secondary pathway for polymer growth. With this method, the solution to the kinetic equations assumes a simple analytic closed form that can easily be used in fitting data. So long as the formation of polymers by the secondary pathway depends linearly on the concentration of monomers polymerized, the form of the solutions is the same. This permits the analysis of augmented growth models with a minimum number of modeling assumptions, and thus makes it readily possible to distinguish between a variety of secondary processes (heterogeneous nucleation, lateral growth, and fragmentation). In addition, the parameters of the homogeneous process, such as the homogeneous nucleus size, can be determined independent of the nature of the secondary mechanism. We describe applications of this method to the polymerization of actin, collagen, and sickle hemoglobin. We present an extensive analysis of data on actin polymerization (Wegner, A., and P. Savko, 1982, Biochemistry, 21:1909-1913) to illustrate the use of the method. Although our conclusions generally agree with theirs, we find that lateral growth describes the secondary pathway better than the fragmentation model originally proposed. We also show how this method can be used to study the degree of polymerization, the parentage of polymers, and the behavior of polymers in cycling experiments. PMID:6498276

  5. The aryl hydrocarbon receptor: a molecular pathway for the environmental control of the immune response.

    PubMed

    Quintana, Francisco J

    2013-03-01

    Environmental factors have significant effects on the development of autoimmune diseases. The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) is controlled by endogenous and environmental small molecules. Hence, AHR provides a molecular pathway by which endogenous and environmental signals can influence the immune response and the development of autoimmune diseases. AHR also provides a target for therapeutic intervention in immune-mediated disorders. In this review, we discuss the role of AHR in the regulation of T-cell differentiation and autoimmunity. PMID:23190340

  6. MYC Is a Major Determinant of Mitotic Cell Fate

    PubMed Central

    Topham, Caroline; Tighe, Anthony; Ly, Peter; Bennett, Ailsa; Sloss, Olivia; Nelson, Louisa; Ridgway, Rachel A.; Huels, David; Littler, Samantha; Schandl, Claudia; Sun, Ying; Bechi, Beatrice; Procter, David J.; Sansom, Owen J.; Cleveland, Don W.; Taylor, Stephen S.

    2015-01-01

    Summary Taxol and other antimitotic agents are frontline chemotherapy agents but the mechanisms responsible for patient benefit remain unclear. Following a genome-wide siRNA screen, we identified the oncogenic transcription factor Myc as a taxol sensitizer. Using time-lapse imaging to correlate mitotic behavior with cell fate, we show that Myc sensitizes cells to mitotic blockers and agents that accelerate mitotic progression. Myc achieves this by upregulating a cluster of redundant pro-apoptotic BH3-only proteins and suppressing pro-survival Bcl-xL. Gene expression analysis of breast cancers indicates that taxane responses correlate positively with Myc and negatively with Bcl-xL. Accordingly, pharmacological inhibition of Bcl-xL restores apoptosis in Myc-deficient cells. These results open up opportunities for biomarkers and combination therapies that could enhance traditional and second-generation antimitotic agents. PMID:26175417

  7. MYC Is a Major Determinant of Mitotic Cell Fate.

    PubMed

    Topham, Caroline; Tighe, Anthony; Ly, Peter; Bennett, Ailsa; Sloss, Olivia; Nelson, Louisa; Ridgway, Rachel A; Huels, David; Littler, Samantha; Schandl, Claudia; Sun, Ying; Bechi, Beatrice; Procter, David J; Sansom, Owen J; Cleveland, Don W; Taylor, Stephen S

    2015-07-13

    Taxol and other antimitotic agents are frontline chemotherapy agents but the mechanisms responsible for patient benefit remain unclear. Following a genome-wide siRNA screen, we identified the oncogenic transcription factor Myc as a taxol sensitizer. Using time-lapse imaging to correlate mitotic behavior with cell fate, we show that Myc sensitizes cells to mitotic blockers and agents that accelerate mitotic progression. Myc achieves this by upregulating a cluster of redundant pro-apoptotic BH3-only proteins and suppressing pro-survival Bcl-xL. Gene expression analysis of breast cancers indicates that taxane responses correlate positively with Myc and negatively with Bcl-xL. Accordingly, pharmacological inhibition of Bcl-xL restores apoptosis in Myc-deficient cells. These results open up opportunities for biomarkers and combination therapies that could enhance traditional and second-generation antimitotic agents. PMID:26175417

  8. Shaping mitotic chromosomes: From classical concepts to molecular mechanisms

    PubMed Central

    Kschonsak, Marc; Haering, Christian H

    2015-01-01

    How eukaryotic genomes are packaged into compact cylindrical chromosomes in preparation for cell divisions has remained one of the major unsolved questions of cell biology. Novel approaches to study the topology of DNA helices inside the nuclei of intact cells, paired with computational modeling and precise biomechanical measurements of isolated chromosomes, have advanced our understanding of mitotic chromosome architecture. In this Review Essay, we discuss – in light of these recent insights – the role of chromatin architecture and the functions and possible mechanisms of SMC protein complexes and other molecular machines in the formation of mitotic chromosomes. Based on the information available, we propose a stepwise model of mitotic chromosome condensation that envisions the sequential generation of intra-chromosomal linkages by condensin complexes in the context of cohesin-mediated inter-chromosomal linkages, assisted by topoisomerase II. The described scenario results in rod-shaped metaphase chromosomes ready for their segregation to the cell poles. PMID:25988527

  9. A requirement for epsin in mitotic membrane and spindle organization

    PubMed Central

    2009-01-01

    Eukaryotic cells possess a sophisticated membrane system to facilitate diverse functions. Whereas much is known about the nature of membrane systems in interphase, the organization and function of the mitotic membrane system are less well understood. In this study, we show that epsin, an endocytic adapter protein, regulates mitotic membrane morphology and spindle integrity in HeLa cells. Using epsin that harbors point mutations in the epsin NH2-terminal homology domain and spindle assembly assays in Xenopus laevis egg extracts, we show that epsin-induced membrane curvature is required for proper spindle morphogenesis, independent of its function in endocytosis during interphase. Although several other membrane-interacting proteins, including clathrin, AP2, autosomal recessive hypercholesterolemia, and GRASP65, are implicated in the regulation of mitosis, whether they participate through regulation of membrane organization is unclear. Our study of epsin provides evidence that mitotic membrane organization influences spindle integrity. PMID:19704019

  10. 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

  11. Shaping mitotic chromosomes: From classical concepts to molecular mechanisms.

    PubMed

    Kschonsak, Marc; Haering, Christian H

    2015-07-01

    How eukaryotic genomes are packaged into compact cylindrical chromosomes in preparation for cell divisions has remained one of the major unsolved questions of cell biology. Novel approaches to study the topology of DNA helices inside the nuclei of intact cells, paired with computational modeling and precise biomechanical measurements of isolated chromosomes, have advanced our understanding of mitotic chromosome architecture. In this Review Essay, we discuss - in light of these recent insights - the role of chromatin architecture and the functions and possible mechanisms of SMC protein complexes and other molecular machines in the formation of mitotic chromosomes. Based on the information available, we propose a stepwise model of mitotic chromosome condensation that envisions the sequential generation of intra-chromosomal linkages by condensin complexes in the context of cohesin-mediated inter-chromosomal linkages, assisted by topoisomerase II. The described scenario results in rod-shaped metaphase chromosomes ready for their segregation to the cell poles. PMID:25988527

  12. The moyamoya disease susceptibility variant RNF213 R4810K (rs112735431) induces genomic instability by mitotic abnormality

    SciTech Connect

    Hitomi, Toshiaki; Habu, Toshiyuki; Kobayashi, Hatasu; Okuda, Hiroko; Harada, Kouji H.; Osafune, Kenji; Taura, Daisuke; Sone, Masakatsu; Asaka, Isao; Ameku, Tomonaga; Watanabe, Akira; Kasahara, Tomoko; Sudo, Tomomi; Shiota, Fumihiko; Hashikata, Hirokuni; Takagi, Yasushi; Morito, Daisuke; Miyamoto, Susumu; Nakao, Kazuwa; Koizumi, Akio

    2013-10-04

    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 cell 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.

  13. The JAK-STAT Pathway Controls Plasmodium vivax Load in Early Stages of Anopheles aquasalis Infection

    PubMed Central

    Bahia, Ana C.; Kubota, Marina S.; Tempone, Antonio J.; Araújo, Helena R. C.; Guedes, Bruno A. M.; Orfanó, Alessandra S.; Tadei, Wanderli P.; Ríos-Velásquez, Claudia M.; Han, Yeon S.; Secundino, Nágila F. C.; Barillas-Mury, Carolina

    2011-01-01

    Malaria affects 300 million people worldwide every year and 450,000 in Brazil. In coastal areas of Brazil, the main malaria vector is Anopheles aquasalis, and Plasmodium vivax is responsible for the majority of malaria cases in the Americas. Insects possess a powerful immune system to combat infections. Three pathways control the insect immune response: Toll, IMD, and JAK-STAT. Here we analyze the immune role of the A. aquasalis JAK-STAT pathway after P. vivax infection. Three genes, the transcription factor Signal Transducers and Activators of Transcription (STAT), the regulatory Protein Inhibitors of Activated STAT (PIAS) and the Nitric Oxide Synthase enzyme (NOS) were characterized. Expression of STAT and PIAS was higher in males than females and in eggs and first instar larvae when compared to larvae and pupae. RNA levels for STAT and PIAS increased 24 and 36 hours (h) after P. vivax challenge. NOS transcription increased 36 h post infection (hpi) while this protein was already detected in some midgut epithelial cells 24 hpi. Imunocytochemistry experiments using specific antibodies showed that in non-infected insects STAT and PIAS were found mostly in the fat body, while in infected mosquitoes the proteins were found in other body tissues. The knockdown of STAT by RNAi increased the number of oocysts in the midgut of A. aquasalis. This is the first clear evidence for the involvement of a specific immune pathway in the interaction of the Brazilian malaria vector A. aquasalis with P. vivax, delineating a potential target for the future development of disease controlling strategies. PMID:22069502

  14. The JAK-STAT pathway controls Plasmodium vivax load in early stages of Anopheles aquasalis infection.

    PubMed

    Bahia, Ana C; Kubota, Marina S; Tempone, Antonio J; Araújo, Helena R C; Guedes, Bruno A M; Orfanó, Alessandra S; Tadei, Wanderli P; Ríos-Velásquez, Claudia M; Han, Yeon S; Secundino, Nágila F C; Barillas-Mury, Carolina; Pimenta, Paulo F P; Traub-Csekö, Yara M

    2011-11-01

    Malaria affects 300 million people worldwide every year and 450,000 in Brazil. In coastal areas of Brazil, the main malaria vector is Anopheles aquasalis, and Plasmodium vivax is responsible for the majority of malaria cases in the Americas. Insects possess a powerful immune system to combat infections. Three pathways control the insect immune response: Toll, IMD, and JAK-STAT. Here we analyze the immune role of the A. aquasalis JAK-STAT pathway after P. vivax infection. Three genes, the transcription factor Signal Transducers and Activators of Transcription (STAT), the regulatory Protein Inhibitors of Activated STAT (PIAS) and the Nitric Oxide Synthase enzyme (NOS) were characterized. Expression of STAT and PIAS was higher in males than females and in eggs and first instar larvae when compared to larvae and pupae. RNA levels for STAT and PIAS increased 24 and 36 hours (h) after P. vivax challenge. NOS transcription increased 36 h post infection (hpi) while this protein was already detected in some midgut epithelial cells 24 hpi. Imunocytochemistry experiments using specific antibodies showed that in non-infected insects STAT and PIAS were found mostly in the fat body, while in infected mosquitoes the proteins were found in other body tissues. The knockdown of STAT by RNAi increased the number of oocysts in the midgut of A. aquasalis. This is the first clear evidence for the involvement of a specific immune pathway in the interaction of the Brazilian malaria vector A. aquasalis with P. vivax, delineating a potential target for the future development of disease controlling strategies. PMID:22069502

  15. The Prefrontal Cortex Achieves Inhibitory Control by Facilitating Subcortical Motor Pathway Connectivity

    PubMed Central

    Hughes, Laura E.; Anderson, Michael C.; Rowe, James B.

    2015-01-01

    Communication between the prefrontal cortex and subcortical nuclei underpins the control and inhibition of behavior. However, the interactions in such pathways remain controversial. Using a stop-signal response inhibition task and functional imaging with analysis of effective connectivity, we show that the lateral prefrontal cortex influences the strength of communication between regions in the frontostriatal motor system. We compared 20 generative models that represented alternative interactions between the inferior frontal gyrus, presupplementary motor area (preSMA), subthalamic nucleus (STN), and primary motor cortex during response inhibition. Bayesian model selection revealed that during successful response inhibition, the inferior frontal gyrus modulates an excitatory influence of the preSMA on the STN, thereby amplifying the downstream polysynaptic inhibition from the STN to the motor cortex. Critically, the strength of the interaction between preSMA and STN, and the degree of modulation by the inferior frontal gyrus, predicted individual differences in participants' stopping performance (stop-signal reaction time). We then used diffusion-weighted imaging with tractography to assess white matter structure in the pathways connecting these three regions. The mean diffusivity in tracts between preSMA and the STN, and between the inferior frontal gyrus and STN, also predicted individual differences in stopping efficiency. Finally, we found that white matter structure in the tract between preSMA and STN correlated with effective connectivity of the same pathway, providing important cross-modal validation of the effective connectivity measures. Together, the results demonstrate the network dynamics and modulatory role of the prefrontal cortex that underpin individual differences in inhibitory control. PMID:25589771

  16. A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size

    SciTech Connect

    Puseenam, Aekkachai; Yoshioka, Yasuhide; Nagai, Rika; Hashimoto, Reina; Suyari, Osamu; Itoh, Masanobu; Enomoto, Atsushi; Takahashi, Masahide; Yamaguchi, Masamitsu

    2009-11-15

    The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. We here identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway.

  17. Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes.

    PubMed

    Cestari, Igor; Stuart, Ken

    2015-05-26

    African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing. PMID:25964327

  18. Distinct protease pathways control cell shape and apoptosis in v-src-transformed quail neuroretina cells

    SciTech Connect

    Neel, Benjamin D.; Gillet, Germain . E-mail: g.gillet@ibcp.fr

    2005-11-15

    Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60 {sup v-src} tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60 {sup v-src}, we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60 {sup v-src} inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin-proteasome pathway is recruited early after p60 {sup v-src} inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells.

  19. Synapses with inhibitory neurons differentiate anterior cingulate from dorsolateral prefrontal pathways associated with cognitive control

    PubMed Central

    Medalla, M.; Barbas, H.

    2009-01-01

    Summary The primate dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC) focus attention on relevant signals and suppress noise in cognitive tasks. However, their synaptic interactions and unique roles in cognitive control are unknown. We report that two distinct pathways to DLPFC area 9, one from the neighboring area 46 and the other from the functionally distinct ACC, similarly innervate excitatory neurons associated with selecting relevant stimuli. However, ACC has more prevalent and larger synapses with inhibitory neurons and preferentially innervates calbindin inhibitory neurons, which reduce noise by inhibiting excitatory neurons. In contrast, area 46 mostly innervates calretinin inhibitory neurons, which disinhibit excitatory neurons. These synaptic specializations suggest that ACC has a greater impact in reducing noise in dorsolateral areas during challenging cognitive tasks involving conflict, error, or reversing decisions, mechanisms that are disrupted in schizophrenia. These observations highlight the unique roles of the DLPFC and ACC in cognitive control. PMID:19249280

  20. Modeling effective transmission pathways and control of the world's most successful parasite.

    PubMed

    Turner, Matthew; Lenhart, Suzanne; Rosenthal, Benjamin; Zhao, Xiaopeng

    2013-06-01

    Toxoplasma gondii(T. gondii) is a single-celled, intracellular protozoan responsible for the disease toxoplasmosis. The parasite is prevalent worldwide, and it infects all warm-blooded vertebrates. Consumption of meats in which this parasite has encysted confers risk of infection to people and other animals, as does ingestion of water or foods contaminated with environmentally resistant oocysts excreted by cats. Vertical transmission (from mother to offspring) is also possible, leading to disease risk and contributing additional means of ensuring perpetuation of transmission. In this work, we adopt a differential equation model to investigate the effective transmission pathways of T. gondii, as well as potential control mechanisms. Detailed analyses are carried out to examine the significance of transmission routes, virulence, vertical transmission, parasite-induced changes in host behavior, and controls based on vaccination and harvesting. Modeling and analysis efforts may shed insights into understanding the complex life cycle of T. gondii. PMID:23624067

  1. CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation

    PubMed Central

    Shuda, Masahiro; Velásquez, Celestino; Cheng, Erdong; Cordek, Daniel G.; Kwun, Hyun Jin; Chang, Yuan; Moore, Patrick S.

    2015-01-01

    Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E–m7GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3S10+ mitotic cell population having higher inactive p4E-BP1T37/T46+ saturation levels than pH3S10– interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling. PMID:25883264

  2. Mitotic catastrophe and cell death induced by depletion of centrosomal proteins

    PubMed Central

    Kimura, M; Yoshioka, T; Saio, M; Banno, Y; Nagaoka, H; Okano, Y

    2013-01-01

    Mitotic catastrophe, which refers to cell death or its prologue triggered by aberrant mitosis, can be induced by a heterogeneous group of stimuli, including chromosome damage or perturbation of the mitotic apparatus. We investigated the mechanism of mitotic catastrophe and cell death induced by depletion of centrosomal proteins that perturbs microtubule organization. We transfected cells harboring wild-type or mutated p53 with siRNAs targeting Aurora A, ninein, TOG, TACC3, γ-tubulin, or pericentriolar material-1, and monitored the effects on cell death. Knockdown of Aurora A, ninein, TOG, and TACC3 led to cell death, regardless of p53 status. Knockdown of Aurora A, ninein, and TOG, led to aberrant spindle formation and subsequent cell death, which was accompanied by several features of apoptosis, including nuclear condensation and Annexin V binding in HeLa cells. During this process, cleavage of poly(ADP-ribose) polymerase-1, caspase-3, and caspase-9 was detected, but cleavage of caspase-8 was not. Cell death, monitored by time-lapse imaging, occurred during both interphase and M phase. In cells depleted of a centrosomal protein (Aurora A, ninein, or TOG), the rate of cell death was higher if the cells were cotransfected with siRNA against BubR1 or Mad2 than if they were transfected with siRNA against Bub1 or a control siRNA. These results suggest that metaphase arrest is necessary for the mitotic catastrophe and cell death caused by depletion of centrosomal proteins. Knockdown of centrosomal proteins led to increased phosphorylation of Chk2. Enhanced p-Chk2 localization was also observed at the centrosome in cells arrested in M phase, as well as in the nuclei of dying cells. Cotransfection of siRNAs against Chk2, in combination with depletion of a centrosomal protein, decreased the amount of cell death. Thus, Chk2 activity is indispensable for apoptosis after mitotic catastrophe induced by depletion of centrosomal proteins that perturbs microtubule organization

  3. [Engineering MEP pathway in Escherichia coli for amorphadiene production and optimizing the bioprocess through glucose feeding control].

    PubMed

    Wang, Jianfeng; Xiong, Zhiqiang; Zhang, Siliang; Wang, Yong

    2014-01-01

    The pathway of 2-methyl-D-erythritol-4-phosphate (MEP) is the exclusive isoprenoid precursor biosynthetic pathway in Escherichia coli, with a higher theoretical yield than mevalonate (MVA) pathway. However, due to lack of information about the regulation of MEP pathway, only engineering MEP pathway in E. coli achieved limited improvement of heterologous isoprenoid production. We used exogenous MEP pathway genes to improve MEP pathway in E. coli and optimized the glucose feeding to release the potential of MEP pathway. The results demonstrate that co-expression of dxs2 from Streptomyces avermitilis and idi from Bacillus subtilis can increase amorphadiene production with 12.2-fold compared with the wild-type strain in shake flask fermentation. Then we established a high-cell density fermentation process for the engineered strain, and found that the phase from 24 to 72 h is important for product biosynthesis. The optimization of glucose feeding rate during 24 to 72 h significantly improved product accumulation, which was improved from 2.5 to 4.85 g/L, within the same process time. Considering the attenuation of strain metabolism after 72 h, this study further modulated the glucose feeding rate during exponential phase to control strain growth and the amorphadiene yield eventually reached to 6.1 g/L. These results provided useful information to develop engineered E. coli for isoprenoid production through MEP pathway engineering. PMID:24818480

  4. Mitotic Spindle Disruption by Alternating Electric Fields Leads to Improper Chromosome Segregation and Mitotic Catastrophe in Cancer Cells

    PubMed Central

    Giladi, Moshe; Schneiderman, Rosa S; Voloshin, Tali; Porat, Yaara; Munster, Mijal; Blat, Roni; Sherbo, Shay; Bomzon, Zeev; Urman, Noa; Itzhaki, Aviran; Cahal, Shay; Shteingauz, Anna; Chaudhry, Aafia; Kirson, Eilon D; Weinberg, Uri; Palti, Yoram

    2015-01-01

    Tumor Treating Fields (TTFields) are low intensity, intermediate frequency, alternating electric fields. TTFields are a unique anti-mitotic treatment modality delivered in a continuous, noninvasive manner to the region of a tumor. It was previously postulated that by exerting directional forces on highly polar intracellular elements during mitosis, TTFields could disrupt the normal assembly of spindle microtubules. However there is limited evidence directly linking TTFields to an effect on microtubules. Here we report that TTFields decrease the ratio between polymerized and total tubulin, and prevent proper mitotic spindle assembly. The aberrant mitotic events induced by TTFields lead to abnormal chromosome segregation, cellular multinucleation, and caspase dependent apoptosis of daughter cells. The effect of TTFields on cell viability and clonogenic survival substantially depends upon the cell division rate. We show that by extending the duration of exposure to TTFields, slowly dividing cells can be affected to a similar extent as rapidly dividing cells. PMID:26658786

  5. Mitotic Spindle Disruption by Alternating Electric Fields Leads to Improper Chromosome Segregation and Mitotic Catastrophe in Cancer Cells.

    PubMed

    Giladi, Moshe; Schneiderman, Rosa S; Voloshin, Tali; Porat, Yaara; Munster, Mijal; Blat, Roni; Sherbo, Shay; Bomzon, Zeev; Urman, Noa; Itzhaki, Aviran; Cahal, Shay; Shteingauz, Anna; Chaudhry, Aafia; Kirson, Eilon D; Weinberg, Uri; Palti, Yoram

    2015-01-01

    Tumor Treating Fields (TTFields) are low intensity, intermediate frequency, alternating electric fields. TTFields are a unique anti-mitotic treatment modality delivered in a continuous, noninvasive manner to the region of a tumor. It was previously postulated that by exerting directional forces on highly polar intracellular elements during mitosis, TTFields could disrupt the normal assembly of spindle microtubules. However there is limited evidence directly linking TTFields to an effect on microtubules. Here we report that TTFields decrease the ratio between polymerized and total tubulin, and prevent proper mitotic spindle assembly. The aberrant mitotic events induced by TTFields lead to abnormal chromosome segregation, cellular multinucleation, and caspase dependent apoptosis of daughter cells. The effect of TTFields on cell viability and clonogenic survival substantially depends upon the cell division rate. We show that by extending the duration of exposure to TTFields, slowly dividing cells can be affected to a similar extent as rapidly dividing cells. PMID:26658786

  6. Complexity of contrasting flow controls on phosphorus flux and transfer pathways

    NASA Astrophysics Data System (ADS)

    Mellander, Per-Erik; Jordan, Phil; Shore, Mairead; Melland, Alice R.; Shortle, Ger

    2015-04-01

    Insights on hydrological processes from 'rain to stream' are important when interpreting the effectiveness of measures for reducing phosphorus (P) losses from agricultural sources to water bodies. A general understanding is that measures for management of P transfers along surface pathways will be consistently effective when applied on a whole territory approach. It is, however, necessary for policies to incorporate an understanding of spatial and temporal variation in hydrological flow controls, associated nutrient transfer pathways and chemical processes along the pathways. This variation is associated with variability in soil drainage, geology, climate and land management between hillslopes and catchments. In this study, four years of hourly stream P flux data from two Irish agricultural catchments were analysed on an annual and event flow basis. The analysis was related to hydrological flow paths in order to help develop a catchment scale (ca. 10 km2) theory of P export and associated processes that could help with specific P mitigation policies in heterogeneous river basin planning zones. A grassland catchment with mostly poorly drained soils and a 'flashy hydrology' had three times higher annual P flux than an arable catchment with mostly well-drained soils and a more buffered hydrology (1.04 kg total P ha-1 compared to 0.34 kg total P ha-1), despite the arable catchment having larger areas with high soil P status and more discharge. Neither of the catchments indicated P supply limitations. The magnitude of the P fluxes from the two catchments were not defined by land use, source pressure or discharge volume, but rather by a more basic rainfall-to-runoff partitioning which influenced the proportions of quickflow and slowflow. Despite the catchments having contrasting flow controls and P transfer pathways, there were larger differences in P loss between the years than between the catchments and the P loss from the arable catchment appeared to be more sensitive

  7. The Emerging Nexus of Active DNA Demethylation and Mitochondrial Oxidative Metabolism in Post-Mitotic Neurons

    PubMed Central

    Meng, Huan; Chen, Guiquan; Gao, Hui-Ming; Song, Xiaoyu; Shi, Yun; Cao, Liu

    2014-01-01

    The variable patterns of DNA methylation in mammals have been linked to a number of physiological processes, including normal embryonic development and disease pathogenesis. Active removal of DNA methylation, which potentially regulates neuronal gene expression both globally and gene specifically, has been recently implicated in neuronal plasticity, learning and memory processes. Model pathways of active DNA demethylation involve ten-eleven translocation (TET) methylcytosine dioxygenases that are dependent on oxidative metabolites. In addition, reactive oxygen species (ROS) and oxidizing agents generate oxidative modifications of DNA bases that can be removed by base excision repair proteins. These potentially link the two processes of active DNA demethylation and mitochondrial oxidative metabolism in post-mitotic neurons. We review the current biochemical understanding of the DNA demethylation process and discuss its potential interaction with oxidative metabolism. We then summarise the emerging roles of both processes and their interaction in neural plasticity and memory formation and the pathophysiology of neurodegeneration. Finally, possible therapeutic approaches for neurodegenerative diseases are proposed, including reprogramming therapy by global DNA demethylation and mitohormesis therapy for locus-specific DNA demethylation in post-mitotic neurons. PMID:25490140

  8. The Flexible Ends of CENP-A Nucleosome Are Required for Mitotic Fidelity.

    PubMed

    Roulland, Yohan; Ouararhni, Khalid; Naidenov, Mladen; Ramos, Lorrie; Shuaib, Muhammad; Syed, Sajad Hussain; Lone, Imtiaz Nizar; Boopathi, Ramachandran; Fontaine, Emeline; Papai, Gabor; Tachiwana, Hiroaki; Gautier, Thierry; Skoufias, Dimitrios; Padmanabhan, Kiran; Bednar, Jan; Kurumizaka, Hitoshi; Schultz, Patrick; Angelov, Dimitar; Hamiche, Ali; Dimitrov, Stefan

    2016-08-18

    CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure of CENP-A nucleosome suggests flexible nucleosomal DNA ends, but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy, we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic, and genetic data reveal that higher flexibility of DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn αN-helix of CENP-A with the 3-turn αN-helix of H3 results in compact particles with rigidified DNA ends, able to bind histone H1. In vivo replacement of CENP-A with H3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins, and significant mitotic and cytokinesis defects. Our data reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway. PMID:27499292

  9. The GTPase regulatory proteins Pix and Git control tissue growth via the Hippo pathway.

    PubMed

    Dent, Lucas G; Poon, Carole L C; Zhang, Xiaomeng; Degoutin, Joffrey L; Tipping, Marla; Veraksa, Alexey; Harvey, Kieran F

    2015-01-01

    The Salvador-Warts-Hippo (Hippo) pathway is a conserved regulator of organ size and is deregulated in human cancers. In epithelial tissues, the Hippo pathway is regulated by fundamental cell biological properties, such as polarity and adhesion, and coordinates these with tissue growth. Despite its importance in disease, development, and regeneration, the complete set of proteins that regulate Hippo signaling remain undefined. To address this, we used proteomics to identify proteins that bind to the Hippo (Hpo) kinase. Prominent among these were PAK-interacting exchange factor (known as Pix or RtGEF) and G-protein-coupled receptor kinase-interacting protein (Git). Pix is a conserved Rho-type guanine nucleotide exchange factor (Rho-GEF) homologous to Beta-PIX and Alpha-PIX in mammals. Git is the single Drosophila melanogaster homolog of the mammalian GIT1 and GIT2 proteins, which were originally identified in the search for molecules that interact with G-protein-coupled receptor kinases. Pix and Git form an oligomeric scaffold to facilitate sterile 20-like kinase activation and have also been linked to GTPase regulation. We show that Pix and Git regulate Hippo-pathway-dependent tissue growth in D. melanogaster and that they do this in parallel to the known upstream regulator Fat cadherin. Pix and Git influence activity of the Hpo kinase by acting as a scaffold complex, rather than enzymes, and promote Hpo dimerization and autophosphorylation of Hpo's activation loop. Therefore, we provide important new insights into an ancient signaling network that controls the growth of metazoan tissues. PMID:25484297

  10. The mammalian molecular clockwork controls rhythmic expression of its own input pathway components.

    PubMed

    Pfeffer, Martina; Müller, Christian M; Mordel, Jérôme; Meissl, Hilmar; Ansari, Nariman; Deller, Thomas; Korf, Horst-Werner; von Gall, Charlotte

    2009-05-13

    The core molecular clockwork in the suprachiasmatic nucleus (SCN) is based on autoregulatory feedback loops of transcriptional activators (CLOCK/NPAS2 and BMAL1) and inhibitors (mPER1-2 and mCRY1-2). To synchronize the phase of the molecular clockwork to the environmental day and night condition, light at dusk and dawn increases mPer expression. However, the signal transduction pathways differ remarkably between the day/night and the night/day transition. Light during early night leads to intracellular Ca(2+) release by neuronal ryanodine receptors (RyRs), resulting in phase delays. Light during late night triggers an increase in guanylyl cyclase activity, resulting in phase advances. To date, it is still unknown how the core molecular clockwork regulates the availability of the respective input pathway components. Therefore, we examined light resetting mechanisms in mice with an impaired molecular clockwork (BMAL1(-/-)) and the corresponding wild type (BMAL1(+/+)) using in situ hybridization, real-time PCR, immunohistochemistry, and a luciferase reporter system. In addition, intracellular calcium concentrations (Ca(2+)(i)) were measured in SCN slices using two-photon microscopy. In the SCN of BMAL1(-/-) mice Ryr mRNA and RyR protein levels were reduced, and light-induced mPer expression was selectively impaired during early night. Transcription assays with NIH3T3 fibroblasts showed that Ryr expression was activated by CLOCK::BMAL1 and inhibited by mCRY1. The Ca(2+)(i) response of SCN cells to the RyR agonist caffeine was reduced in BMAL1(-/-) compared with BMAL1(+/+) mice. Our findings provide the first evidence that the mammalian molecular clockwork influences Ryr expression and thus controls its own photic input pathway components. PMID:19439589

  11. Dynamical modeling of syncytial mitotic cycles in Drosophila embryos.

    PubMed

    Calzone, Laurence; Thieffry, Denis; Tyson, John J; Novak, Bela

    2007-01-01

    Immediately following fertilization, the fruit fly embryo undergoes 13 rapid, synchronous, syncytial nuclear division cycles driven by maternal genes and proteins. During these mitotic cycles, there are barely detectable oscillations in the total level of B-type cyclins. In this paper, we propose a dynamical model for the molecular events underlying these early nuclear division cycles in Drosophila. The model distinguishes nuclear and cytoplasmic compartments of the embryo and permits exploration of a variety of rules for protein transport between the compartments. Numerical simulations reproduce the main features of wild-type mitotic cycles: patterns of protein accumulation and degradation, lengthening of later cycles, and arrest in interphase 14. The model is consistent with mutations that introduce subtle changes in the number of mitotic cycles before interphase arrest. Bifurcation analysis of the differential equations reveals the dependence of mitotic oscillations on cycle number, and how this dependence is altered by mutations. The model can be used to predict the phenotypes of novel mutations and effective ranges of the unmeasured rate constants and transport coefficients in the proposed mechanism. PMID:17667953

  12. A mitotic transcriptional switch in polycystic kidney disease

    PubMed Central

    Verdeguer, Francisco; Corre, Stephanie Le; Fischer, Evelyne; Callens, Celine; Garbay, Serge; Doyen, Antonia; Igarashi, Peter; Terzi, Fabiola; Pontoglio, Marco

    2011-01-01

    Hepatocyte nuclear factor-1β(HNF-1β) is a transcription factor required for the expression of several renal cystic genes and whose prenatal deletion leads to polycystic kidney disease (PKD)1. We show here that inactivation of Hnf1b from postnatal day 10 onward does not elicit cystic dilations in tubules after their proliferative morphogenetic elongation is over. Cystogenic resistance is intrinsically linked to the quiescent state of cells. In fact, when Hnf1b deficient quiescent cells are forced to proliferate by an ischemiareperfusion injury, they give rise to cysts, owing to loss of oriented cell division. Remarkably, in quiescent cells, the transcription of crucial cystogenic target genes is maintained even in the absence of HNF-1β. However, their expression is lost as soon as cells proliferate and the chromatin of target genes acquires heterochromatin marks. These results unveil a previously undescribed aspect of gene regulation. It is well established that transcription is shut off during the mitotic condensation of chromatin2,3. We propose that transcription factors such as HNF-1β might be involved in reprogramming gene expression after transcriptional silencing is induced by mitotic chromatin condensation. Notably, HNF-1β remains associated with the mitotically condensed chromosomal barrels. This association suggests that HNF-1β is a bookmarking factor that is necessary for reopening the chromatin of target genes after mitotic silencing. PMID:19966811

  13. Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets.

    PubMed

    Vleugel, Mathijs; Roth, Sophie; Groenendijk, Celebrity F; Dogterom, Marileen

    2016-01-01

    Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle. PMID:27584979

  14. Cep192 and the generation of the mitotic spindle.

    PubMed

    Gomez-Ferreria, Maria Ana; Sharp, David J

    2008-06-01

    The cellular mechanisms used to generate sufficient microtubule polymer mass to drive the assembly and function of the mitotic spindle remain a matter of great interest. As the primary microtubule nucleating structures in somatic animal cells, centrosomes have been assumed to figure prominently in spindle assembly. At the onset of mitosis, centrosomes undergo a dramatic increase in size and microtubule nucleating capacity, termed maturation, which is likely a key event in mitotic spindle formation. Interestingly, however, spindles can still form in the absence of centrosomes calling into question the specific mitotic role of these organelles. Recent work has shown that the human centrosomal protein, Cep192, is required for both centrosome maturation and spindle assembly thus providing a molecular link between these two processes. In this article, we propose that Cep192 does so by forming a scaffolding on which proteins involved in microtubule nucleation are sequestered and become active in mitotic cells. Normally, this activity is largely confined to centrosomes but in their absence continues to function but is dispersed to other sites within the cell. PMID:18469523

  15. Controlling anoxic tolerance in adult Drosophila via the cGMP–PKG pathway

    PubMed Central

    Dawson-Scully, K.; Bukvic, D.; Chakaborty-Chatterjee, M.; Ferreira, R.; Milton, S. L.; Sokolowski, M. B.

    2010-01-01

    In this study we identify a cGMP-dependent protein kinase (PKG) cascade as a biochemical pathway critical for controlling low-oxygen tolerance in the adult fruit fly, Drosophila melanogaster. Even though adult Drosophila can survive in 0% oxygen (anoxia) environments for hours, air with less than 2% oxygen rapidly induces locomotory failure resulting in an anoxic coma. We use natural genetic variation and an induced mutation in the foraging (for) gene, which encodes a Drosophila PKG, to demonstrate that the onset of anoxic coma is correlated with PKG activity. Flies that have lower PKG activity demonstrate a significant increase in time to the onset of anoxic coma. Further, in vivo pharmacological manipulations reveal that reducing either PKG or protein phosphatase 2A (PP2A) activity increases tolerance of behavior to acute hypoxic conditions. Alternatively, PKG activation and phosphodiesterase (PDE5/6) inhibition significantly reduce the time to the onset of anoxic coma. By manipulating these targets in paired combinations, we characterized a specific PKG cascade, with upstream and downstream components. Further, using genetic variants of PKG expression/activity subjected to chronic anoxia over 6 h, ~50% of animals with higher PKG activity survive, while only ~25% of those with lower PKG activity survive after a 24 h recovery. Therefore, in this report we describe the PKG pathway and the differential protection of function vs survival in a critically low oxygen environment. PMID:20581270

  16. Dynamic control over supramolecular handedness by selecting chiral induction pathways at the solution-solid interface.

    PubMed

    Fang, Yuan; Ghijsens, Elke; Ivasenko, Oleksandr; Cao, Hai; Noguchi, Aya; Mali, Kunal S; Tahara, Kazukuni; Tobe, Yoshito; De Feyter, Steven

    2016-07-01

    A dominant theme within the research on two-dimensional chirality is the sergeant-soldiers principle, wherein a small fraction of chiral molecules (sergeants) is used to skew the handedness of achiral molecules (soldiers) to generate a homochiral surface. Here, we have combined the sergeant-soldiers principle with temperature-dependent molecular self-assembly to unravel a peculiar chiral amplification mechanism at the solution-solid interface in which, depending on the concentration of a sergeant-soldiers solution, the majority handedness of the system can either be amplified or entirely reversed after an annealing step, furnishing a homochiral surface. Two discrete pathways that affect different stages of two-dimensional crystal growth are invoked for rationalizing this phenomenon and we present a set of experiments where the access to each pathway can be precisely controlled. These results demonstrate that a detailed understanding of subtle intermolecular and interfacial interactions can be used to induce drastic changes in the handedness of a supramolecular network. PMID:27325099

  17. Pathway activation profiling reveals new insights into Age-related Macular Degeneration and provides avenues for therapeutic interventions

    PubMed Central

    Makarev, Evgeny; Cantor, Charles; Zhavoronkov, Alex; Buzdin, Anton; Aliper, Alexander; Csoka, Antonei Benjamin

    2014-01-01

    Age-related macular degeneration (AMD) is a major cause of blindness in older people and is caused by loss of the central region of the retinal pigment epithelium (RPE). Conventional methods of gene expression analysis have yielded important insights into AMD pathogenesis, but the precise molecular pathway alterations are still poorly understood. Therefore we developed a new software program, “AMD Medicine”, and discovered differential pathway activation profiles in samples of human RPE/choroid from AMD patients and controls. We identified 29 pathways in RPE-choroid AMD phenotypes: 27 pathways were activated in AMD compared to controls, and 2 pathways were activated in controls compared to AMD. In AMD, we identified a graded activation of pathways related to wound response, complement cascade, and cell survival. Also, there was downregulation of two pathways responsible for apoptosis. Furthermore, significant activation of pro-mitotic pathways is consistent with dedifferentiation and cell proliferation events, which occur early in the pathogenesis of AMD. Significantly, we discovered new global pathway activation signatures of AMD involved in the cell-based inflammatory response: IL-2, STAT3, and ERK. The ultimate aim of our research is to achieve a better understanding of signaling pathways involved in AMD pathology, which will eventually lead to better treatments. PMID:25543336

  18. Cytotoxic effects of cylindrospermopsin in mitotic and non-mitotic Vicia faba cells.

    PubMed

    Garda, Tamás; Riba, Milán; Vasas, Gábor; Beyer, Dániel; M-Hamvas, Márta; Hajdu, Gréta; Tándor, Ildikó; Máthé, Csaba

    2015-02-01

    Cylindrospermopsin (CYN) is a cyanobacterial toxin known as a eukaryotic protein synthesis inhibitor. We aimed to study its effects on growth, stress responses and mitosis of a eukaryotic model, Vicia faba (broad bean). Growth responses depended on exposure time (3 or 6d), cyanotoxin concentration, culture conditions (dark or continuous light) and V. faba cultivar ("Standard" or "ARC Egypt Cross"). At 6d of exposure, CYN had a transient stimulatory effect on root system growth, roots being possibly capable of detoxification. The toxin induced nucleus fragmentation, blebbing and chromosomal breaks indicating double stranded DNA breaks and programmed cell death. Root necrotic tissue was observed at 0.1-20 μg mL(-1) CYN that probably impeded toxin uptake into vascular tissue. Growth and cell death processes observed were general stress responses. In lateral root tip meristems, lower CYN concentrations (0.01-0.1 μg mL(-1)) induced the stimulation of mitosis and distinct mitotic phases, irrespective of culture conditions or the cultivar used. Higher cyanotoxin concentrations inhibited mitosis. Short-term exposure of hydroxylurea-synchronized roots to 5 μg mL(-1) CYN induced delay of mitosis that might have been related to a delay of de novo protein synthesis. CYN induced the formation of double, split and asymmetric preprophase bands (PPBs), in parallel with the alteration of cell division planes, related to the interference of cyanotoxin with protein synthesis, thus it was a plant- and CYN specific alteration. PMID:25016338

  19. Deoxyxylulose 5-Phosphate Synthase Controls Flux through the Methylerythritol 4-Phosphate Pathway in Arabidopsis1[C][W][OPEN

    PubMed Central

    Wright, Louwrance P.; Rohwer, Johann M.; Ghirardo, Andrea; Hammerbacher, Almuth; Ortiz-Alcaide, Miriam; Raguschke, Bettina; Schnitzler, Jörg-Peter; Gershenzon, Jonathan; Phillips, Michael A.

    2014-01-01

    The 2-C-methylerythritol 4-phosphate (MEP) pathway supplies precursors for plastidial isoprenoid biosynthesis including carotenoids, redox cofactor side chains, and biogenic volatile organic compounds. We examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), using metabolic control analysis. Multiple Arabidopsis (Arabidopsis thaliana) lines presenting a range of DXS activities were dynamically labeled with 13CO2 in an illuminated, climate-controlled, gas exchange cuvette. Carbon was rapidly assimilated into MEP pathway intermediates, but not into the mevalonate pathway. A flux control coefficient of 0.82 was calculated for DXS by correlating absolute flux to enzyme activity under photosynthetic steady-state conditions, indicating that DXS is the major controlling enzyme of the MEP pathway. DXS manipulation also revealed a second pool of a downstream metabolite, 2-C-methylerythritol-2,4-cyclodiphosphate (MEcDP), metabolically isolated from the MEP pathway. DXS overexpression led to a 3- to 4-fold increase in MEcDP pool size but to a 2-fold drop in maximal labeling. The existence of this pool was supported by residual MEcDP levels detected in dark-adapted transgenic plants. Both pools of MEcDP are closely modulated by DXS activity, as shown by the fact that the concentration control coefficient of DXS was twice as high for MEcDP (0.74) as for 1-deoxyxylulose 5-phosphate (0.35) or dimethylallyl diphosphate (0.34). Despite the high flux control coefficient for DXS, its overexpression led to only modest increases in isoprenoid end products and in the photosynthetic rate. Diversion of flux via MEcDP may partly explain these findings and suggests new opportunities to engineer the MEP pathway. PMID:24987018

  20. OXIDATIVE STRESS, INFLAMMATION AND CARCINOGENESIS ARE CONTROLLED THROUGH THE PENTOSE PHOSPHATE PATHWAY BY TRANSALDOLASE

    PubMed Central

    Perl, Andras; Hanczko, Robert; Telarico, Tiffany; Oaks, Zachary; Landas, Steve

    2011-01-01

    Metabolism of glucose through the pentose phosphate pathway (PPP) influences the development of diverse pathologies. Hemolytic anemia due to deficiency of PPP enzyme glucose 6-phosphate dehydrogenase is the most common genetic disease in humans. Recently, inactivation of another PPP enzyme, transaldolase (TAL), has been implicated in male infertility and fatty liver progressing to steatohepatitis and cancer. Hepatocarcinogenesis was associated with activation of aldose reductase and redox-sensitive transcription factors and prevented by N-acetylcysteine. Here, we discuss how alternative formulations of the PPP with and without TAL reflect cell type-specific metabolic control of oxidative stress, a critical source of inflammation and carcinogenesis. Ongoing studies of TAL deficiency will identify new molecular targets for diagnosis and treatment in clinical practice. PMID:21376665

  1. Rac1 controls epithelial tube length through the apical secretion and polarity pathways

    PubMed Central

    Sollier, Kévin; Gaudé, Helori-Mael; Chartier, François J.-M.; Laprise, Patrick

    2016-01-01

    ABSTRACT The morphometric parameters of epithelial tubes are critical to the physiology and homeostasis of most organs. In addition, many human diseases are associated with tube-size defects. Here, we show that Rac1 limits epithelial tube elongation in the developing fly trachea by promoting Rab5-dependent endocytosis of the apical determinant Crumbs. Rac1 is also involved in a positive feedback loop with the septate junction protein Coracle. Thereby, Rac1 precludes paracellular diffusion and contributes to the septate junction-dependent secretion of the chitin-modifying enzymes Vermiform and Serpentine, which restrict epithelial tube length independently of Crumbs. Thus, Rac1 is a critical component of two important pathways controlling epithelial tube morphogenesis. PMID:26700724

  2. Rac1 controls epithelial tube length through the apical secretion and polarity pathways.

    PubMed

    Sollier, Kévin; Gaudé, Helori-Mael; Chartier, François J-M; Laprise, Patrick

    2015-01-01

    The morphometric parameters of epithelial tubes are critical to the physiology and homeostasis of most organs. In addition, many human diseases are associated with tube-size defects. Here, we show that Rac1 limits epithelial tube elongation in the developing fly trachea by promoting Rab5-dependent endocytosis of the apical determinant Crumbs. Rac1 is also involved in a positive feedback loop with the septate junction protein Coracle. Thereby, Rac1 precludes paracellular diffusion and contributes to the septate junction-dependent secretion of the chitin-modifying enzymes Vermiform and Serpentine, which restrict epithelial tube length independently of Crumbs. Thus, Rac1 is a critical component of two important pathways controlling epithelial tube morphogenesis. PMID:26700724

  3. Controlling the pathways in molecular decomposition: The vibrationally mediated photodissociation of water

    SciTech Connect

    Vander Wal, R.L.; Crim, F.F. )

    1989-07-13

    Vibrationally mediated photodissociation, in which vibrational overtone excitation prepares a single rovibrational state of water that an ultraviolet photon subsequently dissociates, permits a fully state resolved study of the photodissociation of water. Dissociating water from six different initial rotational states and detecting the OH products by laser-induced fluorescence shows that the distribution of the products among their rotational states depends strongly on the state initially selected in the vibrational overtone excitation step. These measurements also demonstrate the control of the dissociation pathway by selection of different intermediate states. Dissociating water molecules from one vibrational state ({vert bar}04>4{sup {minus}}) produces almost no vibrationally excited OH products, but dissociating another state ({vert bar}13>{sup {minus}}) that corresponds to a different nuclear motion produces roughly comparable amounts of OH(v = 1) and OH(v = O).

  4. RHAMM Promotes Interphase Microtubule Instability and Mitotic Spindle Integrity through MEK1/ERK1/2 Activity*

    PubMed Central

    Tolg, Cornelia; Hamilton, Sara R.; Morningstar, Lyndsey; Zhang, Jing; Zhang, S.; Esguerra, Kenneth V.; Telmer, Patrick G.; Luyt, Len G.; Harrison, Rene; McCarthy, James B.; Turley, Eva A.

    2010-01-01

    An oncogenic form of RHAMM (receptor for hyaluronan-mediated motility, mouse, amino acids 163–794 termed RHAMMΔ163) is a cell surface hyaluronan receptor and mitotic spindle protein that is highly expressed in aggressive human cancers. Its regulation of mitotic spindle integrity is thought to contribute to tumor progression, but the molecular mechanisms underlying this function have not previously been defined. Here, we report that intracellular RHAMMΔ163 modifies the stability of interphase and mitotic spindle microtubules through ERK1/2 activity. RHAMM−/− mouse embryonic fibroblasts exhibit strongly acetylated interphase microtubules, multi-pole mitotic spindles, aberrant chromosome segregation, and inappropriate cytokinesis during mitosis. These defects are rescued by either expression of RHAMM or mutant active MEK1. Mutational analyses show that RHAMMΔ163 binds to α- and β-tubulin protein via a carboxyl-terminal leucine zipper, but in vitro analyses indicate this interaction does not directly contribute to tubulin polymerization/stability. Co-immunoprecipitation and pulldown assays reveal complexes of RHAMMΔ163, ERK1/2-MEK1, and α- and β-tubulin and demonstrate direct binding of RHAMMΔ163 to ERK1 via a D-site motif. In vitro kinase analyses, expression of mutant RHAMMΔ163 defective in ERK1 binding in mouse embryonic fibroblasts, and blocking MEK1 activity collectively confirm that the effect of RHAMMΔ163 on interphase and mitotic spindle microtubules is mediated by ERK1/2 activity. Our results suggest a model wherein intracellular RHAMMΔ163 functions as an adaptor protein to control microtubule polymerization during interphase and mitosis as a result of localizing ERK1/2-MEK1 complexes to their tubulin-associated substrates. PMID:20558733

  5. Alterations in leukocyte transcriptional control pathway activity associated with major depressive disorder and antidepressant treatment.

    PubMed

    Mellon, S H; Wolkowitz, O M; Schonemann, M D; Epel, E S; Rosser, R; Burke, H B; Mahan, L; Reus, V I; Stamatiou, D; Liew, C-C; Cole, S W

    2016-01-01

    Major depressive disorder (MDD) is associated with a significantly elevated risk of developing serious medical illnesses such as cardiovascular disease, immune impairments, infection, dementia and premature death. Previous work has demonstrated immune dysregulation in subjects with MDD. Using genome-wide transcriptional profiling and promoter-based bioinformatic strategies, we assessed leukocyte transcription factor (TF) activity in leukocytes from 20 unmedicated MDD subjects versus 20 age-, sex- and ethnicity-matched healthy controls, before initiation of antidepressant therapy, and in 17 of the MDD subjects after 8 weeks of sertraline treatment. In leukocytes from unmedicated MDD subjects, bioinformatic analysis of transcription control pathway activity indicated an increased transcriptional activity of cAMP response element-binding/activating TF (CREB/ATF) and increased activity of TFs associated with cellular responses to oxidative stress (nuclear factor erythroid-derived 2-like 2, NFE2l2 or NRF2). Eight weeks of antidepressant therapy was associated with significant reductions in Hamilton Depression Rating Scale scores and reduced activity of NRF2, but not in CREB/ATF activity. Several other transcriptional regulation pathways, including the glucocorticoid receptor (GR), nuclear factor kappa-B cells (NF-κB), early growth response proteins 1-4 (EGR1-4) and interferon-responsive TFs, showed either no significant differences as a function of disease or treatment, or activities that were opposite to those previously hypothesized to be involved in the etiology of MDD or effective treatment. Our results suggest that CREB/ATF and NRF2 signaling may contribute to MDD by activating immune cell transcriptome dynamics that ultimately influence central nervous system (CNS) motivational and affective processes via circulating mediators. PMID:27219347

  6. A Protein Turnover Signaling Motif Controls the Stimulus-Sensitivity of Stress Response Pathways

    PubMed Central

    Loriaux, Paul Michael; Hoffmann, Alexander

    2013-01-01

    Stimulus-induced perturbations from the steady state are a hallmark of signal transduction. In some signaling modules, the steady state is characterized by rapid synthesis and degradation of signaling proteins. Conspicuous among these are the p53 tumor suppressor, its negative regulator Mdm2, and the negative feedback regulator of NFκB, IκBα. We investigated the physiological importance of this turnover, or flux, using a computational method that allows flux to be systematically altered independently of the steady state protein abundances. Applying our method to a prototypical signaling module, we show that flux can precisely control the dynamic response to perturbation. Next, we applied our method to experimentally validated models of p53 and NFκB signaling. We find that high p53 flux is required for oscillations in response to a saturating dose of ionizing radiation (IR). In contrast, high flux of Mdm2 is not required for oscillations but preserves p53 sensitivity to sub-saturating doses of IR. In the NFκB system, degradation of NFκB-bound IκB by the IκB kinase (IKK) is required for activation in response to TNF, while high IKK-independent degradation prevents spurious activation in response to metabolic stress or low doses of TNF. Our work identifies flux pairs with opposing functional effects as a signaling motif that controls the stimulus-sensitivity of the p53 and NFκB stress-response pathways, and may constitute a general design principle in signaling pathways. PMID:23468615

  7. Signal transduction pathways induced by GM-CSF in microglia: significance in the control of proliferation.

    PubMed

    Liva, S M; Kahn, M A; Dopp, J M; de Vellis, J

    1999-06-01

    Communication between cells of the central nervous system (CNS) and of the immune system is accomplished by a network of cytokines and growth factors. Certain cytokines and growth factors cause activation of microglia, contributing to inflammatory states in the CNS. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has numerous effects on microglia, ranging from induction of proliferation to changes in morphology. GM-CSF is also a growth factor for cells of the myeloid lineage, and the signal tranduction induced by GM-CSF in these cells has been extensively studied. Most notably, the importance of the Jak/STAT and MAP kinase pathways in mitogenesis has been shown in many different systems. We show here that primary microglia and a microglia cell line, BV-2, have a Jak/STAT expression pattern and GM-CSF inducibility similar to that of monocytes and macrophages. Primary microglia and BV-2 cells expressed identical Jak/STATs: Jakl, Jak2, Jak3, Tyk2, STAT1alpha/beta, STAT3, STAT5A, STAT5B, and STAT6. In addition, GM-CSF induced Jak2, STAT5A, and STAT5B in BV-2 cells, as it does in monocytes and macrophages. Immunocytochemical analysis showed that STAT5 translocates to the nucleus following GM-CSF stimulation of microglia. We also found the MAP kinases, ERK1 and ERK2, to be phosphorylated in microglia and BV-2 cells following induction by GM-CSF. Jak2, STAT5A, STAT5B, and ERKs are known to be important in controlling cellular proliferation. Drugs that block these pathways may become tools to control inflammation in the CNS by limiting microglial proliferation. PMID:10383053

  8. Apoptotic-like death occurs through a caspase-independent route in colon carcinoma cells undergoing mitotic catastrophe.

    PubMed

    Llovera, Laia; Mansilla, Sylvia; Portugal, José

    2012-12-29

    We have examined the relationship between chemotherapy-induced mitotic catastrophe and cell death by apoptosis in both wild-type and p53(-/-) HCT116 human colon carcinoma cells treated with nanomolar concentrations of paclitaxel (PTX), a drug that acts on tubulin altering the normal development of mitosis. After treatment, HCT116 cells entered mitosis regardless of the presence of functional p53, which resulted in changes in the distribution of cells in the different phases of the cell cycle, and in cell death. In the presence of PTX, the percentage of polyploid cells observed was higher in p53-deficient cells, indicating that mitotic slippage was favored compared to wild-type cells, with the presence of large multinucleate cells. PTX caused mitotic catastrophe and about 50-60% cells that were entering an aberrant mitosis died through an apoptotic-like pathway characterized by the presence of phosphatidylserine in the outer cell membrane, which occurred in the absence of significant activation of caspases. Lack of p53 facilitated endoreduplication and polyploidy in PTX-treated cells, but cells were still killed with similar efficacy through the same apoptotic-like mechanism in the absence of caspase activity. PMID:22885806

  9. Live-Cell Imaging Visualizes Frequent Mitotic Skipping During Senescence-Like Growth Arrest in Mammary Carcinoma Cells Exposed to Ionizing Radiation

    SciTech Connect

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

    2012-06-01

    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% and 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.

  10. PAR1 participates in the ability of multidrug resistance and tumorigenesis by controlling Hippo-YAP pathway

    PubMed Central

    Fujimoto, Daisuke; Ueda, Yuki; Hirono, Yasuo; Goi, Takanori; Yamaguchi, Akio

    2015-01-01

    The Hippo pathway significantly correlates with organ size control and tumorigenesis. The activity of YAP/TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in cancer stem cells (CSCs). But, upstream signals that control the mammalian Hippo pathway have not been well understood. Here, we reveal a connection between the Protease-activated receptor 1 (PAR1) signaling pathway and the Hippo-YAP pathway in gastric cancer stem-like cells. The selective PAR1 agonist TFLLR-NH2 induces an increase in the fraction of side population cells which is enriched in CSCs, and promotes tumorigenesis, multi cancer drug resistance, cell morphological change, and cell invasion which are characteristics of CSCs. In addition, PAR1 activation inhibits the Hippo-YAP pathway kinase Lats via Rho GTPase. Lats kinase inhibition in turn results in increased nuclear localization of dephosphorylated YAP. Furthermore, PAR1 activation confers CSCs related traits via the Hippo-YAP pathway, and the Hippo-YAP pathway correlates with epithelial mesenchymal transition which is induced by PAR1 activation. Our research suggests that the PAR1 signaling deeply participates in the ability of multi drug resistance and tumorigenesis through interactions with the Hippo-YAP pathway signaling in gastric cancer stem-like cells. We presume that inhibited YAP is a new therapeutic target in the treatment human gastric cancer invasion and metastasis by dysregulated PAR1 or its agonists. The Hippo pathway significantly correlates with organ size control and tumorigenesis. The activity of YAP/TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in cancer stem cells (CSCs). But, upstream signals that control the mammalian Hippo pathway have not been well understood. Here, we reveal a connection between the Protease-activated receptor 1 (PAR1) signaling pathway and the Hippo-YAP pathway in gastric cancer stem

  11. Autophagy mediates the mitotic senescence transition

    PubMed Central

    Young, Andrew R.J.; Narita, Masako; Ferreira, Manuela; Kirschner, Kristina; Sadaie, Mahito; Darot, Jeremy F.J.; Tavaré, Simon; Arakawa, Satoko; Shimizu, Shigeomi; Watt, Fiona M.; Narita, Masashi

    2009-01-01

    As a stress response, senescence is a dynamic process involving multiple effector mechanisms whose combination determines the phenotypic quality. Here we identify autophagy as a new effector mechanism of senescence. Autophagy is activated during senescence and its activation is correlated with negative feedback in the PI3K–mammalian target of rapamycin (mTOR) pathway. A subset of autophagy-related genes are up-regulated during senescence: Overexpression of one of those genes, ULK3, induces autophagy and senescence. Furthermore, inhibition of autophagy delays the senescence phenotype, including senescence-associated secretion. Our data suggest that autophagy, and its consequent protein turnover, mediate the acquisition of the senescence phenotype. PMID:19279323

  12. Molecular mechanism of APC/C activation by mitotic phosphorylation.

    PubMed

    Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

    2016-05-12

    In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

  13. The stress-activated protein kinases p38α/β and JNK1/2 cooperate with Chk1 to inhibit mitotic entry upon DNA replication arrest

    PubMed Central

    Llopis, Alba; Salvador, Noelia; Ercilla, Amaia; Guaita-Esteruelas, Sandra; Barrantes, Ivan del Barco; Gupta, Jalaj; Gaestel, Matthias; Davis, Roger J.; Nebreda, Angel R.; Agell, Neus

    2012-01-01

    Accurate DNA replication is crucial for the maintenance of genome integrity. To this aim, cells have evolved complex surveillance mechanisms to prevent mitotic entry in the presence of partially replicated DNA. ATR and Chk1 are key elements in the signal transduction pathways of DNA replication checkpoint; however, other kinases also make significant contributions. We show here that the stress kinases p38 and JNK are activated when DNA replication is blocked, and that their activity allows S/M, but not G₂/M, checkpoint maintenance when Chk1 is inhibited. Activation of both kinases by DNA replication inhibition is not mediated by the caffeine-sensitive kinases ATR or ATM. Phosphorylation of MKK3/6 and MKK4, p38 and JNK upstream kinases was also observed upon DNA replication inhibition. Using a genetic approach, we dissected the p38 pathway and showed that both p38α and p38β isoforms collaborate to inhibit mitotic entry. We further defined MKK3/6 and MK2/3 as the key upstream and downstream elements in the p38 signaling cascade after replication arrest. Accordingly, we found that the stress signaling pathways collaborate with Chk1 to keep cyclin B1/Cdk1 complexes inactive when DNA replication is inhibited, thereby preventing cell cycle progression when DNA replication is stalled. Our results show a complex response to replication stress, where multiple pathways are activated and fulfill overlapping roles to prevent mitotic entry with unreplicated DNA. PMID:22935704

  14. Cdc2 phosphorylation of nucleolin demarcates mitotic stages and Alzheimer's disease pathology.

    PubMed

    Dranovsky, A; Vincent, I; Gregori, L; Schwarzman, A; Colflesh, D; Enghild, J; Strittmatter, W; Davies, P; Goldgaber, D

    2001-01-01

    Nucleolin is a major multifunctional nuclear phosphoprotein that is phosphorylated by Cdc2 kinase in mitosis and that participates in a number of cellular processes. The monoclonal antibody TG-3 generated against neurofibrillary tangles (NFT) found in Alzheimer's disease (AD) is highly specific for mitotic cells in culture. We here demonstrate that phosphorylation of nucleolin by Cdc2 kinase generates the TG-3 epitope. The unique pool of TG-3 immunoreactive nucleolin appears abruptly during the prophase. It is associated with chromosomes through the metaphase and it gradually disappears during separation of chromosomes and exit from mitosis. In the brain, nucleolin was localized not only to nuclei but also to neuronal cytoplasm, and it is a marker for early NFT. In patients with AD, Cdc2 phosphorylated nucleolin was present in NFT. These findings suggest that phosphorylation of nucleolin by Cdc2 kinase is a critical event and the point of convergence of two distinct pathways, mitosis and neurodegeneration. PMID:11445251

  15. Quantum optimal control pathways of ozone isomerization dynamics subject to competing dissociation: A two-state one-dimensional model

    NASA Astrophysics Data System (ADS)

    Kurosaki, Yuzuru; Ho, Tak-San; Rabitz, Herschel

    2014-02-01

    We construct a two-state one-dimensional reaction-path model for ozone open → cyclic isomerization dynamics. The model is based on the intrinsic reaction coordinate connecting the cyclic and open isomers with the O2 + O asymptote on the ground-state 1A' potential energy surface obtained with the high-level ab initio method. Using this two-state model time-dependent wave packet optimal control simulations are carried out. Two possible pathways are identified along with their respective band-limited optimal control fields; for pathway 1 the wave packet initially associated with the open isomer is first pumped into a shallow well on the excited electronic state potential curve and then driven back to the ground electronic state to form the cyclic isomer, whereas for pathway 2 the corresponding wave packet is excited directly to the primary well of the excited state potential curve. The simulations reveal that the optimal field for pathway 1 produces a final yield of nearly 100% with substantially smaller intensity than that obtained in a previous study [Y. Kurosaki, M. Artamonov, T.-S. Ho, and H. Rabitz, J. Chem. Phys. 131, 044306 (2009)] using a single-state one-dimensional model. Pathway 2, due to its strong coupling to the dissociation channel, is less effective than pathway 1. The simulations also show that nonlinear field effects due to molecular polarizability and hyperpolarizability are small for pathway 1 but could become significant for pathway 2 because much higher field intensity is involved in the latter. The results suggest that a practical control may be feasible with the aid of a few lowly excited electronic states for ozone isomerization.

  16. Quantum optimal control pathways of ozone isomerization dynamics subject to competing dissociation: A two-state one-dimensional model

    SciTech Connect

    Kurosaki, Yuzuru; Ho, Tak-San Rabitz, Herschel

    2014-02-28

    We construct a two-state one-dimensional reaction-path model for ozone open → cyclic isomerization dynamics. The model is based on the intrinsic reaction coordinate connecting the cyclic and open isomers with the O{sub 2} + O asymptote on the ground-state {sup 1}A{sup ′} potential energy surface obtained with the high-level ab initio method. Using this two-state model time-dependent wave packet optimal control simulations are carried out. Two possible pathways are identified along with their respective band-limited optimal control fields; for pathway 1 the wave packet initially associated with the open isomer is first pumped into a shallow well on the excited electronic state potential curve and then driven back to the ground electronic state to form the cyclic isomer, whereas for pathway 2 the corresponding wave packet is excited directly to the primary well of the excited state potential curve. The simulations reveal that the optimal field for pathway 1 produces a final yield of nearly 100% with substantially smaller intensity than that obtained in a previous study [Y. Kurosaki, M. Artamonov, T.-S. Ho, and H. Rabitz, J. Chem. Phys. 131, 044306 (2009)] using a single-state one-dimensional model. Pathway 2, due to its strong coupling to the dissociation channel, is less effective than pathway 1. The simulations also show that nonlinear field effects due to molecular polarizability and hyperpolarizability are small for pathway 1 but could become significant for pathway 2 because much higher field intensity is involved in the latter. The results suggest that a practical control may be feasible with the aid of a few lowly excited electronic states for ozone isomerization.

  17. Mitotic fidelity requires transgenerational action of a testis-restricted HP1

    PubMed Central

    Levine, Mia T; Vander Wende, Helen M; Malik, Harmit S

    2015-01-01

    Sperm-packaged DNA must undergo extensive reorganization to ensure its timely participation in embryonic mitosis. Whereas maternal control over this remodeling is well described, paternal contributions are virtually unknown. In this study, we show that Drosophila melanogaster males lacking Heterochromatin Protein 1E (HP1E) sire inviable embryos that undergo catastrophic mitosis. In these embryos, the paternal genome fails to condense and resolve into sister chromatids in synchrony with the maternal genome. This delay leads to a failure of paternal chromosomes, particularly the heterochromatin-rich sex chromosomes, to separate on the first mitotic spindle. Remarkably, HP1E is not inherited on mature sperm chromatin. Instead, HP1E primes paternal chromosomes during spermatogenesis to ensure faithful segregation post-fertilization. This transgenerational effect suggests that maternal control is necessary but not sufficient for transforming sperm DNA into a mitotically competent pronucleus. Instead, paternal action during spermiogenesis exerts post-fertilization control to ensure faithful chromosome segregation in the embryo. DOI: http://dx.doi.org/10.7554/eLife.07378.001 PMID:26151671

  18. Multiple independent regulatory pathways control UBI4 expression after heat shock in Saccharomyces cerevisiae.

    PubMed

    Simon, J R; Treger, J M; McEntee, K

    1999-02-01

    Transcription of the polyubiquitin gene UBI4 of Saccharomyces cerevisiae is strongly induced by a variety of environmental stresses, such as heat shock, nutrient depletion and exposure to DNA-damaging agents. This transcriptional response of UBI4 is likely to be the primary mechanism for increasing the pool of ubiquitin for degradation of stress-damaged proteins. Deletion and promoter fusion studies of the 5' regulatory sequences indicated that two different elements, heat shock elements (HSEs) and stress response element (STREs), contributed independently to heat shock regulation of the UBI4 gene. In the absence of HSEs, STRE sequences localized to the intervals -264 to -238 and -215 to -183 were needed for stress control of transcription after heat shock. Site-directed mutagenesis of the STRE (AG4) at -252 to -248 abolished heat shock induction of UBI4 transcription. Northern analysis demonstrated that cells containing either a temperature-sensitive HSF or non-functional Msn2p/Msn4p transcription factors induced high levels of UBI4 transcripts after heat shock. In cells deficient in both heat stress pathways, heat-induced UBI4 transcript levels were considerably lower but not abolished, suggesting a role for another factor(s) in stress control of its expression. PMID:10048026

  19. Best strategies to implement clinical pathways in an emergency department setting: study protocol for a cluster randomized controlled trial

    PubMed Central

    2013-01-01

    Background The clinical pathway is a tool that operationalizes best evidence recommendations and clinical practice guidelines in an accessible format for ‘point of care’ management by multidisciplinary health teams in hospital settings. While high-quality, expert-developed clinical pathways have many potential benefits, their impact has been limited by variable implementation strategies and suboptimal research designs. Best strategies for implementing pathways into hospital settings remain unknown. This study will seek to develop and comprehensively evaluate best strategies for effective local implementation of externally developed expert clinical pathways. Design/methods We will develop a theory-based and knowledge user-informed intervention strategy to implement two pediatric clinical pathways: asthma and gastroenteritis. Using a balanced incomplete block design, we will randomize 16 community emergency departments to receive the intervention for one clinical pathway and serve as control for the alternate clinical pathway, thus conducting two cluster randomized controlled trials to evaluate this implementation intervention. A minimization procedure will be used to randomize sites. Intervention sites will receive a tailored strategy to support full clinical pathway implementation. We will evaluate implementation strategy effectiveness through measurement of relevant process and clinical outcomes. The primary process outcome will be the presence of an appropriately completed clinical pathway on the chart for relevant patients. Primary clinical outcomes for each clinical pathway include the following: Asthma—the proportion of asthmatic patients treated appropriately with corticosteroids in the emergency department and at discharge; and Gastroenteritis—the proportion of relevant patients appropriately treated with oral rehydration therapy. Data sources include chart audits, administrative databases, environmental scans, and qualitative interviews. We will

  20. Autocrine and exogenous transforming growth factor beta control cell cycle inhibition through pathways with different sensitivity.

    PubMed

    Wang, Jing; Sergina, Natalia; Ko, Tien C; Gong, Jiangeng; Brattain, Michael G

    2004-09-17

    Human colon carcinoma cells HCT116 that lack transforming growth factor beta (TGF-beta) type II receptor (RII) demonstrated restoration of autocrine TGF-beta activity upon reexpression of RII without restoring inhibitory responses to exogenous TGF-beta treatment. RII transfectants (designated RII Cl 37) had a longer lag phase relative to NEO-transfected control cells (designated NEO pool) before entering exponential growth in tissue culture. The prolonged growth arrest of RII Cl 37 cells was associated with markedly reduced cyclin-dependent kinase (CDK)2 activity. Our results demonstrate that p21 induction by autocrine TGF-beta is responsible for reduced CDK2 activity, which at least partially contributes to prolonged growth arrest and reduced cell proliferation in RII Cl 37 cells. In contrast to RII transfectants, HCT116 cells transfected with chromosome 3 (designated HCT116Ch3), which bears the RII gene, restored the response to exogenous TGF-beta as well as autocrine TGF-beta activity. Autocrine TGF-beta activity in HCT116Ch3 cells induced p21 expression as seen in RII Cl 37 cells; however, in addition to autocrine activity, HCT116Ch3 cells responded to exogenous TGF-beta as decreased CDK4 expression and reduced pRb phosphorylation mediated a TGF-beta inhibitory response in these cells. These results indicate that autocrine TGF-beta regulates the cell cycle through a pathway different from exogenous TGF-beta in the sense that p21 is a more sensitive effector of the TGF-beta signaling pathway, which can be induced and saturated by autocrine TGF-beta, whereas CDK4 inhibition is a less sensitive effector, which can only be activated by high levels of exogenous TGF-beta PMID:15271980

  1. Final Technical Report: Genetic and Molecular Analysis of a new control pathway in assimilate partitioning.

    SciTech Connect

    Bush, Daniel, R.

    2009-03-10

    Assimilate partitioning refers to the systemic distribution of photoassimilate from sites of primary assimilation (source tissue) to import-dependent tissues and organs (sinks). One of the defining questions in this area is how plants balance source productivity with sink demand. We discovered a sucrose-sensing signal transduction pathway that controls the activity of BvSUT1, a proton-sucrose symporter in sugar beet leaf tissue. Sucrose symporters are responsible for sucrose accumulation in the phloem of many plants and, therefore, they mediate the pivotal step in the long-distance transport of photoassimilate to non-photosynthetic tissues, such as roots and seed. We previously showed that sucrose transport activity is directly proportional to the transcription rate of BvSUT1 and that symporter mRNA and protein have high rates of turnover with half-lives on the order of 2 h. We further demonstrated that symporter transcription is regulated by sucrose levels in the leaf and that sucrose-dependent regulation of BvSUT1 transcription is mediated, at least in part, by a protein phosphorylation relay pathway. The goal of the experiments during this current grant were to use genetic and molecular approaches to identify essential components of this vital regulatory system. The initial objectives were to: (1) to characterize Arabidopsis mutants we've isolated that are resistant to growth inhibition by sucrose analogues that are recognized by the sucrose-sensor, (2) to screen for loss of function mutants in BvSUT1-promoter:luciferase transgenic plants that no longer respond to sucrose accumulation in the leaf using non-destructive visualization of luciferase activity, (3) to use gel mobility-shift assays and nuclease protection experiments to identify cis elements in the symporter promoter and DNA-binding proteins that are involved in sucrose regulation of symporter expression.

  2. Signaling Pathways That Control Rho Kinase Activity Maintain the Embryonic Epicardial Progenitor State

    PubMed Central

    Artamonov, Mykhaylo V.; Jin, Li; Franke, Aaron S.; Momotani, Ko; Ho, Ruoya; Dong, Xiu Rong; Majesky, Mark W.; Somlyo, Avril V.

    2015-01-01

    This study identifies signaling pathways that play key roles in the formation and maintenance of epicardial cells, a source of progenitors for coronary smooth muscle cells (SMCs). After epithelial to mesenchymal transition (EMT), mesenchymal cells invade the myocardium to form coronary SMCs. RhoA/Rho kinase activity is required for EMT and for differentiation into coronary SMCs, whereas cAMP activity is known to inhibit EMT in epithelial cells by an unknown mechanism. We use outgrowth of epicardial cells from E9.5 isolated mouse proepicardium (PE) explants, wild type and Epac1 null E12.5 mouse heart explants, adult rat epicardial cells, and immortalized mouse embryonic epicardial cells as model systems to identify signaling pathways that regulate RhoA activity to maintain the epicardial progenitor state. We demonstrate that RhoA activity is suppressed in the epicardial progenitor state, that the cAMP-dependent Rap1 GTP exchange factor (GEF), Epac, known to down-regulate RhoA activity through activation of Rap1 GTPase activity increased, that Rap1 activity increased, and that expression of the RhoA antagonistic Rnd proteins known to activate p190RhoGAP increased and associated with p190RhoGAP. Finally, EMT is associated with increased p63RhoGEF and RhoGEF-H1 protein expression, increased GEF-H1 activity, with a trend in increased p63RhoGEF activity. EMT is suppressed by partial silencing of p63RhoGEF and GEF-H1. In conclusion, we have identified new signaling molecules that act together to control RhoA activity and play critical roles in the maintenance of coronary smooth muscle progenitor cells in the embryonic epicardium. We suggest that their eventual manipulation could promote revascularization after myocardial injury. PMID:25733666

  3. Mitotic Checkpoint Regulators Control Insulin Signaling and Metabolic Homeostasis.

    PubMed

    Choi, Eunhee; Zhang, Xiangli; Xing, Chao; Yu, Hongtao

    2016-07-28

    Insulin signaling regulates many facets of animal physiology. Its dysregulation causes diabetes and other metabolic disorders. The spindle checkpoint proteins MAD2 and BUBR1 prevent precocious chromosome segregation and suppress aneuploidy. The MAD2 inhibitory protein p31(comet) promotes checkpoint inactivation and timely chromosome segregation. Here, we show that whole-body p31(comet) knockout mice die soon after birth and have reduced hepatic glycogen. Liver-specific ablation of p31(comet) causes insulin resistance, hyperinsulinemia, glucose intolerance, and hyperglycemia and diminishes the plasma membrane localization of the insulin receptor (IR) in hepatocytes. MAD2 directly binds to IR and facilitates BUBR1-dependent recruitment of the clathrin adaptor AP2 to IR. p31(comet) blocks the MAD2-BUBR1 interaction and prevents spontaneous clathrin-mediated IR endocytosis. BUBR1 deficiency enhances insulin sensitivity in mice. BUBR1 depletion in hepatocytes or the expression of MAD2-binding-deficient IR suppresses the metabolic phenotypes of p31(comet) ablation. Our findings establish a major IR regulatory mechanism and link guardians of chromosome stability to nutrient metabolism. PMID:27374329

  4. Kalanchoe tubiflora extract inhibits cell proliferation by affecting the mitotic apparatus

    PubMed Central

    2012-01-01

    Background Kalanchoe tubiflora (KT) is a succulent plant native to Madagascar, and is commonly used as a medicinal agent in Southern Brazil. The underlying mechanisms of tumor suppression are largely unexplored. Methods Cell viability and wound-healing were analyzed by MTT assay and scratch assay respectively. Cell cycle profiles were analyzed by FACS. Mitotic defects were analyzed by indirect immunofluoresence images. Results An n-Butanol-soluble fraction of KT (KT-NB) was able to inhibit cell proliferation. After a 48 h treatment with 6.75 μg/ml of KT, the cell viability was less than 50% of controls, and was further reduced to less than 10% at higher concentrations. KT-NB also induced an accumulation of cells in the G2/M phase of the cell cycle as well as an increased level of cells in the subG1 phase. Instead of disrupting the microtubule network of interphase cells, KT-NB reduced cell viability by inducing multipolar spindles and defects in chromosome alignment. KT-NB inhibits cell proliferation and reduces cell viability by two mechanisms that are exclusively involved with cell division: first by inducing multipolarity; second by disrupting chromosome alignment during metaphase. Conclusion KT-NB reduced cell viability by exclusively affecting formation of the proper structure of the mitotic apparatus. This is the main idea of the new generation of anti-mitotic agents. All together, KT-NB has sufficient potential to warrant further investigation as a potential new anticancer agent candidate. PMID:22963191

  5. Phyllanthus emblica Fruit Extract Activates Spindle Assembly Checkpoint, Prevents Mitotic Aberrations and Genomic Instability in Human Colon Epithelial NCM460 Cells.

    PubMed

    Guo, Xihan; Wang, Xu

    2016-01-01

    The fruit of Phyllanthus emblica Linn. (PE) has been widely consumed as a functional food and folk medicine in Southeast Asia due to its remarkable nutritional and pharmacological effects. Previous research showed PE delays mitotic progress and increases genomic instability (GIN) in human colorectal cancer cells. This study aimed to investigate the similar effects of PE by the biomarkers related to spindle assembly checkpoint (SAC), mitotic aberrations and GIN in human NCM460 normal colon epithelial cells. Cells were treated with PE and harvested differently according to the biomarkers observed. Frequencies of micronuclei (MN), nucleoplasmic bridge (NPB) and nuclear bud (NB) in cytokinesis-block micronucleus assay were used as indicators of GIN. Mitotic aberrations were assessed by the biomarkers of chromosome misalignment, multipolar division, chromosome lagging and chromatin bridge. SAC activity was determined by anaphase-to- metaphase ratio (AMR) and the expression of core SAC gene budding uninhibited by benzimidazoles related 1 (BubR1). Compared with the control, PE-treated cells showed (1) decreased incidences of MN, NPB and NB (p < 0.01); (2) decreased frequencies of all mitotic aberration biomarkers (p < 0.01); and (3) decreased AMR (p < 0.01) and increased BubR1 expression (p < 0.001). The results revealed PE has the potential to protect human normal colon epithelial cells from mitotic and genomic damages partially by enhancing the function of SAC. PMID:27598149

  6. Hippo pathway genes developed varied exon numbers and coevolved functional domains in metazoans for species specific growth control

    PubMed Central

    2013-01-01

    Background The Hippo pathway controls growth by mediating cell proliferation and apoptosis. Dysregulation of Hippo signaling causes abnormal proliferation in both healthy and cancerous cells. The Hippo pathway receives inputs from multiple developmental pathways and interacts with many tissue-specific transcription factors, but how genes in the pathway have evolved remains inadequately revealed. Results To explore the origin and evolution of Hippo pathway, we have extensively examined 16 Hippo pathway genes, including upstream regulators and downstream targets, in 24 organisms covering major metazoan phyla. From simple to complex organisms, these genes are varied in the length and number of exons but encode conserved domains with similar higher-order organization. The core of the pathway is more conserved than its upstream regulators and downstream targets. Several components, despite existing in the most basal metazoan sponges, cannot be convincingly identified in other species. Potential recombination breakpoints were identified in some genes. Coevolutionary analysis reveals that most functional domains in Hippo genes have coevolved with interacting functional domains in other genes. Conclusions The two essential upstream regulators cadherins fat and dachsous may have originated in the unicellular organism Monosiga brevicollis and evolved more significantly than the core of the pathway. Genes having varied numbers of exons in different species, recombination events, and the gain and loss of some genes indicate alternative splicing and species-specific evolution. Coevolution signals explain some species-specific loss of functional domains. These results significantly unveil the structure and evolution of the Hippo pathway in distant phyla and provide valuable clues for further examination of Hippo signaling. PMID:23547742

  7. The Hippo signalling pathway maintains quiescence in Drosophila neural stem cells

    PubMed Central

    Ding, Rouven; Weynans, Kevin; Bossing, Torsten; Barros, Claudia S.; Berger, Christian

    2016-01-01

    Stem cells control their mitotic activity to decide whether to proliferate or to stay in quiescence. Drosophila neural stem cells (NSCs) are quiescent at early larval stages, when they are reactivated in response to metabolic changes. Here we report that cell-contact inhibition of growth through the canonical Hippo signalling pathway maintains NSC quiescence. Loss of the core kinases hippo or warts leads to premature nuclear localization of the transcriptional co-activator Yorkie and initiation of growth and proliferation in NSCs. Yorkie is necessary and sufficient for NSC reactivation, growth and proliferation. The Hippo pathway activity is modulated via inter-cellular transmembrane proteins Crumbs and Echinoid that are both expressed in a nutrient-dependent way in niche glial cells and NSCs. Loss of crumbs or echinoid in the niche only is sufficient to reactivate NSCs. Finally, we provide evidence that the Hippo pathway activity discriminates quiescent from non-quiescent NSCs in the Drosophila nervous system. PMID:26821647

  8. Regulation of mitotic progression by the spindle assembly checkpoint

    PubMed Central

    Lischetti, Tiziana; Nilsson, Jakob

    2015-01-01

    Equal segregation of sister chromatids during mitosis requires that pairs of kinetochores establish proper attachment to microtubules emanating from opposite poles of the mitotic spindle. The spindle assembly checkpoint (SAC) protects against errors in segregation by delaying sister separation in response to improper kinetochore–microtubule interactions, and certain checkpoint proteins help to establish proper attachments. Anaphase entry is inhibited by the checkpoint through assembly of the mitotic checkpoint complex (MCC) composed of the 2 checkpoint proteins, Mad2 and BubR1, bound to Cdc20. The outer kinetochore acts as a catalyst for MCC production through the recruitment and proper positioning of checkpoint proteins and recently there has been remarkable progress in understanding how this is achieved. Here, we highlight recent advances in our understanding of kinetochore–checkpoint protein interactions and inhibition of the anaphase promoting complex by the MCC. PMID:27308407

  9. Mitotic activity in dorsal epidermis of Rana pipiens.

    NASA Technical Reports Server (NTRS)

    Garcia-Arce, H.; Mizell, S.

    1972-01-01

    Study of statistically significant rhythms of mitotic division in dorsal epidermis of frogs, Rana pipiens, exposed to a 12:12 light:dark environment for 14 days. The results include the findings that (1) male animals have a primary period of 22 hr in summer and 18 hr in winter, (2) female animals have an 18 hr period, and (3) parapinealectomy and blinding abolish the rhythm.

  10. Different cell fates after mitotic slippage: From aneuploidy to polyploidy.

    PubMed

    Ohashi, Akihiro

    2016-03-01

    The molecular mechanism responsible for cell fate after mitotic slippage remains unclear. We investigated the different postmitotic effects of aneuploidy versus polyploidy using chemical inhibitors of centromere-associated protein-E (CENP-E) and kinesin family member 11 (KIF11, also known as Eg5). Aneuploidy caused substantial proteotoxic stress and DNA damage accompanied by p53-mediated postmitotic apoptosis, whereas polyploidy did not induce these antiproliferative effects. PMID:27308610

  11. Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo.

    PubMed

    Percival, Stefanie M; Parant, John M

    2016-01-01

    Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraB(hi1045) (,) and esco2(hi2865) mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease. PMID:27501381

  12. Mitotic exit: Determining the PP2A dephosphorylation program.

    PubMed

    Pereira, Gislene; Schiebel, Elmar

    2016-08-29

    In mitotic exit, proteins that were highly phosphorylated are sequentially targeted by the phosphatase PP2A-B55, but what underlies substrate selection is unclear. In this issue, Cundell et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201606033) identify the determinants of PP2A-B55's dephosphorylation program, thereby influencing spindle disassembly, nuclear envelope reformation, and cytokinesis. PMID:27551057

  13. PTEN Tumor Suppressor Network in PI3K-Akt Pathway Control.

    PubMed

    Georgescu, Maria-Magdalena

    2010-12-01

    The PI3K-Akt pathway is a major survival pathway activated in cancer. Efforts to develop targeted therapies have not been fully successful, mainly because of extensive internal intrapathway or external interpathway negative feedback loops or because of networking between pathway suppressors. The PTEN tumor suppressor is the major brake of the pathway and a common target for inactivation in somatic cancers. This review will highlight the networking of PTEN with other inhibitors of the pathway, relevant to cancer progression. PTEN constitutes the main node of the inhibitory network, and a series of convergences at different levels in the PI3K-Akt pathway, starting from those with growth factor receptors, will be described. As PTEN exerts enzymatic activity as a phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) phosphatase, thus opposing the activity of PI3K, the concerted actions to increase the availability of PIP(3) in cancer cells, relying either on other phosphoinositide enzymes or on the intrinsic regulation of PTEN activity by other molecules, will be discussed. In particular, the synergy between PTEN and the circle of its direct interacting proteins will be brought forth in an attempt to understand both the activation of the PI3K-Akt pathway and the connections with other parallel oncogenic pathways. The understanding of the interplay between the modulators of the PI3K-Akt pathway in cancer should eventually lead to the design of therapeutic approaches with increased efficacy in the clinic. PMID:21779440

  14. SUMOylation inhibits FOXM1 activity and delays mitotic transition.

    PubMed

    Myatt, S S; Kongsema, M; Man, C W-Y; Kelly, D J; Gomes, A R; Khongkow, P; Karunarathna, U; Zona, S; Langer, J K; Dunsby, C W; Coombes, R C; French, P M; Brosens, J J; Lam, E W-F

    2014-08-21

    The forkhead box transcription factor FOXM1 is an essential effector of G2/M-phase transition, mitosis and the DNA damage response. As such, it is frequently deregulated during tumorigenesis. Here we report that FOXM1 is dynamically modified by SUMO1 but not by SUMO2/3 at multiple sites. We show that FOXM1 SUMOylation is enhanced in MCF-7 breast cancer cells in response to treatment with epirubicin and mitotic inhibitors. Mutation of five consensus conjugation motifs yielded a SUMOylation-deficient mutant FOXM1. Conversely, fusion of the E2 ligase Ubc9 to FOXM1 generated an auto-SUMOylating mutant (FOXM1-Ubc9). Analysis of wild-type FOXM1 and mutants revealed that SUMOylation inhibits FOXM1 activity, promotes translocation to the cytoplasm and enhances APC/Cdh1-mediated ubiquitination and degradation. Further, expression of the SUMOylation-deficient mutant enhanced cell proliferation compared with wild-type FOXM1, whereas the FOXM1-Ubc9 fusion protein resulted in persistent cyclin B1 expression and slowed the time from mitotic entry to exit. In summary, our findings suggest that SUMOylation attenuates FOXM1 activity and causes mitotic delay in cytotoxic drug response. PMID:24362530

  15. On the molecular mechanisms of mitotic kinase activation.

    PubMed

    Bayliss, Richard; Fry, Andrew; Haq, Tamanna; Yeoh, Sharon

    2012-11-01

    During mitosis, human cells exhibit a peak of protein phosphorylation that alters the behaviour of a significant proportion of proteins, driving a dramatic transformation in the cell's shape, intracellular structures and biochemistry. These mitotic phosphorylation events are catalysed by several families of protein kinases, including Auroras, Cdks, Plks, Neks, Bubs, Haspin and Mps1/TTK. The catalytic activities of these kinases are activated by phosphorylation and through protein-protein interactions. In this review, we summarize the current state of knowledge of the structural basis of mitotic kinase activation mechanisms. This review aims to provide a clear and comprehensive primer on these mechanisms to a broad community of researchers, bringing together the common themes, and highlighting specific differences. Along the way, we have uncovered some features of these proteins that have previously gone unreported, and identified unexplored questions for future work. The dysregulation of mitotic kinases is associated with proliferative disorders such as cancer, and structural biology will continue to play a critical role in the development of chemical probes used to interrogate disease biology and applied to the treatment of patients. PMID:23226601

  16. On the molecular mechanisms of mitotic kinase activation

    PubMed Central

    Bayliss, Richard; Fry, Andrew; Haq, Tamanna; Yeoh, Sharon

    2012-01-01

    During mitosis, human cells exhibit a peak of protein phosphorylation that alters the behaviour of a significant proportion of proteins, driving a dramatic transformation in the cell's shape, intracellular structures and biochemistry. These mitotic phosphorylation events are catalysed by several families of protein kinases, including Auroras, Cdks, Plks, Neks, Bubs, Haspin and Mps1/TTK. The catalytic activities of these kinases are activated by phosphorylation and through protein–protein interactions. In this review, we summarize the current state of knowledge of the structural basis of mitotic kinase activation mechanisms. This review aims to provide a clear and comprehensive primer on these mechanisms to a broad community of researchers, bringing together the common themes, and highlighting specific differences. Along the way, we have uncovered some features of these proteins that have previously gone unreported, and identified unexplored questions for future work. The dysregulation of mitotic kinases is associated with proliferative disorders such as cancer, and structural biology will continue to play a critical role in the development of chemical probes used to interrogate disease biology and applied to the treatment of patients. PMID:23226601

  17. A Genome Scale Screen for Mutants with Delayed Exit from Mitosis: Ire1-Independent Induction of Autophagy Integrates ER Homeostasis into Mitotic Lifespan

    PubMed Central

    Ghavidel, Ata; Baxi, Kunal; Ignatchenko, Vladimir; Prusinkiewicz, Martin; Arnason, Terra G.; Kislinger, Thomas; Carvalho, Carlos E.; Harkness, Troy A. A.

    2015-01-01

    Proliferating eukaryotic cells undergo a finite number of cell divisions before irreversibly exiting mitosis. Yet pathways that normally limit the number of cell divisions remain poorly characterized. Here we describe a screen of a collection of 3762 single gene mutants in the yeast Saccharomyces cerevisiae, accounting for 2/3 of annotated yeast ORFs, to search for mutants that undergo an atypically high number of cell divisions. Many of the potential longevity genes map to cellular processes not previously implicated in mitotic senescence, suggesting that regulatory mechanisms governing mitotic exit may be broader than currently anticipated. We focused on an ER-Golgi gene cluster isolated in this screen to determine how these ubiquitous organelles integrate into mitotic longevity. We report that a chronic increase in ER protein load signals an expansion in the assembly of autophagosomes in an Ire1-independent manner, accelerates trafficking of high molecular weight protein aggregates from the cytoplasm to the vacuoles, and leads to a profound enhancement of daughter cell production. We demonstrate that this catabolic network is evolutionarily conserved, as it also extends reproductive lifespan in the nematode Caenorhabditis elegans. Our data provide evidence that catabolism of protein aggregates, a natural byproduct of high protein synthesis and turn over in dividing cells, is among the drivers of mitotic longevity in eukaryotes. PMID:26247883

  18. Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase.

    PubMed

    Cornish, Adam J; Ginovska, Bojana; Thelen, Adam; da Silva, Julio C S; Soares, Thereza A; Raugei, Simone; Dupuis, Michel; Shaw, Wendy J; Hegg, Eric L

    2016-06-01

    The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a water molecule. A computational analysis of this pathway in the [FeFe]-hydrogenase from Clostridium pasteurianum revealed that the solvent-exposed residue of the pathway (Glu282) forms hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implying that these residues could function in regulating proton transfer. In this study, we show that substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in an ∼3-fold enhancement of H2 production activity when methyl viologen is used as an electron donor, suggesting that Arg286 may help control the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate ∼5-fold, implying that it either acts as a member of the pathway or influences Glu282 to permit proton transfer. Interestingly, quantum mechanics/molecular mechanics and molecular dynamics calculations indicate that Ser320 does not play a structural role or indirectly influence the barrier for proton movement at the entrance of the channel. Rather, it may act as an additional proton acceptor for the pathway or serve in a regulatory role. While further studies are needed to elucidate the role of Ser320, collectively these data provide insights into the complex proton transport process. PMID:27186945

  19. The general amino acid control pathway regulates mTOR and autophagy during serum/glutamine starvation

    PubMed Central

    Chen, Rui; Zou, Yilong; Mao, Dongxue; Sun, Daxiao; Gao, Guanguang; Shi, Jingwen; Liu, Xiaoqing; Zhu, Chen; Yang, Mingyu; Ye, Wanlu; Hao, Qianqian

    2014-01-01

    Organisms have evolved elaborate mechanisms to adjust intracellular nutrient levels in response to fluctuating availability of exogenous nutrients. During starvation, cells can enhance amino acid uptake and synthesis through the general amino acid control (GAAC) pathway, whereas nonessential cellular contents are recycled by autophagy. How these two pathways are coordinated in response to starvation is currently unknown. Here we show that the GAAC pathway couples exogenous amino acid availability with autophagy. Starvation caused deactivation of mTOR, which then activated autophagy. In parallel, serum/glutamine starvation activated the GAAC pathway, which up-regulated amino acid transporters, leading to increased amino acid uptake. This elevated the intracellular amino acid level, which in turn reactivated mTOR and suppressed autophagy. Knockdown of activating transcription factor 4, the major transcription factor in the GAAC pathway, or of SLC7A5, a leucine transporter, caused impaired mTOR reactivation and much higher levels of autophagy. Thus, the GAAC pathway modulates autophagy by regulating amino acid uptake and mTOR reactivation during serum/glutamine starvation. PMID:25049270

  20. Prep1 directly regulates the intrinsic apoptotic pathway by controlling Bcl-XL levels.

    PubMed

    Micali, Nicola; Ferrai, Carmelo; Fernandez-Diaz, Luis C; Blasi, Francesco; Crippa, Massimo P

    2009-03-01

    The Prep1 homeodomain transcription factor is essential in embryonic development. Prep1 hypomorphic mutant mouse (Prep1(i/i)) embryos (embryonic day 9.5) display an increased terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction compared to wild-type (WT) littermates. Prep1(i/i) mouse embryo fibroblasts (MEFs) show an increased basal level of annexin V binding activity, reduction of the mitochondrial-membrane potential, and increased caspase 9 and 3 activation, indicating increased apoptosis. Prep1(i/i) MEFs also respond faster than WT MEFs to genotoxic stress, indicating increased activation of the intrinsic apoptotic pathways. We did not observe an increase in p53 or an abnormal p53 response to apoptotic stimuli. However, hypomorphic MEFs have decreased endogenous levels of antiapoptotic Bcl-X(L) mRNA and protein, and Bcl-x overexpression rescues the defect of Prep1(i/i) MEFs. Using transient transfections and chromatin immunoprecipitation, we identified the Bcl-x promoter as a novel target of Prep1. Thus, Prep1 directly controls mitochondrial homeostasis (and the apoptotic potential) by modulating Bcl-x gene expression. PMID:19103748

  1. Dis3l2-Mediated Decay Is a Quality Control Pathway for Noncoding RNAs.

    PubMed

    Pirouz, Mehdi; Du, Peng; Munafò, Marzia; Gregory, Richard I

    2016-08-16

    Mutations in the 3'-5' exonuclease DIS3L2 are associated with Perlman syndrome and hypersusceptibility to Wilms tumorigenesis. Previously, we found that Dis3l2 specifically recognizes and degrades uridylated pre-let-7 microRNA. However, the widespread relevance of Dis3l2-mediated decay of uridylated substrates remains unknown. Here, we applied an unbiased RNA immunoprecipitation strategy to identify Dis3l2 targets in mouse embryonic stem cells. The disease-associated long noncoding RNA (lncRNA) Rmrp, 7SL, as well as several other Pol III-transcribed noncoding RNAs (ncRNAs) were among the most highly enriched Dis3l2-bound RNAs. 3'-Uridylated Rmrp, 7SL, and small nuclear RNA (snRNA) species were highly stabilized in the cytoplasm of Dis3l2-depleted cells. Deep sequencing analysis of Rmrp 3' ends revealed extensive oligouridylation mainly on transcripts with imprecise ends. We implicate the terminal uridylyl transferases (TUTases) Zcchc6/11 in the uridylation of these ncRNAs, and biochemical reconstitution assays demonstrate the sufficiency of TUTase-Dis3l2 for Rmrp decay. This establishes Dis3l2-mediated decay (DMD) as a quality-control pathway that eliminates aberrant ncRNAs. PMID:27498873

  2. Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway

    PubMed Central

    Mah, In Kyoung; Soloff, Rachel; Hedrick, Stephen M.; Mariani, Francesca V.

    2015-01-01

    Summary The number of stem/progenitor cells available can profoundly impact tissue homeostasis and the response to injury or disease. Here, we propose that an atypical PKC, Prkci, is a key player in regulating the switch from an expansion to a differentiation/maintenance phase via regulation of Notch, thus linking the polarity pathway with the control of stem cell self-renewal. Prkci is known to influence symmetric cell division in invertebrates; however a definitive role in mammals has not yet emerged. Using a genetic approach, we find that loss of Prkci results in a marked increase in the number of various stem/progenitor cells. The mechanism used likely involves inactivation and symmetric localization of NUMB, leading to the activation of NOTCH1 and its downstream effectors. Inhibition of atypical PKCs may be useful for boosting the production of pluripotent stem cells, multipotent stem cells, or possibly even primordial germ cells by promoting the stem cell/progenitor fate. PMID:26527382

  3. Cooperative control of Drosophila immune responses by the JNK and NF-κB signaling pathways

    PubMed Central

    Delaney, Joseph R; Stöven, Svenja; Uvell, Hanna; Anderson, Kathryn V; Engström, Ylva; Mlodzik, Marek

    2006-01-01

    Jun N-terminal kinase (JNK) signaling is a highly conserved pathway that controls both cytoskeletal remodeling and transcriptional regulation in response to a wide variety of signals. Despite the importance of JNK in the mammalian immune response, and various suggestions of its importance in Drosophila immunity, the actual contribution of JNK signaling in the Drosophila immune response has been unclear. Drosophila TAK1 has been implicated in the NF-κB/Relish-mediated activation of antimicrobial peptide genes. However, we demonstrate that Relish activation is intact in dTAK1 mutant animals, and that the immune response in these mutant animals was rescued by overexpression of a downstream JNKK. The expression of a JNK inhibitor and induction of JNK loss-of-function clones in immune responsive tissue revealed a general requirement for JNK signaling in the expression of antimicrobial peptides. Our data indicate that dTAK1 is not required for Relish activation, but instead is required in JNK signaling for antimicrobial peptide gene expression. PMID:16763552

  4. A photoactivable multi-inhibitor nanoliposome for tumour control and simultaneous inhibition of treatment escape pathways

    PubMed Central

    Spring, Bryan Q.; Sears, R. Bryan; Zheng, Lei Zak; Mai, Zhiming; Watanabe, Reika; Sherwood, Margaret E.; Schoenfeld, David A.; Pogue, Brian W.; Pereira, Stephen P.; Villa, Elizabeth; Hasan, Tayyaba

    2015-01-01

    Nanoscale drug delivery vehicles can facilitate multimodal therapies of cancer by promoting tumour-selective drug release. However, few are effective because cancer cells develop ways to resist and evade treatment. Here, we introduce a photoactivatable multi-inhibitor nanoliposome (PMIL) that imparts light-induced cytotoxicity in synchrony with photo-initiated and sustained release of inhibitors that suppress tumour regrowth and treatment escape signalling pathways. The PMIL consists of a nanoliposome doped with a photoactivatable chromophore (benzoporphyrin derivative, BPD) in the lipid bilayer, and a nanoparticle containing cabozantinib (XL184)—a multikinase inhibitor—encapsulated inside. Near infrared tumour irradiation, following intravenous PMIL administration, triggers photodynamic damage of tumour cells and microvessels, and simultaneously initiates release of XL184 inside the tumour. A single PMIL treatment achieves prolonged tumour reduction in two mouse models and suppresses metastatic escape in an orthotopic pancreatic tumour model. The PMIL offers new prospects for cancer therapy by enabling spatiotemporal control of drug release whilst reducing systemic drug exposure and associated toxicities. PMID:26780659

  5. The Diversity of the Pollen Tube Pathway in Plants: Toward an Increasing Control by the Sporophyte

    PubMed Central

    Lora, Jorge; Hormaza, José I.; Herrero, María

    2016-01-01

    Plants, unlike animals, alternate multicellular diploid, and haploid generations in their life cycle. While this is widespread all along the plant kingdom, the size and autonomy of the diploid sporophyte and the haploid gametophyte generations vary along evolution. Vascular plants show an evolutionary trend toward a reduction of the gametophyte, reflected both in size and lifespan, together with an increasing dependence from the sporophyte. This has resulted in an overlooking of the importance of the gametophytic phase in the evolution of higher plants. This reliance on the sporophyte is most notorious along the pollen tube journey, where the male gametophytes have to travel a long way inside the sporophyte to reach the female gametophyte. Along evolution, there is a change in the scenery of the pollen tube pathway that favors pollen competition and selection. This trend, toward apparently making complicated what could be simple, appears to be related to an increasing control of the sporophyte over the gametophyte with implications for understanding plant evolution. PMID:26904071

  6. Regulation of renin processing and secretion: chemiosmotic control and novel secretory pathway.

    PubMed

    King, J A; Lush, D J; Fray, J C

    1993-08-01

    The renin-angiotensin-aldosterone system (RAAS) plays an important role in cardiovascular and electrolyte regulation in health and disease. Juxtaglomerular cells in the kidney regulate endocrine RAAS by physiologically controlling conversion of prorenin and secretion of renin. The classical baroceptor, neurogenic, and macula densa mechanisms regulate renin expression at the cellular level by Ca2+, adenosine 3',5'-cyclic monophosphate (cAMP), and chemiosmotic forces (K+, Cl-, and water flux coupled to H+ movement). The baroceptor mechanism (through Ca2+) activates K+ and Cl- channels in the surface membrane and deactivates a KCl-H+ exchange chemiosmotic transporter in the secretory granular membrane. The neurogenic mechanism (through cAMP) promotes prorenin processing to renin. The macula densa mechanism (through K+ and Cl-) involves the processing of prorenin to renin. Ca2+, by inhibiting the KCl-H+ exchange transporter, prevents secretory granules from engaging in chemiosmotically mediated exocytosis. cAMP, on the other hand, by stimulating H+ influx, provides the acidic granular environment for prorenin processing to renin. It is concluded that, in the presence of a favorable chemiosmotic environment, prorenin is processed to renin, which may then be secreted by regulative degranulation or divergence translocation, a novel secretory pathway used by several secretory proteins, including renin. PMID:7690183

  7. Targeting pleiotropic signaling pathways to control adult cardiac stem cell fate and function

    PubMed Central

    Pagliari, Stefania; Jelinek, Jakub; Grassi, Gabriele; Forte, Giancarlo

    2014-01-01

    The identification of different pools of cardiac progenitor cells resident in the adult mammalian heart opened a new era in heart regeneration as a means to restore the loss of functional cardiac tissue and overcome the limited availability of donor organs. Indeed, resident stem cells are believed to participate to tissue homeostasis and renewal in healthy and damaged myocardium although their actual contribution to these processes remain unclear. The poor outcome in terms of cardiac regeneration following tissue damage point out at the need for a deeper understanding of the molecular mechanisms controlling CPC behavior and fate determination before new therapeutic strategies can be developed. The regulation of cardiac resident stem cell fate and function is likely to result from the interplay between pleiotropic signaling pathways as well as tissue- and cell-specific regulators. Such a modular interaction—which has already been described in the nucleus of a number of different cells where transcriptional complexes form to activate specific gene programs—would account for the unique responses of cardiac progenitors to general and tissue-specific stimuli. The study of the molecular determinants involved in cardiac stem/progenitor cell regulatory mechanisms may shed light on the processes of cardiac homeostasis in health and disease and thus provide clues on the actual feasibility of cardiac cell therapy through tissue-specific progenitors. PMID:25071583

  8. A photoactivable multi-inhibitor nanoliposome for tumour control and simultaneous inhibition of treatment escape pathways

    NASA Astrophysics Data System (ADS)

    Spring, Bryan Q.; Bryan Sears, R.; Zheng, Lei Zak; Mai, Zhiming; Watanabe, Reika; Sherwood, Margaret E.; Schoenfeld, David A.; Pogue, Brian W.; Pereira, Stephen P.; Villa, Elizabeth; Hasan, Tayyaba

    2016-04-01

    Nanoscale drug delivery vehicles can facilitate multimodal therapies of cancer by promoting tumour-selective drug release. However, few are effective because cancer cells develop ways to resist and evade treatment. Here, we introduce a photoactivable multi-inhibitor nanoliposome (PMIL) that imparts light-induced cytotoxicity in synchrony with a photoinitiated and sustained release of inhibitors that suppress tumour regrowth and treatment escape signalling pathways. The PMIL consists of a nanoliposome doped with a photoactivable chromophore (benzoporphyrin derivative, BPD) in the lipid bilayer, and a nanoparticle containing cabozantinib (XL184)—a multikinase inhibitor—encapsulated inside. Near-infrared tumour irradiation, following intravenous PMIL administration, triggers photodynamic damage of tumour cells and microvessels, and simultaneously initiates release of XL184 inside the tumour. A single PMIL treatment achieves prolonged tumour reduction in two mouse models and suppresses metastatic escape in an orthotopic pancreatic tumour model. The PMIL offers new prospects for cancer therapy by enabling spatiotemporal control of drug release while reducing systemic drug exposure and associated toxicities.

  9. From Rabbit Reticulocytes to Clam Oocytes: In Search of the System That Targets Mitotic Cyclins for Degradation

    PubMed Central

    2010-01-01

    By the late 1980s, the basic biochemistry of ubiquitin-mediated protein degradation had already been elucidated by studies that used reticulocyte lysates. However, the scope and biological functions of this system remained largely obscure. Therefore, I became interested at that time in the mechanisms by which mitotic cyclins are degraded in exit from mitosis. Using a cell-free system from clam oocytes that faithfully reproduced cell cycle stage–specific degradation of cyclins, we identified in 1995 a large ubiquitin ligase complex that targets mitotic cyclins for degradation. Subsequent studies in many laboratories showed that this ubiquitin ligase, now called the anaphase-promoting complex/cyclosome, has centrally important roles in many aspects of cell cycle control. PMID:20335505

  10. Maternal embryonic leucine zipper kinase is stabilized in mitosis by phosphorylation and is partially degraded upon mitotic exit

    SciTech Connect

    Badouel, Caroline; Chartrain, Isabelle; Blot, Joelle; Tassan, Jean-Pierre

    2010-08-01

    MELK (maternal embryonic leucine zipper kinase) is a cell cycle dependent protein kinase involved in diverse cell processes including cell proliferation, apoptosis, cell cycle and mRNA processing. Noticeably, MELK expression is increased in cancerous tissues, upon cell transformation and in mitotically-blocked cells. The question of how MELK protein level is controlled is therefore important. Here, we show that MELK protein is restricted to proliferating cells derived from either cancer or normal tissues and that MELK protein level is severely decreased concomitantly with other cell cycle proteins in cells which exit the cell cycle. Moreover, we demonstrate in human HeLa cells and Xenopus embryos that approximately half of MELK protein is degraded upon mitotic exit whereas another half remains stable during interphase. We show that the stability of MELK protein in M-phase is dependent on its phosphorylation state.

  11. Pathway-Based Genome-Wide Association Studies for Plasma Triglycerides in Obese Females and Normal-Weight Controls

    PubMed Central

    Grant, Struan F. A.; Hakonarson, Hakon; Price, R. Arlen; Li, Wei-Dong

    2015-01-01

    Pathway-based analysis as an alternative approach can provide complementary information to single-marker genome-wide association studies (GWASs), which always ignore the epistasis and does not have sufficient power to find rare variants. In this study, using genotypes from a genome-wide association study (GWAS), pathway-based association studies were carried out by a modified Gene Set Enrichment Algorithm (GSEA) method (GenGen) for triglyceride in 1028 unrelated European-American extremely obese females (BMI≥35kg/m2) and normal-weight controls (BMI<25kg/m2), and another pathway association analysis (ICSNPathway) was also used to verify the GenGen result in the same data. The GO0009110 pathway (vitamin anabolism) was among the strongest associations with triglyceride (empirical P<0.001); the result remained significant after FDR correction (P = 0.022). MMAB, an obesity-related locus, included in this pathway. The ABCG1 and BCL6 gene was found in several triglyceride-related pathways (empirical P<0.05), which were also replicated by ICSNPathway (empirical P<0.05, FDR<0.05). We also performed single-marked GWAS using PLINK for TG levels (log-transformed). Significant associations were found between ASTN2 gene SNPs and plasma triglyceride levels (rs7035794, P = 2.24×10−10). Our study suggested that vitamin anabolism pathway, BCL6 gene pathways and ASTN2 gene may contribute to the genetic variation of plasma triglyceride concentrations. PMID:26308950

  12. The Drosophila Toll pathway controls but does not clear Candida glabrata infections.

    PubMed

    Quintin, Jessica; Asmar, Joelle; Matskevich, Alexey A; Lafarge, Marie-Céline; Ferrandon, Dominique

    2013-03-15

    The pathogenicity of Candida glabrata to patients remains poorly understood for lack of convenient animal models to screen large numbers of mutants for altered virulence. In this study, we explore the minihost model Drosophila melanogaster from the dual perspective of host and pathogen. As in vertebrates, wild-type flies contain C. glabrata systemic infections yet are unable to kill the injected yeasts. As for other fungal infections in Drosophila, the Toll pathway restrains C. glabrata proliferation. Persistent C. glabrata yeasts in wild-type flies do not appear to be able to take shelter in hemocytes from the action of the Toll pathway, the effectors of which remain to be identified. Toll pathway mutant flies succumb to injected C. glabrata. In this immunosuppressed background, cellular defenses provide a residual level of protection. Although both the Gram-negative binding protein 3 pattern recognition receptor and the Persephone protease-dependent detection pathway are required for Toll pathway activation by C. glabrata, only GNBP3, and not psh mutants, are susceptible to the infection. Both Candida albicans and C. glabrata are restrained by the Toll pathway, yet the comparative study of phenoloxidase activation reveals a differential activity of the Toll pathway against these two fungal pathogens. Finally, we establish that the high-osmolarity glycerol pathway and yapsins are required for virulence of C. glabrata in this model. Unexpectedly, yapsins do not appear to be required to counteract the cellular immune response but are needed for the colonization of the wild-type host. PMID:23401590

  13. 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. PMID:25664600

  14. 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

  15. MASTL(Greatwall) regulates DNA damage responses by coordinating mitotic entry after checkpoint recovery and APC/C activation

    PubMed Central

    Wong, Po Yee; Ma, Hoi Tang; Lee, Hyun-jung; Poon, Randy Y. C.

    2016-01-01

    The G2 DNA damage checkpoint is one of the most important mechanisms controlling G2–mitosis transition. The kinase Greatwall (MASTL in human) promotes normal G2–mitosis transition by inhibiting PP2A via ARPP19 and ENSA. In this study, we demonstrate that MASTL is critical for maintaining genome integrity after DNA damage. Although MASTL did not affect the activation of DNA damage responses and subsequent repair, it determined the timing of entry into mitosis and the subsequent fate of the recovering cells. Constitutively active MASTL promoted dephosphorylation of CDK1Tyr15 and accelerated mitotic entry after DNA damage. Conversely, downregulation of MASTL or ARPP19/ENSA delayed mitotic entry. Remarkably, APC/C was activated precociously, resulting in the damaged cells progressing from G2 directly to G1 and skipping mitosis all together. Collectively, these results established that precise control of MASTL is essential to couple DNA damage to mitosis through the rate of mitotic entry and APC/C activation. PMID:26923777

  16. Impaired angiogenesis and tumor development by inhibition of the mitotic kinesin Eg5

    PubMed Central

    Exertier, Prisca; Javerzat, Sophie; Wang, Baigang; Franco, Mélanie; Herbert, John; Platonova, Natalia; Winandy, Marie; Pujol, Nadège; Nivelles, Olivier; Ormenese, Sandra; Godard, Virginie; Becker, Jürgen; Bicknell, Roy; Pineau, Raphael; Wilting, Jörg; Bikfalvi, Andreas; Hagedorn, Martin

    2013-01-01

    Kinesin motor proteins exert essential cellular functions in all eukaryotes. They control mitosis, migration and intracellular transport through interaction with microtubules. Small molecule inhibitors of the mitotic kinesin KiF11/Eg5 are a promising new class of anti-neoplastic agents currently evaluated in clinical cancer trials for solid tumors and hematological malignancies. Here we report induction of Eg5 and four other mitotic kinesins including KIF20A/Mklp2 upon stimulation of in vivo angiogenesis with vascular endothelial growth factor-A (VEGF-A). Expression analyses indicate up-regulation of several kinesin-encoding genes predominantly in lymphoblasts and endothelial cells. Chemical blockade of Eg5 inhibits endothelial cell proliferation and migration in vitro. Mitosis-independent vascular outgrowth in aortic ring cultures is strongly impaired after Eg5 or Mklp2 protein inhibition. In vivo, interfering with KIF11/Eg5 function causes developmental and vascular defects in zebrafish and chick embryos and potent inhibition of tumor angiogenesis in experimental tumor models. Besides blocking tumor cell proliferation, impairing endothelial function is a novel mechanism of action of kinesin inhibitors. PMID:24327603

  17. αTAT1 downregulation induces mitotic catastrophe in HeLa and A549 cells

    PubMed Central

    Chien, J-Y; Tsen, S-D; Chien, C-C; Liu, H-W; Tung, C-Y; Lin, C-H

    2016-01-01

    α-Tubulin acetyltransferase 1 (αTAT1) controls reversible acetylation on Lys40 of α-tubulin and modulates multiple cellular functions. αTAT1 depletion induced morphological defects of touch receptor neurons in Caenorhabditis elegans and impaired cell adhesion and contact inhibition in mouse embryonic fibroblasts, however, no morphological or proliferation defects in human RPE-hTERT cells were found after αTAT1-specific siRNA treatment. Here, we compared the effect of three αTAT1-specific shRNAs on proliferation and morphology in two human cell lines, HeLa and A549. The more efficient two shRNAs induced mitotic catastrophe in both cell lines and the most efficient one also decreased F-actin and focal adhesions. Further analysis revealed that αTAT1 downregulation increased γ-H2AX, but not other DNA damage markers p-CHK1 and p-CHK2, along with marginal change in microtubule outgrowth speed and inter-kinetochore distance. Overexpression of αTAT1 could not precisely mimic the distribution and concentration of endogenous acetylated α-tubulin (Ac-Tu), although no overt phenotype change was observed, meanwhile, this could not completely prevent αTAT1 downregulation-induced deficiencies. We therefore conclude that efficient αTAT1 downregulation could impair actin architecture and induce mitotic catastrophe in HeLa and A549 cells through mechanisms partly independent of Ac-Tu. PMID:27551500

  18. αTAT1 downregulation induces mitotic catastrophe in HeLa and A549 cells.

    PubMed

    Chien, J-Y; Tsen, S-D; Chien, C-C; Liu, H-W; Tung, C-Y; Lin, C-H

    2016-01-01

    α-Tubulin acetyltransferase 1 (αTAT1) controls reversible acetylation on Lys40 of α-tubulin and modulates multiple cellular functions. αTAT1 depletion induced morphological defects of touch receptor neurons in Caenorhabditis elegans and impaired cell adhesion and contact inhibition in mouse embryonic fibroblasts, however, no morphological or proliferation defects in human RPE-hTERT cells were found after αTAT1-specific siRNA treatment. Here, we compared the effect of three αTAT1-specific shRNAs on proliferation and morphology in two human cell lines, HeLa and A549. The more efficient two shRNAs induced mitotic catastrophe in both cell lines and the most efficient one also decreased F-actin and focal adhesions. Further analysis revealed that αTAT1 downregulation increased γ-H2AX, but not other DNA damage markers p-CHK1 and p-CHK2, along with marginal change in microtubule outgrowth speed and inter-kinetochore distance. Overexpression of αTAT1 could not precisely mimic the distribution and concentration of endogenous acetylated α-tubulin (Ac-Tu), although no overt phenotype change was observed, meanwhile, this could not completely prevent αTAT1 downregulation-induced deficiencies. We therefore conclude that efficient αTAT1 downregulation could impair actin architecture and induce mitotic catastrophe in HeLa and A549 cells through mechanisms partly independent of Ac-Tu. PMID:27551500

  19. Proteins related to the spindle and checkpoint mitotic emphasize the different pathogenesis of hypoplastic MDS.

    PubMed

    Heredia, Fabiola Fernandes; de Sousa, Juliana Cordeiro; Ribeiro Junior, Howard Lopes; Carvalho, Alex Fiorini; Magalhaes, Silvia Maria Meira; Pinheiro, Ronald Feitosa

    2014-02-01

    Some studies show that alterations in expression of proteins related to mitotic spindle (AURORAS KINASE A and B) and mitotic checkpoint (CDC20 and MAD2L1) are involved in chromosomal instability and tumor progression in various solid and hematologic malignancies. This study aimed to evaluate these genes in MDS patients. The cytogenetics analysis was carried out by G-banding, AURKA and AURKB amplification was performed using FISH, and AURKA, AURKB, CDC20 and MAD2L1 gene expression was performed by qRT-PCR in 61 samples of bone marrow from MDS patients. AURKA gene amplification was observed in 10% of the cases, which also showed higher expression levels than the control group (p=0.038). Patients with normo/hypercellular BM presented significantly higher expression levels than hypocellular BM patients, but normo and hypercellular BM groups did not differ. After logistic regression analysis, our results showed that HIGH expression levels were associated with increased risk of developing normo/hypercellular MDS. It also indicated that age is associated with AURKA, CDC20 and MAD2L1 HIGH expression levels. The distinct expression of hypocellular patients emphasizes the prognostic importance of cellularity to MDS. The amplification/high expression of AURKA suggests that the increased expression of this gene may be related to the pathogenesis of disease. PMID:24314588

  20. Exploring the Altered Dynamics of Mammalian Central Carbon Metabolic Pathway in Cancer Cells: A Classical Control Theoretic Approach

    PubMed Central

    Paul, Debjyoti; Dasgupta, Abhijit; De, Rajat K.

    2015-01-01

    Background In contrast with normal cells, most of the cancer cells depend on aerobic glycolysis for energy production in the form of adenosine triphosphate (ATP) bypassing mitochondrial oxidative phosphorylation. Moreover, compared to normal cells, cancer cells exhibit higher consumption of glucose with higher production of lactate. Again, higher rate of glycolysis provides the necessary glycolytic intermediary precursors for DNA, protein and lipid synthesis to maintain high active proliferation of the tumor cells. In this scenario, classical control theory based approach may be useful to explore the altered dynamics of the cancer cells. Since the dynamics of the cancer cells is different from that of the normal cells, understanding their dynamics may lead to development of novel therapeutic strategies. Method We have developed a model based on the state space equations of classical control theory along with an order reduction technique to mimic the actual dynamic behavior of mammalian central carbon metabolic (CCM) pathway in normal cells. Here, we have modified Michaelis Menten kinetic equation to incorporate feedback mechanism along with perturbations and cross talks associated with a metabolic pathway. Furthermore, we have perturbed the proposed model to reduce the mitochondrial oxidative phosphorylation. Thereafter, we have connected proportional-integral (PI) controller(s) with the model for tuning it to behave like the CCM pathway of a cancer cell. This methodology allows one to track the altered dynamics mediated by different enzymes. Results and Discussions The proposed model successfully mimics all the probable dynamics of the CCM pathway in normal cells. Moreover, experimental results demonstrate that in cancer cells, a coordination among enzymes catalyzing pentose phosphate pathway and intermediate glycolytic enzymes along with switching of pyruvate kinase (M2 isoform) plays an important role to maintain their altered dynamics. PMID:26367460

  1. The alternative complement pathway control protein H binds to immune complexes and serves their detection

    SciTech Connect

    Nydegger, U.E.; Corvetta, A.; Spaeth, P.J.; Spycher, M.

    1983-01-01

    During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound /sup 125/I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of /sup 125/I-H; when fresh serum was chelated with 10 mM EDTA, /sup 125/I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samples from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), /sup 125/I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while /sup 125/I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.

  2. Interphase APC/C–Cdc20 inhibition by cyclin A2–Cdk2 ensures efficient mitotic entry

    PubMed Central

    Hein, Jamin B.; Nilsson, Jakob

    2016-01-01

    Proper cell-cycle progression requires tight temporal control of the Anaphase Promoting Complex/Cyclosome (APC/C), a large ubiquitin ligase that is activated by one of two co-activators, Cdh1 or Cdc20. APC/C and Cdc20 are already present during interphase but APC/C–Cdc20 regulation during this window of the cell cycle, if any, is unknown. Here we show that cyclin A2–Cdk2 binds and phosphorylates Cdc20 in interphase and this inhibits APC/C–Cdc20 activity. Preventing Cdc20 phosphorylation results in pre-mature activation of the APC/C–Cdc20 and several substrates, including cyclin B1 and A2, are destabilized which lengthens G2 and slows mitotic entry. Expressing non-degradable cyclin A2 but not cyclin B1 restores mitotic entry in these cells. We have thus uncovered a novel positive feedback loop centred on cyclin A2–Cdk2 inhibition of interphase APC/C–Cdc20 to allow further cyclin A2 accumulation and mitotic entry. PMID:26960431

  3. Enhancement of spontaneous mitotic recombination by the meiotic mutant spo11-1 in Saccharomyces cerevisiae

    SciTech Connect

    Bruschi, C.V.; Esposito, M.S.

    1983-12-01

    Both nonreciprocal and reciprocal mitotic recombination are enhanced by the recessive mutant spo11-1, which was previously shown to affect meiosis by decreasing recombination and increasing nondisjunction. The mitotic effects are not distributed equally in all chromosomal regions. The genotypes of mitotic recombinants in spo11-1/spo11-1 diploid cells provide further evidence that widely spaced chromosomal markers undergo coincident conversion in mitosis.

  4. mTOR Controls Ovarian Follicle Growth by Regulating Granulosa Cell Proliferation

    PubMed Central

    Yu, James; Yaba, Aylin; Kasiman, Corinna; Thomson, Travis; Johnson, Joshua

    2011-01-01

    We have shown that inhibition of mTOR in granulosa cells and ovarian follicles results in compromised granulosa proliferation and reduced follicle growth. Further analysis here using spontaneously immortalized rat granulosa cells has revealed that mTOR pathway activity is enhanced during M-phase of the cell cycle. mTOR specific phosphorylation of p70S6 kinase and 4E-BP, and expression of Raptor are all enhanced during M-phase. The predominant effect of mTOR inhibition by the specific inhibitor Rapamycin (RAP) was a dose-responsive arrest in the G1 cell cycle stage. The fraction of granulosa cells that continued to divide in the presence of RAP exhibited a dose-dependent increase in aberrant mitotic figures known as anaphase bridges. Strikingly, estradiol consistently decreased the incidence of aberrant mitotic figures. In mice treated with RAP, the mitotic index was reduced compared to controls, and a similar increase in aberrant mitotic events was noted. RAP injected during a superovulation regime resulted in a dose-dependent reduction in the numbers of eggs ovulated. Implications for the real-time regulation of follicle growth and dominance, including the consequences of increased numbers of aneuploid granulosa cells, are discussed. PMID:21750711

  5. Delaying mitotic exit downregulates FLIP expression and strongly sensitizes tumor cells to TRAIL.

    PubMed

    Sánchez-Pérez, T; Medema, R H; López-Rivas, A

    2015-01-29

    Many of the current antitumor therapeutic strategies are based on the perturbation of the cell cycle, especially during mitosis. Antimitotic drugs trigger mitotic checkpoint activation, mitotic arrest and eventually cell death. However, mitotic slippage represents a major mechanism of resistance to these treatments. In an attempt to circumvent the process of slippage, targeting mitotic exit has been proposed as a better strategy to kill tumor cells. In this study, we show that treatments that induce mitotic checkpoint activation and mitotic arrest downregulate FLICE-like inhibitory protein (FLIP) levels and sensitize several tumor cell lines to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. Interestingly, we also demonstrate that in absence of mitotic checkpoint activation, mitotic arrest induced either by Cdc20 knockdown or overexpression of nondegradable cyclin B is sufficient to induce both FLIP downregulation and sensitivity to TRAIL. In summary, our data suggest that a combination of antimitotic drugs targeting cyclin B degradation and TRAIL might prevent mitotic slippage and allow tumor cells to reach the threshold for apoptosis induction, thereby facilitating tumor suppression. PMID:24488010

  6. Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation

    PubMed Central

    Chen, Long; Li, Zhiguo; Ahmad, Nihal; Liu, Xiaoqi

    2016-01-01

    Insulin receptor substrate (IRS) proteins play important roles by acting as a platform in transducing signals from transmembrane receptors upon growth factor stimulation. Although tyrosine phosphorylation on IRS proteins plays critical roles in signal transduction, phosphorylation of IRS proteins on serine/threonine residues are believed to play various regulatory roles on IRS protein function. However, studies on serine/threonine phosphorylation of IRS proteins are very limited, especially for insulin receptor substrate 2 (IRS2), one member of the IRS protein family. In this study, we identify Polo-like kinase 1 (Plk1) as the responsible kinase for phosphorylation of IRS2 on two serine residues, Ser 556 and Ser 1098. Phosphorylation of IRS2 on these two serine residues by Plk1 prevents the activation of the PI3K pathway upon growth factor stimulation by inhibiting the binding between IRS2 and the PI3K pathway components and increasing IRS2 protein degradation. Of significance, we show that IRS2 phosphorylation is cell cycle regulated and that Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation. PMID:25830382

  7. Cyto-3D-print to attach mitotic cells.

    PubMed

    Castroagudin, Michelle R; Zhai, Yujia; Li, Zhi; Marnell, Michael G; Glavy, Joseph S

    2016-08-01

    The Cyto-3D-print is an adapter that adds cytospin capability to a standard centrifuge. Like standard cytospinning, Cyto-3D-print increases the surface attachment of mitotic cells while giving a higher degree of adaptability to other slide chambers than available commercial devices. The use of Cyto-3D-print is cost effective, safe, and applicable to many slide designs. It is durable enough for repeated use and made of biodegradable materials for environment-friendly disposal. PMID:26464272

  8. Role of Direct vs. Indirect Pathways from the Motor Cortex to Spinal Motoneurons in the Control of Hand Dexterity

    PubMed Central

    Isa, Tadashi; Kinoshita, Masaharu; Nishimura, Yukio

    2013-01-01

    Evolutionally, development of the direct connection from the motor cortex to spinal motoneurons [corticomotoneuronal (CM) pathway] parallels the ability of hand dexterity. Damage to the corticofugal fibers in higher primates resulted in deficit of fractionated digit movements. Based on such observations, it was generally believed that the CM pathway plays a critical role in the control of hand dexterity. On the other hand, a number of “phylogenetically older” indirect pathways from the motor cortex to motoneurons still exist in primates. The indirect pathways are mediated by intercalated neurons such as segmental interneurons (sINs), propriospinal neurons (PNs) reticulospinal neurons (RSNs), or rubrospinal neurons (RuSNs). However, their contribution to hand dexterity remains elusive. Lesion of the brainstem pyramid sparing the transmission through the RuSNs and RSNs, resulted in permanent deficit of fractionated digit movements in macaque monkeys. On the other hand, in our recent study, after lesion of the dorsolateral funiculus (DLF) at the C5 segment, which removed the lateral corticospinal tract (l-CST) including the CM pathway and the transmission through sINs and RuSNs but spared the processing through the PNs and RSNs, fractionated digit movements recovered within several weeks. These results suggest that the PNs can be involved in the recovery of fractionated digit movements, but the RSNs and RuSNs have less capacity in this regard. However, on closer inspection, it was found that the activation pattern of hand and arm muscles considerably changed after the C5 lesion, suggesting limitation of PNs for the compensation of hand dexterity. Altogether, it is suggested that PNs, RSNs RuSNs, and the CM pathway (plus sINs) make a different contribution to the hand dexterity and appearance of motor deficit of the hand dexterity caused by damage to the corticofugal fibers and potential of recovery varies depending on the rostrocaudal level of the lesion. PMID

  9. The Opa1-Dependent Mitochondrial Cristae Remodeling Pathway Controls Atrophic, Apoptotic, and Ischemic Tissue Damage

    PubMed Central

    Varanita, Tatiana; Soriano, Maria Eugenia; Romanello, Vanina; Zaglia, Tania; Quintana-Cabrera, Rubén; Semenzato, Martina; Menabò, Roberta; Costa, Veronica; Civiletto, Gabriele; Pesce, Paola; Viscomi, Carlo; Zeviani, Massimo; Di Lisa, Fabio; Mongillo, Marco; Sandri, Marco; Scorrano, Luca

    2015-01-01

    Summary Mitochondrial morphological and ultrastructural changes occur during apoptosis and autophagy, but whether they are relevant in vivo for tissue response to damage is unclear. Here we investigate the role of the optic atrophy 1 (OPA1)-dependent cristae remodeling pathway in vivo and provide evidence that it regulates the response of multiple tissues to apoptotic, necrotic, and atrophic stimuli. Genetic inhibition of the cristae remodeling pathway in vivo does not affect development, but protects mice from denervation-induced muscular atrophy, ischemic heart and brain damage, as well as hepatocellular apoptosis. Mechanistically, OPA1-dependent mitochondrial cristae stabilization increases mitochondrial respiratory efficiency and blunts mitochondrial dysfunction, cytochrome c release, and reactive oxygen species production. Our results indicate that the OPA1-dependent cristae remodeling pathway is a fundamental, targetable determinant of tissue damage in vivo. PMID:26039448

  10. Sensitivity Analysis of Intracellular Signaling Pathway Kinetics Predicts Targets for Stem Cell Fate Control

    PubMed Central

    Mahdavi, Alborz; Davey, Ryan E; Bhola, Patrick; Yin, Ting; Zandstra, Peter W

    2007-01-01

    Directing stem cell fate requires knowledge of how signaling networks integrate temporally and spatially segregated stimuli. We developed and validated a computational model of signal transducer and activator of transcription-3 (Stat3) pathway kinetics, a signaling network involved in embryonic stem cell (ESC) self-renewal. Our analysis identified novel pathway responses; for example, overexpression of the receptor glycoprotein-130 results in reduced pathway activation and increased ESC differentiation. We used a systematic in silico screen to identify novel targets and protein interactions involved in Stat3 activation. Our analysis demonstrates that signaling activation and desensitization (the inability to respond to ligand restimulation) is regulated by balancing the activation state of a distributed set of parameters including nuclear export of Stat3, nuclear phosphatase activity, inhibition by suppressor of cytokine signaling, and receptor trafficking. This knowledge was used to devise a temporally modulated ligand delivery strategy that maximizes signaling activation and leads to enhanced ESC self-renewal. PMID:17616983

  11. The cAMP pathway and the control of adrenocortical development and growth

    PubMed Central

    de Joussineau, Cyrille; Sahut-Barnola, Isabelle; Levy, Isaac; Saloustros, Emmanouil; Val, Pierre; Stratakis, Constantine A.; Martinez, Antoine

    2013-01-01

    In the last 10 years, extensive studies showed that the cAMP pathway is deregulated in patients suffering from adrenocortical tumours, and particularly in primary pigmented nodular adrenocortical disease (PPNAD). Here we describe how evidence arising from the analysis of patients’ data, mouse models and in vitro experiments, have shed light on the cAMP pathway as a central player in adrenal physiopathology. We also show how novel data generated from mouse models may point to new targets for potential therapies. PMID:22019902

  12. A Set of Activators and Repressors Control Peripheral Glucose Pathways in Pseudomonas putida To Yield a Common Central Intermediate▿

    PubMed Central

    del Castillo, Teresa; Duque, Estrella; Ramos, Juan L.

    2008-01-01

    Pseudomonas putida KT2440 channels glucose to the central Entner-Doudoroff intermediate 6-phosphogluconate through three convergent pathways. The genes for these convergent pathways are clustered in three independent regions on the host chromosome. A number of monocistronic units and operons coexist within each of these clusters, favoring coexpression of catabolic enzymes and transport systems. Expression of the three pathways is mediated by three transcriptional repressors, HexR, GnuR, and PtxS, and by a positive transcriptional regulator, GltR-2. In this study, we generated mutants in each of the regulators and carried out transcriptional assays using microarrays and transcriptional fusions. These studies revealed that HexR controls the genes that encode glucokinase/glucose 6-phosphate dehydrogenase that yield 6-phosphogluconate; the genes for the Entner-Doudoroff enzymes that yield glyceraldehyde-3-phosphate and pyruvate; and gap-1, which encodes glyceraldehyde-3-phosphate dehydrogenase. GltR-2 is the transcriptional regulator that controls specific porins for the entry of glucose into the periplasmic space, as well as the gtsABCD operon for glucose transport through the inner membrane. GnuR is the repressor of gluconate transport and gluconokinase responsible for the conversion of gluconate into 6-phosphogluconate. PtxS, however, controls the enzymes for oxidation of gluconate to 2-ketogluconate, its transport and metabolism, and a set of genes unrelated to glucose metabolism. PMID:18245293

  13. Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling

    PubMed Central

    Li, Renfeng; Pinto, Sneha M.; Shaw, Patrick G.; Huang, Tai-Chung; Wan, Jun; Qian, Jiang; Gowda, Harsha; Wu, Xinyan; Lv, Dong-Wen; Zhang, Kun; Manda, Srikanth S.; Pandey, Akhilesh; Hayward, S. Diane

    2015-01-01

    Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in

  14. Induction of morphological and biochemical apoptosis following prolonged mitotic blockage by halichondrin B macrocyclic ketone analog E7389.

    PubMed

    Kuznetsov, Galina; Towle, Murray J; Cheng, Hongsheng; Kawamura, Takanori; TenDyke, Karen; Liu, Diana; Kishi, Yoshito; Yu, Melvin J; Littlefield, Bruce A

    2004-08-15

    E7389, a macrocyclic ketone analog of the marine natural product halichondrin B, currently is undergoing clinical trials for cancer. This fully synthetic agent exerts its highly potent in vitro and in vivo anticancer effects via tubulin-based antimitotic mechanisms, which are similar or identical to those of parental halichondrin B. In an attempt to understand the impressive potency of E7389 in animal models of human cancer, its ability to induce apoptosis following prolonged mitotic blockage was evaluated. Treatment of U937 human histiocytic lymphoma cells with E7389 led to time-dependent collection of cells in the G2-M phase of the cell cycle, beginning as early as 2 h and becoming maximal by 12 h. Increased numbers of hypodiploid events were seen beginning at 12 h, suggesting initiation of apoptosis after prolonged E7389-induced mitotic blockage. The identity of hypodiploid events as apoptotic cells under these conditions was confirmed by two additional morphologic criteria: green to orange/yellow shifts on acridine orange/ethidium bromide staining, and cell surface annexin V binding as assessed by flow cytometry. Several biochemical correlates of apoptosis also were seen following E7389 treatment, including phosphorylation of the antiapoptotic protein Bcl-2, cytochrome c release from mitochondria, proteolytic activation of caspase-3 and -9, and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). In LNCaP human prostate cancer cells, treatment with E7389 also led to generation of hypodiploid cells, activation of caspase-3 and -9, and appearance of cleaved PARP, indicating that E7389 can activate cellular apoptosis pathways under anchorage-independent and -dependent cell culture conditions. These results show that prolonged mitotic blockage by E7389 can lead to apoptotic cell death of human cancer cells in vitro and can provide a mechanistic basis for the significant in vivo anticancer efficacy of E7389. PMID:15313917

  15. Rhn1, a Nuclear Protein, Is Required for Suppression of Meiotic mRNAs in Mitotically Dividing Fission Yeast

    PubMed Central

    Hada, Kazumasa; Niwa, Ryusuke

    2012-01-01

    In the fission yeast Schizosaccharomyces pombe, many meiotic mRNAs are transcribed during mitosis and meiosis and selectively eliminated in mitotic cells. However, this pathway for mRNA decay, called the determinant of selective removal (DSR)-Mmi1 system, targets only some of the numerous meiotic mRNAs that are transcribed in mitotic cells. Here we describe Rhn1, a nuclear protein involved in meiotic mRNA suppression in vegetative fission yeast. Rhn1 is homologous to budding yeast Rtt103 and localizes to one or a few discrete nuclear dots in growing vegetative cells. Rhn1 colocalizes with a pre-mRNA 3′-end processing factor, Pcf11, and with the 5′–3′ exoribonuclease, Dhp1; moreover, Rhn1 coimmunoprecipitates with Pcf11. Loss of rhn1 results in elevated sensitivity to high temperature, to thiabendazole (TBZ), and to UV. Interestingly, meiotic mRNAs—including moa1+, mcp5+, and mug96+—accumulate in mitotic rhn1Δ cells. Accumulation of meiotic mRNAs also occurs in strains lacking Lsk1, a kinase that phosphorylates serine 2 (Ser-2) in the C-terminal domain (CTD) of RNA polymerase II (Pol II), and in strains lacking Sen1, an ATP-dependent 5′–3′ RNA/DNA helicase: notably, both Lsk1 and Sen1 have been implicated in termination of Pol II-dependent transcription. Furthermore, RNAi knockdown of cids-2, a Caenorhabditis elegans ortholog of rhn1+, leads to elevated expression of a germline-specific gene, pgl-1, in somatic cells. These results indicate that Rhn1 contributes to the suppression of meiotic mRNAs in vegetative fission yeast and that the mechanism by which Rhn1 downregulates germline-specific transcripts may be conserved in unicellular and multicellular organisms. PMID:22912768

  16. Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling.

    PubMed

    Li, Renfeng; Liao, Gangling; Nirujogi, Raja Sekhar; Pinto, Sneha M; Shaw, Patrick G; Huang, Tai-Chung; Wan, Jun; Qian, Jiang; Gowda, Harsha; Wu, Xinyan; Lv, Dong-Wen; Zhang, Kun; Manda, Srikanth S; Pandey, Akhilesh; Hayward, S Diane

    2015-12-01

    Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in

  17. Mitotic internalization of planar cell polarity proteins preserves tissue polarity.

    PubMed

    Devenport, Danelle; Oristian, Daniel; Heller, Evan; Fuchs, Elaine

    2011-08-01

    Planar cell polarity (PCP) is the collective polarization of cells along the epithelial plane, a process best understood in the terminally differentiated Drosophila wing. Proliferative tissues such as mammalian skin also show PCP, but the mechanisms that preserve tissue polarity during proliferation are not understood. During mitosis, asymmetrically distributed PCP components risk mislocalization or unequal inheritance, which could have profound consequences for the long-range propagation of polarity. Here, we show that when mouse epidermal basal progenitors divide PCP components are selectively internalized into endosomes, which are inherited equally by daughter cells. Following mitosis, PCP proteins are recycled to the cell surface, where asymmetry is re-established by a process reliant on neighbouring PCP. A cytoplasmic dileucine motif governs mitotic internalization of atypical cadherin Celsr1, which recruits Vang2 and Fzd6 to endosomes. Moreover, embryos transgenic for a Celsr1 that cannot mitotically internalize exhibit perturbed hair-follicle angling, a hallmark of defective PCP. This underscores the physiological relevance and importance of this mechanism for regulating polarity during cell division. PMID:21743464

  18. Evidence for mitotic recombination in W sup ei /+ heterozygous mice

    SciTech Connect

    Panthier, J.J.; Condamine, H.; Jacob, F. ); Guenet, J.L. )

    1990-05-01

    A number of alleles at coat color loci of the house mouse give rise to areas of wild-type pigmentation on the coats of otherwise mutant animals. Such unstable alleles include both recessive and dominant mutations. Among the latter are several alleles at the W locus. In this report, phenotypic reversions of the W{sup ei} allele at the W locus were studied. Mice heterozygous in repulsion for both W{sup ei} and buff (bf) (i.e. W{sup ei}+/+bf) were examined for the occurrence of phenotypic reversion events. Buff (bf) is a recessive mutation, which lies 21 cM from W on the telomeric side of chromosome 5 and is responsible for the khaki colored coat of nonagouti buff homozygotes (a/a; bf/bf). Two kinds of fully pigmented reversion spots were recovered on the coats of a/a; W{sup ei}+/+bf mice: either solid black or khaki colored. Furthermore phenotypic reversions of W{sup ei}/+ were enhanced significantly following X-irradiation of 9.25-day-old W{sup ei}/+ embryos (P < 0.04). These observations are consistent with the suggestion of a role for mitotic recombination in the origin of these phenotypic reversions. In addition these results raise the intriguing possibility that some W mutations may enhance mitotic recombination in the house mouse.

  19. Towards a quantitative understanding of mitotic spindle assembly and mechanics

    PubMed Central

    Mogilner, Alex; Craig, Erin

    2010-01-01

    The ‘simple’ view of the mitotic spindle is that it self-assembles as a result of microtubules (MTs) randomly searching for chromosomes, after which the spindle length is maintained by a balance of outward tension exerted by molecular motors on the MTs connecting centrosomes and chromosomes, and compression generated by other motors on the MTs connecting the spindle poles. This picture is being challenged now by mounting evidence indicating that spindle assembly and maintenance rely on much more complex interconnected networks of microtubules, molecular motors, chromosomes and regulatory proteins. From an engineering point of view, three design principles of this molecular machine are especially important: the spindle assembles quickly, it assembles accurately, and it is mechanically robust – yet malleable. How is this design achieved with randomly interacting and impermanent molecular parts? Here, we review recent interdisciplinary studies that have started to shed light on this question. We discuss cooperative mechanisms of spindle self-assembly, error correction and maintenance of its mechanical properties, speculate on analogy between spindle and lamellipodial dynamics, and highlight the role of quantitative approaches in understanding the mitotic spindle design. PMID:20930139

  20. A molecular mechanism of mitotic centrosome assembly in Drosophila

    PubMed Central

    Conduit, Paul T; Richens, Jennifer H; Wainman, Alan; Holder, James; Vicente, Catarina C; Pratt, Metta B; Dix, Carly I; Novak, Zsofia A; Dobbie, Ian M; Schermelleh, Lothar; Raff, Jordan W

    2014-01-01

    Centrosomes comprise a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, but it is unclear how this occurs. In this study, we show that the centriole protein Asl initiates the recruitment of DSpd-2 and Cnn to mother centrioles; both proteins then assemble into co-dependent scaffold-like structures that spread outwards from the mother centriole and recruit most, if not all, other PCM components. In the absence of either DSpd-2 or Cnn, mitotic PCM assembly is diminished; in the absence of both proteins, it appears to be abolished. We show that DSpd-2 helps incorporate Cnn into the PCM and that Cnn then helps maintain DSpd-2 within the PCM, creating a positive feedback loop that promotes robust PCM expansion around the mother centriole during mitosis. These observations suggest a surprisingly simple mechanism of mitotic PCM assembly in flies. DOI: http://dx.doi.org/10.7554/eLife.03399.001 PMID:25149451

  1. Unconventional Functions of Mitotic Kinases in Kidney Tumorigenesis

    PubMed Central

    Hascoet, Pauline; Chesnel, Franck; Le Goff, Cathy; Le Goff, Xavier; Arlot-Bonnemains, Yannick

    2015-01-01

    Human tumors exhibit a variety of genetic alterations, including point mutations, translocations, gene amplifications and deletions, as well as aneuploid chromosome numbers. For carcinomas, aneuploidy is associated with poor patient outcome for a large variety of tumor types, including breast, colon, and renal cell carcinoma. The Renal cell carcinoma (RCC) is a heterogeneous carcinoma consisting of different histologic types. The clear renal cell carcinoma (ccRCC) is the most common subtype and represents 85% of the RCC. Central to the biology of the ccRCC is the loss of function of the Von Hippel–Lindau gene, but is also associated with genetic instability that could be caused by abrogation of the cell cycle mitotic spindle checkpoint and may involve the Aurora kinases, which regulate centrosome maturation. Aneuploidy can also result from the loss of cell–cell adhesion and apical–basal cell polarity that also may be regulated by the mitotic kinases (polo-like kinase 1, casein kinase 2, doublecortin-like kinase 1, and Aurora kinases). In this review, we describe the “non-mitotic” unconventional functions of these kinases in renal tumorigenesis. PMID:26579493

  2. Differential Mitotic Stability of Yeast Disomes Derived from Triploid Meiosis

    PubMed Central

    Campbell, Douglas; Doctor, John S.; Feuersanger, Jeane H.; Doolittle, Mark M.

    1981-01-01

    The frequencies of recovered disomy among the meiotic segregants of yeast (Saccharomyces cerevisiae) triploids were assessed under conditions in which all 17 yeast chromosomes were monitored simultaneously. The studies employed inbred triploids, in which all homologous centromeres were identical by descent, and single haploid testers carrying genetic markers for all 17 linkage groups. The principal results include: (1) Ascospores from triploid meiosis germinate at frequencies comparable to those from normal diploids, but most fail to produce visible colonies due to the growth-retarding effects of high multiple disomy. (2) The probability of disome formation during triploid meiosis is the same for all chromosomes; disomy for any given chromosome does not exclude simultaneous disomy for any other chromosome. (3) The 17 yeast chromosomes fall into three frequency classes in terms of disome recovery. The results support the idea that multiply disomic meiotic segregants of the triploid experience repeated, nonrandom, post-germination mitotic chromosome losses (N+1→N) and that the observed variations in individual disome recovery are wholly attributable to inherent differences in disome mitotic stability. PMID:7035289

  3. Small Molecule Approach to Study the Function of Mitotic Kinesins.

    PubMed

    Al-Obaidi, Naowras; Kastl, Johanna; Mayer, Thomas U

    2016-01-01

    Mitotic motor proteins of the kinesin superfamily are critical for the faithful segregation of chromosomes and the formation of the two daughter cells during meiotic and mitotic M-phase. Of the 45 human kinesins, roughly a dozen are involved in the assembly of the bipolar spindle, alignment of chromosomes at the spindle equator, chromosome segregation, and cytokinesis. The functions of kinesins in these processes are highly diverse and include the transport of cargo molecules, sliding and bundling of microtubules, and regulation of microtubule dynamics. In light of this multitude of diverse functions and the complex functional interplay of different kinesins during M-phase, it is not surprising that one of the greatest challenges in cell biology is the functional dissection of individual motor proteins. Reversible and fast acting small molecules are powerful tools to accomplish this challenge. However, the validity of conclusions drawn from small molecule studies strictly depends on compound specificity. In this chapter, we present methods for the identification of small molecule inhibitors of a motor protein of interest. In particular, we focus on a protein-based large throughput screen to identify inhibitors of the ATPase activity of kinesins. Furthermore, we provide protocols and guidelines for secondary screens to validate hits and select for specific inhibitors. PMID:27193856

  4. Polyoma small T antigen triggers cell death via mitotic catastrophe.

    PubMed

    Pores Fernando, A T; Andrabi, S; Cizmecioglu, O; Zhu, C; Livingston, D M; Higgins, J M G; Schaffhausen, B S; Roberts, T M

    2015-05-01

    Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, thereby resulting in the activation of the spindle assembly checkpoint. Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed, that PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors. PMID:24998850

  5. AllR Controls the Expression of Streptomyces coelicolor Allantoin Pathway Genes

    PubMed Central

    Navone, Laura; Macagno, Juan Pablo; Licona-Cassani, Cuauhtémoc; Marcellin, Esteban; Nielsen, Lars K.; Gramajo, Hugo

    2015-01-01

    Streptomyces species are native inhabitants of soil, a natural environment where nutrients can be scarce and competition fierce. They have evolved ways to metabolize unusual nutrients, such as purines and its derivatives, which are highly abundant in soil. Catabolism of these uncommon carbon and nitrogen sources needs to be tightly regulated in response to nutrient availability and environmental stimulus. Recently, the allantoin degradation pathway was characterized in Streptomyces coelicolor. However, there are questions that remained unanswered, particularly regarding pathway regulation. Here, using a combination of proteomics and genetic approaches, we identified the negative regulator of the allantoin pathway, AllR. In vitro studies confirmed that AllR binds to the promoter regions of allantoin catabolic genes and determined the AllR DNA binding motif. In addition, effector studies showed that allantoic acid, and glyoxylate, to a lesser extent, inhibit the binding of AllR to the DNA. Inactivation of AllR repressor leads to the constitutive expression of the AllR regulated genes and intriguingly impairs actinorhodin and undecylprodigiosin production. Genetics and proteomics analysis revealed that among all genes from the allantoin pathway that are upregulated in the allR mutant, the hyi gene encoding a hydroxypyruvate isomerase (Hyi) is responsible of the impairment of antibiotic production. PMID:26187964

  6. Fragipan controls on nitrogen loss by surface and subsurface flow pathways in an upland agricultural watershed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Improved understanding of nutrient transport by surface and subsurface flow pathways is critical to protecting water quality in agricultural watersheds. We sought to compare nitrogen loss in overland and subsurface flow on two opposing hillslopes (north versus south facing), each with contrasting so...

  7. Metabolomic profiling and genomic analysis of wheat aneuploid lines to identify genes controlling biochemical pathways in mature grain.

    PubMed

    Francki, Michael G; Hayton, Sarah; Gummer, Joel P A; Rawlinson, Catherine; Trengove, Robert D

    2016-02-01

    Metabolomics is becoming an increasingly important tool in plant genomics to decipher the function of genes controlling biochemical pathways responsible for trait variation. Although theoretical models can integrate genes and metabolites for trait variation, biological networks require validation using appropriate experimental genetic systems. In this study, we applied an untargeted metabolite analysis to mature grain of wheat homoeologous group 3 ditelosomic lines, selected compounds that showed significant variation between wheat lines Chinese Spring and at least one ditelosomic line, tracked the genes encoding enzymes of their biochemical pathway using the wheat genome survey sequence and determined the genetic components underlying metabolite variation. A total of 412 analytes were resolved in the wheat grain metabolome, and principal component analysis indicated significant differences in metabolite profiles between Chinese Spring and each ditelosomic lines. The grain metabolome identified 55 compounds positively matched against a mass spectral library where the majority showed significant differences between Chinese Spring and at least one ditelosomic line. Trehalose and branched-chain amino acids were selected for detailed investigation, and it was expected that if genes encoding enzymes directly related to their biochemical pathways were located on homoeologous group 3 chromosomes, then corresponding ditelosomic lines would have a significant reduction in metabolites compared with Chinese Spring. Although a proportion showed a reduction, some lines showed significant increases in metabolites, indicating that genes directly and indirectly involved in biosynthetic pathways likely regulate the metabolome. Therefore, this study demonstrated that wheat aneuploid lines are suitable experimental genetic system to validate metabolomics-genomics networks. PMID:26032167

  8. Essential role of the m2R-RGS6-IKACh pathway in controlling intrinsic heart rate variability.

    PubMed

    Posokhova, Ekaterina; Ng, David; Opel, Aaisha; Masuho, Ikuo; Tinker, Andrew; Biesecker, Leslie G; Wickman, Kevin; Martemyanov, Kirill A

    2013-01-01

    Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood. Here we examined the contribution of the m2R-I(KACh) intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m2R-I(KACh) signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the RGS6 gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m2R-I(KACh) signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis. PMID:24204714

  9. Essential Role of the m2R-RGS6-IKACh Pathway in Controlling Intrinsic Heart Rate Variability

    PubMed Central

    Posokhova, Ekaterina; Ng, David; Opel, Aaisha; Masuho, Ikuo; Tinker, Andrew; Biesecker, Leslie G.; Wickman, Kevin; Martemyanov, Kirill A.

    2013-01-01

    Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood. Here we examined the contribution of the m2R-IKACh intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m2R-IKACh signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the RGS6 gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m2R-IKACh signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis. PMID:24204714

  10. Aurora Kinase A is critical for the Nkx6.1 mediated β-cell proliferation pathway

    PubMed Central

    Hobson, Amanda; Draney, Carrie; Stratford, Andrew; Becker, Thomas C; Lu, Danhong; Arlotto, Michelle; Tessem, Jeffery S

    2015-01-01

    Type 1 and type 2 diabetes are ultimately characterized by depleted β-cell mass. Characterization of the molecular pathways that control β-cell proliferation could be harnessed to restore these cells. The homeobox β-cell transcription factor Nkx6.1 induces β-cell proliferation by activating the orphan nuclear receptors Nr4a1 and Nr4a3. Here, we demonstrate that Nkx6.1 localizes to the promoter of the mitotic kinase AURKA (Aurora Kinase A) and induces its expression. Adenovirus mediated overexpression of AURKA is sufficient to induce proliferation in primary rat islets while maintaining glucose stimulated insulin secretion. Furthermore, AURKA is necessary for Nkx6.1 mediated β-cell proliferation as demonstrated by shRNA mediated knock down and pharmacological inhibition of AURKA kinase activity. AURKA preferentially induces DNA replication in β-cells as measured by BrdU incorporation, and enhances the rate of histone H3 phosphorylation in primary β-cells, demonstrating that AURKA induces the replicative and mitotic cell cycle phases in rat β-cells. Finally, overexpression of AURKA results in phosphorylation of the cell cycle regulator p53, which targets p53 for degradation and permits cell cycle progression. These studies define a pathway by which AURKA upregulation by Nkx6.1 results in phosphorylation and degradation of p53, thus removing a key inhibitory factor and permitting engagement of the β-cell proliferation pathway. PMID:26030060

  11. Coordinated control of volume regulatory Na+/H+ and K+/H+ exchange pathways in Amphiuma red blood cells.

    PubMed

    Ortiz-Acevedo, Alejandro; Rigor, Robert R; Maldonado, Hector M; Cala, Peter M

    2010-03-01

    The Na(+)/H(+) and K(+)/H(+) exchange pathways of Amphiuma tridactylum red blood cells (RBCs) are quiescent at normal resting cell volume yet are selectively activated in response to cell shrinkage and swelling, respectively. These alkali metal/H(+) exchangers are activated by net kinase activity and deactivated by net phosphatase activity. We employed relaxation kinetic analyses to gain insight into the basis for coordinated control of these volume regulatory ion flux pathways. This approach enabled us to develop a model explaining how phosphorylation/dephosphorylation-dependent events control and coordinate the activity of the Na(+)/H(+) and K(+)/H(+) exchangers around the cell volume set point. We found that the transition between initial and final steady state for both activation and deactivation of the volume-induced Na(+)/H(+) and K(+)/H(+) exchange pathways in Amphiuma RBCs proceed as a single exponential function of time. The rate of Na(+)/H(+) exchange activation increases with cell shrinkage, whereas the rate of Na(+)/H(+) exchange deactivation increases as preshrunken cells are progressively swollen. Similarly, the rate of K(+)/H(+) exchange activation increases with cell swelling, whereas the rate of K(+)/H(+) exchange deactivation increases as preswollen cells are progressively shrunken. We propose a model in which the activities of the controlling kinases and phosphatases are volume sensitive and reciprocally regulated. Briefly, the activity of each kinase-phosphatase pair is reciprocally related, as a function of volume, and the volume sensitivities of kinases and phosphatases controlling K(+)/H(+) exchange are reciprocally related to those controlling Na(+)/H(+) exchange. PMID:19940069

  12. Timely Endocytosis of Cytokinetic Enzymes Prevents Premature Spindle Breakage during Mitotic Exit

    PubMed Central

    Onishi, Masayuki; Yeong, Foong May

    2016-01-01

    Cytokinesis requires the spatio-temporal coordination of membrane deposition and primary septum (PS) formation at the division site to drive acto-myosin ring (AMR) constriction. It has been demonstrated that AMR constriction invariably occurs only after the mitotic spindle disassembly. It has also been established that Chitin Synthase II (Chs2p) neck localization precedes mitotic spindle disassembly during mitotic exit. As AMR constriction depends upon PS formation, the question arises as to how chitin deposition is regulated so as to prevent premature AMR constriction and mitotic spindle breakage. In this study, we propose that cells regulate the coordination between spindle disassembly and AMR constriction via timely endocytosis of cytokinetic enzymes, Chs2p, Chs3p, and Fks1p. Inhibition of endocytosis leads to over accumulation of cytokinetic enzymes during mitotic exit, which accelerates the constriction of the AMR, and causes spindle breakage that eventually could contribute to monopolar spindle formation in the subsequent round of cell division. Intriguingly, the mitotic spindle breakage observed in endocytosis mutants can be rescued either by deleting or inhibiting the activities of, CHS2, CHS3 and FKS1, which are involved in septum formation. The findings from our study highlight the importance of timely endocytosis of cytokinetic enzymes at the division site in safeguarding mitotic spindle integrity during mitotic exit. PMID:27447488

  13. Redundancy control in pathway databases (ReCiPa): an application for improving gene-set enrichment analysis in Omics studies and "Big data" biology.

    PubMed

    Vivar, Juan C; Pemu, Priscilla; McPherson, Ruth; Ghosh, Sujoy

    2013-08-01

    Abstract Unparalleled technological advances have fueled an explosive growth in the scope and scale of biological data and have propelled life sciences into the realm of "Big Data" that cannot be managed or analyzed by conventional approaches. Big Data in the life sciences are driven primarily via a diverse collection of 'omics'-based technologies, including genomics, proteomics, metabolomics, transcriptomics, metagenomics, and lipidomics. Gene-set enrichment analysis is a powerful approach for interrogating large 'omics' datasets, leading to the identification of biological mechanisms associated with observed outcomes. While several factors influence the results from such analysis, the impact from the contents of pathway databases is often under-appreciated. Pathway databases often contain variously named pathways that overlap with one another to varying degrees. Ignoring such redundancies during pathway analysis can lead to the designation of several pathways as being significant due to high content-similarity, rather than truly independent biological mechanisms. Statistically, such dependencies also result in correlated p values and overdispersion, leading to biased results. We investigated the level of redundancies in multiple pathway databases and observed large discrepancies in the nature and extent of pathway overlap. This prompted us to develop the application, ReCiPa (Redundancy Control in Pathway Databases), to control redundancies in pathway databases based on user-defined thresholds. Analysis of genomic and genetic datasets, using ReCiPa-generated overlap-controlled versions of KEGG and Reactome pathways, led to a reduction in redundancy among the top-scoring gene-sets and allowed for the inclusion of additional gene-sets representing possibly novel biological mechanisms. Using obesity as an example, bioinformatic analysis further demonstrated that gene-sets identified from overlap-controlled pathway databases show stronger evidence of prior association

  14. Noncanonical control of C. elegans germline apoptosis by the insulin/IGF-1 and Ras/MAPK signaling pathways.

    PubMed

    Perrin, A J; Gunda, M; Yu, B; Yen, K; Ito, S; Forster, S; Tissenbaum, H A; Derry, W B

    2013-01-01

    The insulin/IGF-1 pathway controls a number of physiological processes in the nematode worm Caenorhabditis elegans, including development, aging and stress response. We previously found that the Akt/PKB ortholog AKT-1 dampens the apoptotic response to genotoxic stress in the germline by negatively regulating the p53-like transcription factor CEP-1. Here, we report unexpected rearrangements to the insulin/IGF-1 pathway, whereby the insulin-like receptor DAF-2 and 3-phosphoinositide-dependent protein kinase PDK-1 oppose AKT-1 to promote DNA damage-induced apoptosis. While DNA damage does not affect phosphorylation at the PDK-1 site Thr350/Thr308 of AKT-1, it increased phosphorylation at Ser517/Ser473. Although ablation of daf-2 or pdk-1 completely suppressed akt-1-dependent apoptosis, the transcriptional activation of CEP-1 was unaffected, suggesting that daf-2 and pdk-1 act independently or downstream of cep-1 and akt-1. Ablation of the akt-1 paralog akt-2 or the downstream target of the insulin/IGF-1 pathway daf-16 (a FOXO transcription factor) restored sensitivity to damage-induced apoptosis in daf-2 and pdk-1 mutants. In addition, daf-2 and pdk-1 mutants have reduced levels of phospho-MPK-1/ERK in their germ cells, indicating that the insulin/IGF-1 pathway promotes Ras signaling in the germline. Ablation of the Ras effector gla-3, a negative regulator of mpk-1, restored sensitivity to apoptosis in daf-2 mutants, suggesting that gla-3 acts downstream of daf-2. In addition, the hypersensitivity of let-60/Ras gain-of-function mutants to damage-induced apoptosis was suppressed to wild-type levels by ablation of daf-2. Thus, insulin/IGF-1 signaling selectively engages AKT-2/DAF-16 to promote DNA damage-induced germ cell apoptosis downstream of CEP-1 through the Ras pathway. PMID:22935616

  15. Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in Catharanthus roseus

    PubMed Central

    Han, Mei; Heppel, Simon C.; Su, Tao; Bogs, Jochen; Zu, Yuangang; An, Zhigang; Rausch, Thomas

    2013-01-01

    In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation. PMID:23650515

  16. Ligand-independent pathway that controls stability of interferon alpha receptor

    SciTech Connect

    Liu Jianghuai; Plotnikov, Alexander; Banerjee, Anamika; Suresh Kumar, K.G.; Ragimbeau, Josiane; Marijanovic, Zrinka; Baker, Darren P.; Pellegrini, Sandra; Fuchs, Serge Y.

    2008-03-07

    Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination, and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance.

  17. A spindle-independent cleavage pathway controls germ cell formation in Drosophila.

    PubMed

    Cinalli, Ryan M; Lehmann, Ruth

    2013-07-01

    The primordial germ cells (PGCs) are the first cells to form during Drosophila melanogaster embryogenesis. Whereas the process of somatic cell formation has been studied in detail, the mechanics of PGC formation are poorly understood. Here, using four-dimensional multi-photon imaging combined with genetic and pharmacological manipulations, we find that PGC formation requires an anaphase spindle-independent cleavage pathway. In addition to using core regulators of cleavage, including the small GTPase RhoA (Drosophila rho1) and the Rho-associated kinase, ROCK (Drosophila drok), we show that this pathway requires Germ cell-less (GCL), a conserved BTB-domain protein not previously implicated in cleavage mechanics. This alternative form of cell formation suggests that organisms have evolved multiple molecular strategies for regulating the cytoskeleton during cleavage. PMID:23728423

  18. Snail Coordinately Regulates Downstream Pathways to Control Multiple Aspects of Mammalian Neural Precursor Development

    PubMed Central

    Zander, Mark A.; Burns, Sarah E.; Yang, Guang; Kaplan, David R.

    2014-01-01

    The Snail transcription factor plays a key role in regulating diverse developmental processes but is not thought to play a role in mammalian neural precursors. Here, we have examined radial glial precursor cells of the embryonic murine cortex and demonstrate that Snail regulates their survival, self-renewal, and differentiation into intermediate progenitors and neurons via two distinct and separable target pathways. First, Snail promotes cell survival by antagonizing a p53-dependent death pathway because coincident p53 knockdown rescues survival deficits caused by Snail knockdown. Second, we show that the cell cycle phosphatase Cdc25b is regulated by Snail in radial precursors and that Cdc25b coexpression is sufficient to rescue the decreased radial precursor proliferation and differentiation observed upon Snail knockdown. Thus, Snail acts via p53 and Cdc25b to coordinately regulate multiple aspects of mammalian embryonic neural precursor biology. PMID:24719096

  19. Bidentate Ligands on Osmium(VI) Nitrido Complexes Control Intracellular Targeting and Cell Death Pathways

    PubMed Central

    Suntharalingam, Kogularamanan; Johnstone, Timothy C.; Bruno, Peter M.; Lin, Wei; Hemann, Michael T.; Lippard, Stephen J.

    2013-01-01

    The cellular response evoked by anti-proliferating osmium(VI) nitrido compounds of general formula OsN(N^N)Cl3 (N^N = 2,2′-bipyridine 1, 1,10-phenanthroline 2, 3,4,7,8-tetramethyl-1,10-phenanthroline 3, or 4,7-diphenyl-1,10-phenanthroline 4) can be tuned by subtle ligand modifications. Complex 2 induces DNA damage, resulting in activation of the p53 pathway, cell cycle arrest at the G2/M phase, and caspase-dependent apoptotic cell death. In contrast, 4 evokes ER stress leading to the upregulation of proteins of the unfolded protein response pathway, increase in ER size, and p53-independent apoptotic cell death. To the best of our knowledge, 4 is the first osmium compound to induce ER stress in cancer cells. PMID:24041161

  20. Regulation of cancer cell survival by BCL2 family members upon prolonged mitotic arrest: opportunities for anticancer therapy.

    PubMed

    Barillé-Nion, Sophie; Bah, Nourdine; Véquaud, Eloïse; Juin, Philippe

    2012-10-01

    Attacking cancer cell survival defense by targeting B-Cell Lymphoma 2 (BCL2) family of anti-apoptotic proteins may provide a powerful means to improve chemotherapy efficiency. This could be particularly relevant to anti-mitotic-based therapy, where tumor response relates to a competing network between mitotic cell death signaling and mitotic slippage as an adaptative response to a leaky mitotic checkpoint. In this review, we focus on recent findings that point out the major role played by BCL2 family members in response to anti-mitotic agents, which reveal dependence of cancer cell survival on BCL2 homologs during mitotic arrest and after mitotic slippage. Finally, we discuss pre-clinical data combining anti-mitotic agents with BCL2 inhibitors. PMID:23060542

  1. Multiple signaling pathways control nitrogen-mediated root elongation in maize

    PubMed Central

    Chen, Fanjun; Zhang, Fusuo

    2008-01-01

    Response of root system architecture to nutrient availability is an essential way for plants to adapt to soil environments. Nitrogen can affect root development either as a result of changes in the external concentration, or through changes in the internal nutrient status of the plant. Low soil N stimulates root elongation in maize. Recent evidence suggests that plant hormones auxin and cytokinin, as well as NO signaling pathway, are involved in the regulation of root elongation by low nitrogen nutrition. PMID:19704443

  2. Protein Phosphatase 2A Inhibition with LB100 Enhances Radiation-Induced Mitotic Catastrophe and Tumor Growth Delay in Glioblastoma.

    PubMed

    Gordon, Ira K; Lu, Jie; Graves, Christian A; Huntoon, Kristin; Frerich, Jason M; Hanson, Ryan H; Wang, Xiaoping; Hong, Christopher S; Ho, Winson; Feldman, Michael J; Ikejiri, Barbara; Bisht, Kheem; Chen, Xiaoyuan S; Tandle, Anita; Yang, Chunzhang; Arscott, W Tristram; Ye, Donald; Heiss, John D; Lonser, Russell R; Camphausen, Kevin; Zhuang, Zhengping

    2015-07-01

    Protein phosphatase 2A (PP2A) is a tumor suppressor whose function is lost in many cancers. An emerging, though counterintuitive, therapeutic approach is inhibition of PP2A to drive damaged cells through the cell cycle, sensitizing them to radiotherapy. We investigated the effects of PP2A inhibition on U251 glioblastoma cells following radiation treatment in vitro and in a xenograft mouse model in vivo. Radiotherapy alone augmented PP2A activity, though this was significantly attenuated with combination LB100 treatment. LB100 treatment yielded a radiation dose enhancement factor of 1.45 and increased the rate of postradiation mitotic catastrophe at 72 and 96 hours. Glioblastoma cells treated with combination LB100 and radiotherapy maintained increased γ-H2AX expression at 24 hours, diminishing cellular repair of radiation-induced DNA double-strand breaks. Combination therapy significantly enhanced tumor growth delay and mouse survival and decreased p53 expression 3.68-fold, compared with radiotherapy alone. LB100 treatment effectively inhibited PP2A activity and enhanced U251 glioblastoma radiosensitivity in vitro and in vivo. Combination treatment with LB100 and radiation significantly delayed tumor growth, prolonging survival. The mechanism of radiosensitization appears to be related to increased mitotic catastrophe, decreased capacity for repair of DNA double-strand breaks, and diminished p53 DNA-damage response pathway activity. PMID:25939762

  3. The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry.

    PubMed

    Lee, Kyung-Jong; Shang, Zeng-Fu; Lin, Yu-Fen; Sun, Jingxin; Morotomi-Yano, Keiko; Saha, Debabrata; Chen, Benjamin P C

    2015-04-01

    DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the key regulator of the non-homologous end joining pathway of DNA double-strand break repair. We have previously reported that DNA-PKcs is required for maintaining chromosomal stability and mitosis progression. Our further investigations reveal that deficiency in DNA-PKcs activity caused a delay in mitotic entry due to dysregulation of cyclin-dependent kinase 1 (Cdk1), the key driving force for cell cycle progression through G2/M transition. Timely activation of Cdk1 requires polo-like kinase 1 (Plk1), which affects modulators of Cdk1. We found that DNA-PKcs physically interacts with Plk1 and could facilitate Plk1 activation both in vitro and in vivo. Further, DNA-PKcs-deficient cells are highly sensitive to Plk1 inhibitor BI2536, suggesting that the coordination between DNA-PKcs and Plk1 is not only crucial to ensure normal cell cycle progression through G2/M phases but also required for cellular resistance to mitotic stress. On the basis of the current study, it is predictable that combined inhibition of DNA-PKcs and Plk1 can be employed in cancer therapy strategy for synthetic lethality. PMID:25925375

  4. The budding yeast nuclear envelope adjacent to the nucleolus serves as a membrane sink during mitotic delay

    PubMed Central

    Witkin, Keren L.; Chong, Yolanda; Shao, Sichen; Webster, Micah T.; Lahiri, Sujoy; Walters, Alison D.; Lee, Brandon; Koh, Judice L.Y.; Prinz, William A.; Andrews, Brenda J.; Cohen-Fix, Orna

    2012-01-01

    Summary The mechanisms that dictate nuclear shape are largely unknown. Here we screened the budding yeast deletion collection for mutants with abnormal nuclear shape. A common phenotype was the appearance of a nuclear extension, particularly in mutants in DNA repair and chromosome segregation genes. Our data suggest that these mutations led to the abnormal nuclear morphology indirectly, by causing a checkpoint-induced cell cycle delay. Indeed, delaying cells in mitosis by other means also led to the appearance of nuclear extensions, while inactivating the DNA damage checkpoint pathway in a DNA repair mutant reduced the fraction of cells with nuclear extensions. Formation of a nuclear extension was specific to a mitotic delay, as cells arrested in S or G2 had round nuclei. Moreover, the nuclear extension always coincided with the nucleolus, while the morphology of DNA mass remained largely unchanged. Finally, we found that phospholipid synthesis continues unperturbed when cells delay in mitosis, and inhibiting phospholipid synthesis abolished the formation of nuclear extensions. Our data suggest a mechanism that promotes nuclear envelope expansion during mitosis. When mitotic progression is delayed, cells sequester the added membrane to the nuclear envelope associated with the nucleolus, possibly to avoid disruption of intra-nuclear organization. PMID:22658600

  5. Partitioning the effects of an ecosystem engineer: kangaroo rats control community structure via multiple pathways.

    PubMed

    Prugh, Laura R; Brashares, Justin S

    2012-05-01

    1. Ecosystem engineers impact communities by altering habitat conditions, but they can also have strong effects through consumptive, competitive and other non-engineering pathways. 2. Engineering effects can lead to fundamentally different community dynamics than non-engineering effects, but the relative strengths of these interactions are seldom quantified. 3. We combined structural equation modelling and exclosure experiments to partition the effects of a keystone engineer, the giant kangaroo rat (Dipodomys ingens), on plants, invertebrates and vertebrates in a semi-arid California grassland. 4. We separated the effects of burrow creation from kangaroo rat density and found that kangaroo rats increased the diversity and abundance of other species via both engineering and non-engineering pathways. 5. Engineering was the primary factor structuring plant and small mammal communities, whereas non-engineering effects structured invertebrate communities and increased lizard abundance. 6. These results highlight the importance of the non-engineering effects of ecosystem engineers and shed new light on the multiple pathways by which strong-interactors shape communities. PMID:22098534

  6. Competing pathways control host resistance to virus via tRNA modification and programmed ribosomal frameshifting.

    PubMed

    Maynard, Nathaniel D; Macklin, Derek N; Kirkegaard, Karla; Covert, Markus W

    2012-01-01

    Viral infection depends on a complex interplay between host and viral factors. Here, we link host susceptibility to viral infection to a network encompassing sulfur metabolism, tRNA modification, competitive binding, and programmed ribosomal frameshifting (PRF). We first demonstrate that the iron-sulfur cluster biosynthesis pathway in Escherichia coli exerts a protective effect during lambda phage infection, while a tRNA thiolation pathway enhances viral infection. We show that tRNA(Lys) uridine 34 modification inhibits PRF to influence the ratio of lambda phage proteins gpG and gpGT. Computational modeling and experiments suggest that the role of the iron-sulfur cluster biosynthesis pathway in infection is indirect, via competitive binding of the shared sulfur donor IscS. Based on the universality of many key components of this network, in both the host and the virus, we anticipate that these findings may have broad relevance to understanding other infections, including viral infection of humans. PMID:22294093

  7. The RanGTP Pathway: From Nucleo-Cytoplasmic Transport to Spindle Assembly and Beyond

    PubMed Central

    Cavazza, Tommaso; Vernos, Isabelle

    2016-01-01

    The small GTPase Ran regulates the interaction of transport receptors with a number of cellular cargo proteins. The high affinity binding of the GTP-bound form of Ran to import receptors promotes cargo release, whereas its binding to export receptors stabilizes their interaction with the cargo. This basic mechanism linked to the asymmetric distribution of the two nucleotide-bound forms of Ran between the nucleus and the cytoplasm generates a switch like mechanism controlling nucleo-cytoplasmic transport. Since 1999, we have known that after nuclear envelope breakdown (NEBD) Ran and the above transport receptors also provide a local control over the activity of factors driving spindle assembly and regulating other aspects of cell division. The identification and functional characterization of RanGTP mitotic targets is providing novel insights into mechanisms essential for cell division. Here we review our current knowledge on the RanGTP system and its regulation and we focus on the recent advances made through the characterization of its mitotic targets. We then briefly review the novel functions of the pathway that were recently described. Altogether, the RanGTP system has moonlighting functions exerting a spatial control over protein interactions that drive specific functions depending on the cellular context. PMID:26793706

  8. The European quality of care pathways (EQCP) study on the impact of care pathways on interprofessional teamwork in an acute hospital setting: study protocol: for a cluster randomised controlled trial and evaluation of implementation processes

    PubMed Central

    2012-01-01

    Background Although care pathways are often said to promote teamwork, high-level evidence that supports this statement is lacking. Furthermore, knowledge on conditions and facilitators for successful pathway implementation is scarce. The objective of the European Quality of Care Pathway (EQCP) study is therefore to study the impact of care pathways on interprofessional teamwork and to build up understanding on the implementation process. Methods/design An international post-test-only cluster Randomised Controlled Trial (cRCT), combined with process evaluations, will be performed in Belgium, Ireland, Italy, and Portugal. Teams caring for proximal femur fracture (PFF) patients and patients hospitalized with an exacerbation of chronic obstructive pulmonary disease (COPD) will be randomised into an intervention and control group. The intervention group will implement a care pathway for PFF or COPD containing three active components: a formative evaluation of the actual teams’ performance, a set of evidence-based key interventions, and a training in care pathway-development. The control group will provide usual care. A set of team input, process and output indicators will be used as effect measures. The main outcome indicator will be relational coordination. Next to these, process measures during and after pathway development will be used to evaluate the implementation processes. In total, 132 teams have agreed to participate, of which 68 were randomly assigned to the intervention group and 64 to the control group. Based on power analysis, a sample of 475 team members per arm is required. To analyze results, multilevel analysis will be performed. Discussion Results from our study will enhance understanding on the active components of care pathways. Through this, preferred implementation strategies can be defined. Trail registration NCT01435538 PMID:22607698

  9. Aurora A's Functions During Mitotic Exit: The Guess Who Game.

    PubMed

    Reboutier, David; Benaud, Christelle; Prigent, Claude

    2015-01-01

    Until recently, the knowledge of Aurora A kinase functions during mitosis was limited to pre-metaphase events, particularly centrosome maturation, G2/M transition, and mitotic spindle assembly. However, an involvement of Aurora A in post-metaphase events was also suspected, but not clearly demonstrated due to the technical difficulty to perform the appropriate experiments. Recent developments of both an analog-specific version of Aurora A and small molecule inhibitors have led to the first demonstration that Aurora A is required for the early steps of cytokinesis. As in pre-metaphase, Aurora A plays diverse functions during anaphase, essentially participating in astral microtubules dynamics and central spindle assembly and functioning. The present review describes the experimental systems used to decipher new functions of Aurora A during late mitosis and situate these functions into the context of cytokinesis mechanisms. PMID:26734572

  10. FTO influences adipogenesis by regulating mitotic clonal expansion

    PubMed Central

    Merkestein, Myrte; Laber, Samantha; McMurray, Fiona; Andrew, Daniel; Sachse, Gregor; Sanderson, Jeremy; Li, Mengdi; Usher, Samuel; Sellayah, Dyan; Ashcroft, Frances M.; Cox, Roger D.

    2015-01-01

    The fat mass and obesity-associated (FTO) gene plays a pivotal role in regulating body weight and fat mass; however, the underlying mechanisms are poorly understood. Here we show that primary adipocytes and mouse embryonic fibroblasts (MEFs) derived from FTO overexpression (FTO-4) mice exhibit increased potential for adipogenic differentiation, while MEFs derived from FTO knockout (FTO-KO) mice show reduced adipogenesis. As predicted from these findings, fat pads from FTO-4 mice fed a high-fat diet show more numerous adipocytes. FTO influences adipogenesis by regulating events early in adipogenesis, during the process of mitotic clonal expansion. The effect of FTO on adipogenesis appears to be mediated via enhanced expression of the pro-adipogenic short isoform of RUNX1T1, which enhanced adipocyte proliferation, and is increased in FTO-4 MEFs and reduced in FTO-KO MEFs. Our findings provide novel mechanistic insight into how upregulation of FTO leads to obesity. PMID:25881961

  11. Mitotic wavefronts mediated by mechanical signaling in early Drosophila embryos

    NASA Astrophysics Data System (ADS)

    Kang, Louis; Idema, Timon; Liu, Andrea; Lubensky, Tom

    2013-03-01

    Mitosis in the early Drosophila embryo demonstrates spatial and temporal correlations in the form of wavefronts that travel across the embryo in each cell cycle. This coordinated phenomenon requires a signaling mechanism, which we suggest is mechanical in origin. We have constructed a theoretical model that supports nonlinear wavefront propagation in a mechanically-excitable medium. Previously, we have shown that this model captures quantitatively the wavefront speed as it varies with cell cycle number, for reasonable values of the elastic moduli and damping coefficient of the medium. Now we show that our model also captures the displacements of cell nuclei in the embryo in response to the traveling wavefront. This new result further supports that mechanical signaling may play an important role in mediating mitotic wavefronts.

  12. SSZ-13 Crystallization by Particle Attachment and Deterministic Pathways to Crystal Size Control.

    PubMed

    Kumar, Manjesh; Luo, Helen; Román-Leshkov, Yuriy; Rimer, Jeffrey D

    2015-10-14

    Many synthetic and natural crystalline materials are either known or postulated to grow via nonclassical pathways involving the initial self-assembly of precursors that serve as putative growth units for crystallization. Elucidating the pathway(s) by which precursors attach to crystal surfaces and structurally rearrange (postattachment) to incorporate into the underlying crystalline lattice is an active and expanding area of research comprising many unanswered fundamental questions. Here, we examine the crystallization of SSZ-13, which is an aluminosilicate zeolite that possesses exceptional physicochemical properties for applications in separations and catalysis (e.g., methanol upgrading to chemicals and the environmental remediation of NO(x)). We show that SSZ-13 grows by two concerted mechanisms: nonclassical growth involving the attachment of amorphous aluminosilicate particles to crystal surfaces and classical layer-by-layer growth via the incorporation of molecules to advancing steps on the crystal surface. A facile, commercially viable method of tailoring SSZ-13 crystal size and morphology is introduced wherein growth modifiers are used to mediate precursor aggregation and attachment to crystal surfaces. We demonstrate that small quantities of polymers can be used to tune crystal size over 3 orders of magnitude (0.1-20 μm), alter crystal shape, and introduce mesoporosity. Given the ubiquitous presence of amorphous precursors in a wide variety of microporous crystals, insight of the SSZ-13 growth mechanism may prove to be broadly applicable to other materials. Moreover, the ability to selectively tailor the physical properties of SSZ-13 crystals through molecular design offers new routes to optimize their performance in a wide range of commercial applications. PMID:26376337

  13. CD47 signaling pathways controlling cellular differentiation and responses to stress

    PubMed Central

    Soto-Pantoja, David R.; Kaur, Sukhbir; Roberts, David D.

    2016-01-01

    CD47 is a widely expressed integral membrane protein that serves as the counter-receptor for the inhibitory phagocyte receptor signal-regulatory protein-α (SIRPα) and as a signaling receptor for the secreted matricellular protein thrombospondin-1. Recent studies employing mice and somatic cells lacking CD47 have revealed important pathophysiological functions of CD47 in cardiovascular homeostasis, immune regulation, resistance of cells and tissues to stress, and chronic diseases of aging including cancer. With the emergence of experimental therapeutics targeting CD47, a more thorough understanding of CD47 signal transduction is essential. CD47 lacks a substantial cytoplasmic signaling domain, but several cytoplasmic binding partners have been identified, and lateral interactions of CD47 with other membrane receptors play important roles in mediating signaling resulting from the binding of thrombospondin-1. This review addresses recent advances in identifying the lateral binding partners, signal transduction pathways, and downstream transcription networks regulated through CD47 in specific cell lineages. Major pathways regulated by CD47 signaling include calcium homeostasis, cyclic nucleotide signaling, nitric oxide and hydrogen sulfide biosynthesis and signaling, and stem cell transcription factors. These pathways and other undefined proximal mediators of CD47 signaling regulate cell death and protective autophagy responses, mitochondrial biogenesis, cell adhesion and motility, and stem cell self-renewal. Although thrombospondin-1 is the best characterized agonist of CD47, the potential roles of other members of the thrombospondin family, SIRPα and SIRPγ binding, and homotypic CD47 interactions as agonists or antagonists of signaling through CD47 should also be considered. PMID:25708195

  14. Determination of genotoxic effects of Imazethapyr herbicide in Allium cepa root cells by mitotic activity, chromosome aberration, and comet assay.

    PubMed

    Liman, Recep; Ciğerci, İbrahim Hakkı; Öztürk, Nur Serap

    2015-02-01

    Imazethapyr (IM) is an imidazolinone herbicide that is currently used for broad-spectrum weed control in soybean and other legume crops. In this study, cytotoxic and genotoxic effects of IM were investigated by using mitotic index (MI), mitotic phases, chromosomal abnormalities (CAs) and DNA damage on the root meristem cells of Allium cepa. In Allium root growth inhibition test, EC50 value was determined as 20 ppm, and 0.5xEC50, EC50 and 2xEC50 concentrations of IM herbicide were introduced to onion tuber roots. Distilled water and methyl methane sulfonate (MMS, 10 mg/L) were used as a negative and positive control, respectively. As A. cepa cell cycle is 24 hours, so, application process was carried out for 24, 48, 72 and 96 hours. All the applied doses decreased MIs compared to control group and these declines were found to be statistically meaningful. Analysis of the chromosomes showed that 10 ppm IM except for 48 h induced CAs but 40 ppm IM except for 72 h decreased CAs. DNA damage was found significantly higher in 20 and 40 ppm of IM compared to the control in comet assay. These results indicated that IM herbicide exhibits cytotoxic activity but not genotoxic activity (except 10 ppm) and induced DNA damage in a dose dependent manner in A. cepa root meristematic cells. PMID:25752428

  15. Spatial rule-based modeling: a method and its application to the human mitotic kinetochore.

    PubMed

    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

  16. Non-anti-mitotic concentrations of taxol reduce breast cancer cell invasiveness

    SciTech Connect

    Tran, Truong-An; Gillet, Ludovic; Roger, Sebastien; Besson, Pierre; White, Edward; Le Guennec, Jean-Yves

    2009-02-06

    Taxol is widely used in breast cancer chemotherapy. Its effects are primarily attributed to its anti-mitotic activity. Microtubule perturbators also exert antimetastatic activities which cannot be explained solely by the inhibition of proliferation. Voltage-dependent sodium channels (Na{sub V}) are abnormally expressed in the highly metastatic breast cancer cell line MDA-MB-231 and not in MDA-MB-468 cell line. Inhibiting Na{sub V} activity with tetrodotoxin is responsible for an approximately 0.4-fold reduction of MDA-MB-231 cell invasiveness. In this study, we focused on the effect of a single, 2-h application of 10 nM taxol on the two cell lines MDA-MB-231 and MDA-MB-468. At this concentration, taxol had no effect on proliferation after 7 days and on migration in any cell line. However it led to a 40% reduction of transwell invasion of MDA-MB-231 cells. There was no additive effect when taxol and tetrodotoxin were simultaneously applied. Na{sub V} activity, as assessed by patch-clamp, indicates that it was changed by taxol pre-treatment. We conclude that taxol can exert anti-tumoral activities, in cells expressing Na{sub V}, at low doses that have no effect on cell proliferation. This effect might be due to a modulation of signalling pathways involving sodium channels.

  17. 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

  18. An okadaic acid-sensitive phosphatase negatively controls the cyclin degradation pathway in amphibian eggs.

    PubMed Central

    Lorca, T; Fesquet, D; Zindy, F; Le Bouffant, F; Cerruti, M; Brechot, C; Devauchelle, G; Dorée, M

    1991-01-01

    Inhibition of okadaic acid-sensitive phosphatases released the cyclin degradation pathway from its inhibited state in extracts prepared from unfertilized Xenopus eggs arrested at the second meiotic metaphase. It also switched on cyclin protease activity in a permanent fashion in interphase extracts prepared from activated eggs. Even after cdc2 kinase inactivation, microinjection of okadaic acid-treated interphase extracts pushed G2-arrested recipient oocytes into the M phase, suggesting that the phosphatase inhibitor stabilizes the activity of an unidentified factor which shares in common with cdc2 kinase the maturation-promoting factor activity. Images PMID:1846666

  19. Coherent Control Protocol for Separating Energy-Transfer Pathways in Photosynthetic Complexes by Chiral Multidimensional Signals†

    PubMed Central

    Abramavicius, Darius; Mukamel, Shaul

    2013-01-01

    Adaptive optimizations performed using a genetic algorithm are employed to construct optimal laser pulse configurations that separate spectroscopic features associated with the two main energy-transfer pathways in the third-order nonlinear optical response simulated for the Fenna–Matthews–Olson (FMO) photosynthetic complex from the green sulfur bacterium Chlorobium tepidum. Superpositions of chirality-induced tensor components in both collinear and noncollinear pulse configurations are analyzed. The optimal signals obtained by manipulating the ratios of various 2D spectral peaks reveal detailed information about the excitation dynamics. PMID:21495702

  20. Neuroendocrine Control of the Gut During Stress: Corticotropin-Releasing Factor Signaling Pathways in the Spotlight

    PubMed Central

    Stengel, Andreas; Taché, Yvette

    2009-01-01

    Stress affects the gastrointestinal tract as part of the visceral response. Various stressors induce similar profiles of gut motor function alterations, including inhibition of gastric emptying, stimulation of colonic propulsive motility, and hypersensitivity to colorectal distension. In recent years, substantial progress has been made in our understanding of the underlying mechanisms of stress’s impact on gut function. Activation of corticotropin-releasing factor (CRF) signaling pathways mediates both the inhibition of upper gastrointestinal (GI) and the stimulation of lower GI motor function through interaction with different CRF receptor subtypes. Here, we review how various stressors affect the gut, with special emphasis on the central and peripheral CRF signaling systems. PMID:18928406

  1. Neuroendocrine control of the gut during stress: corticotropin-releasing factor signaling pathways in the spotlight.

    PubMed

    Stengel, Andreas; Taché, Yvette

    2009-01-01

    Stress affects the gastrointestinal tract as part of the visceral response. Various stressors induce similar profiles of gut motor function alterations, including inhibition of gastric emptying, stimulation of colonic propulsive motility, and hypersensitivity to colorectal distension. In recent years, substantial progress has been made in our understanding of the underlying mechanisms of stress's impact on gut function. Activation of corticotropin-releasing factor (CRF) signaling pathways mediates both the inhibition of upper gastrointestinal (GI) and the stimulation of lower GI motor function through interaction with different CRF receptor subtypes. Here, we review how various stressors affect the gut, with special emphasis on the central and peripheral CRF signaling systems. PMID:18928406

  2. A splicing alteration of 4.1R pre-mRNA generates 2 protein isoforms with distinct assembly to spindle poles in mitotic cells.

    PubMed

    Delhommeau, François; Vasseur-Godbillon, Corinne; Leclerc, Philippe; Schischmanoff, Pierre-Olivier; Croisille, Laure; Rince, Patricia; Morinière, Madeleine; Benz, Edward J; Tchernia, Gil; Tamagnini, Gabriel; Ribeiro, Leticia; Delaunay, Jean; Baklouti, Faouzi

    2002-10-01

    The C-terminal region of erythroid cytoskeletal protein 4.1R, encoded by exons 20 and 21, contains a binding site for nuclear mitotic apparatus protein (NuMA), a protein needed for the formation and stabilization of the mitotic spindle. We have previously described a splicing mutation of 4.1R that yields 2 isoforms: One, CO.1, lacks most of exon 20-encoded peptide and carries a missense C-terminal sequence. The other, CO.2, lacks exon 20-encoded C-terminal sequence, but retains the normal exon 21-encoded C-terminal sequence. Knowing that both shortened proteins are expressed in red cells and assemble to the membrane skeleton, we asked whether they would ensure 4.1R mitotic function in dividing cells. We show here that CO.2, but not CO.1, assembles to spindle poles, and colocalizes with NuMA in erythroid and lymphoid mutated cells, but none of these isoforms interact with NuMA in vitro. In microtubule-destabilizing conditions, again only CO.2 localizes to the centrosomes. These data suggest that the stability of 4.1R association with centrosomes requires an intact C-terminal end, either for a proper conformation of the protein, for a direct binding to an unknown centrosome-cytoskeletal network, or for both. We also found that 4.1G, a ubiquitous homolog of 4.1R, is present in mutated as well as control cells and that its C-terminal region binds efficiently to NuMA, suggesting that in fact mitotic spindles host a mixture of the two 4.1 family members. These findings led to the postulate that the coexpression at the spindle poles of 2 related proteins, 4.1R and 4.1G, might reflect a functional redundancy in mitotic cells. PMID:12239178

  3. Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function.

    PubMed

    Golsteyn, R M; Mundt, K E; Fry, A M; Nigg, E A

    1995-06-01

    Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation. PMID:7790358

  4. Windpipe Controls Drosophila Intestinal Homeostasis by Regulating JAK/STAT Pathway via Promoting Receptor Endocytosis and Lysosomal Degradation

    PubMed Central

    Li, Min; Wu, Longfei; Wang, Guolun; Baeg, Gyeong-Hun; You, Jia; Li, Zhouhua; Lin, Xinhua

    2015-01-01

    The adult intestinal homeostasis is tightly controlled by proper proliferation and differentiation of intestinal stem cells. The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is essential for the regulation of adult stem cell activities and maintenance of intestinal homeostasis. Currently, it remains largely unknown how JAK/STAT signaling activities are regulated in these processes. Here we have identified windpipe (wdp) as a novel component of the JAK/STAT pathway. We demonstrate that Wdp is positively regulated by JAK/STAT signaling in Drosophila adult intestines. Loss of wdp activity results in the disruption of midgut homeostasis under normal and regenerative conditions. Conversely, ectopic expression of Wdp inhibits JAK/STAT signaling activity. Importantly, we show that Wdp interacts with the receptor Domeless (Dome), and promotes its internalization for subsequent lysosomal degradation. Together, these data led us to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis. PMID:25923769

  5. Metabolic control analysis of L-lactate synthesis pathway in Rhizopus oryzae As 3.2686.

    PubMed

    Ke, Wei; Chang, Shu; Chen, Xiaoju; Luo, Shuizhong; Jiang, Shaotong; Yang, Peizhou; Wu, Xuefeng; Zheng, Zhi

    2015-11-01

    The relationship between the metabolic flux and the activities of the pyruvate branching enzymes of Rhizopus oryzae As 3.2686 during L-lactate fermentation was investigated using the perturbation method of aeration. The control coefficients for five enzymes, pyruvate dehydrogenase (PDH), pyruvate carboxylase (PC), pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH), were calculated. Our results indicated significant correlations between PDH and PC, PDC and LDH, PDC and ADH, LDH and ADH, and PDC and PC. It also appeared that PDH, PC, and LDH strongly controlled the L-lactate flux; PDH and ADH strongly controlled the ethanol flux; while PDH and PC strongly controlled the acetyl coenzyme A flux and the oxaloacetate flux. Further, the flux control coefficient curves indicated that the control of the system gradually transferred from PDC to PC during the steady state. Therefore, PC was the key rate-limiting enzyme that controls the flux distribution. PMID:26288952

  6. The SUMO-targeted ubiquitin ligase RNF4 localizes to etoposide-exposed mitotic chromosomes: implication for a novel DNA damage response during mitosis.

    PubMed

    Saito, Masayuki; Fujimitsu, Yuka; Sasano, Takeshi; Yoshikai, Yushi; Ban-Ishihara, Reiko; Nariai, Yuko; Urano, Takeshi; Saitoh, Hisato

    2014-04-25

    RNF4, a SUMO-targeted ubiquitin ligase (STUbL), localizes to the nucleus and functions in the DNA damage response during interphase of the cell cycle. RNF4 also exists in cells undergoing mitosis, where its regulation and function remain poorly understood. Here we showed that administration of etoposide, an anticancer DNA topoisomerase II poison, to mitotic human cervical cancer HeLa cells induced SUMO-2/3-dependent localization of RNF4 to chromosomes. The FK2 antibody signals, indicative of poly/multi-ubiquitin assembly, were detected on etoposide-exposed mitotic chromosomes, whereas the signals were negligible in cells depleted for RNF4 by RNA interference. This suggests that RNF4 functions as a STUbL in the etoposide-induced damage response during mitosis. Indeed, RNF4-depletion sensitized mitotic HeLa cells to etoposide and increased cells with micronuclei. These results indicate the importance of the RNF4-mediated STUbL pathway during mitosis for the maintenance of chromosome integrity and further implicate RNF4 as a target for topo II poison-based therapy for cancer patients. PMID:24695317

  7. rasiRNA pathway controls antisense expression of Drosophila telomeric retrotransposons in the nucleus

    PubMed Central

    Shpiz, Sergey; Kwon, Dmitry; Rozovsky, Yakov; Kalmykova, Alla

    2009-01-01

    Telomeres in Drosophila are maintained by the specialized telomeric retrotransposons HeT-A, TART and TAHRE. Sense transcripts of telomeric retroelements were shown to be the targets of a specialized RNA-interference mechanism, a repeat-associated short interfering (rasi)RNA-mediated system. Antisense rasiRNAs play a key role in this mechanism, highlighting the importance of antisense expression in retrotransposon silencing. Previously, bidirectional transcription was reported for the telomeric element TART. Here, we show that HeT-A is also bidirectionally transcribed, and HeT-A antisense transcription in ovaries is regulated by a promoter localized within its 3′ untranslated region. A remarkable feature of noncoding HeT-A antisense transcripts is the presence of multiple introns. We demonstrate that sense and antisense HeT-A-specific rasiRNAs are present in the same tissue, indicating that transcripts of both directions may be considered as natural targets of the rasiRNA pathway. We found that the expression of antisense transcripts of telomeric elements is regulated by the RNA silencing machinery, suggesting rasiRNA-mediated interplay between sense and antisense transcripts in the cell. Finally, this regulation occurs in the nucleus since disruption of the rasiRNA pathway leads to an accumulation of TART and HeT-A transcripts in germ cell nuclei. PMID:19036789

  8. Deubiquitylating enzyme USP9x regulates hippo pathway activity by controlling angiomotin protein turnover

    PubMed Central

    Thanh Nguyen, Hung; Andrejeva, Diana; Gupta, Rajat; Choudhary, Chunaram; Hong, Xin; Eichhorn, Pieter J A; Loya, Anand C; Cohen, Stephen M

    2016-01-01

    The Hippo pathway has been identified as a key barrier for tumorigenesis, acting through downregulation of YAP/TAZ activity. Elevated YAP/TAZ activity has been documented in many human cancers. Ubiquitylation has been shown to play a key role in regulating YAP/TAZ activity through downregulation of a number of Hippo pathway components. Several ubiquitin ligase complexes have been implicated in this process, however, little is known about the deubiquitylating enzymes that counteract these activities to regulate YAP/TAZ. Here we identify the deubiquitylating enzyme USP9x as a regulator of YAP/TAZ activity. We demonstrate that USPx regulates ubiquitin-mediated turnover of the YAP inhibitor, Angiomotin. USP9x acts to deubiquitylate Angiomotin at lysine 496, resulting in stabilization of Angiomotin and lower YAP/TAZ activity. USP9x mRNA levels were reduced in several cancers. Clinically, USP9x mRNA levels were reduced in several cancers with low USPx expression correlating with poor prognosis in renal clear cell carcinoma. Our data indicate that USP9x may be a useful biomarker for renal clear cell carcinoma. PMID:27462448

  9. A direct anterior cingulate pathway to the primate primary olfactory cortex may control attention to olfaction.

    PubMed

    García-Cabezas, Miguel Á; Barbas, Helen

    2014-09-01

    Behavioral and functional studies in humans suggest that attention plays a key role in activating the primary olfactory cortex through an unknown circuit mechanism. We report that a novel pathway from the anterior cingulate cortex, an area which has a key role in attention, projects directly to the primary olfactory cortex in rhesus monkeys, innervating mostly the anterior olfactory nucleus. Axons from the anterior cingulate cortex formed synapses mostly with spines of putative excitatory pyramidal neurons and with a small proportion of a neurochemical class of inhibitory neurons that are thought to have disinhibitory effect on excitatory neurons. This novel pathway from the anterior cingulate is poised to exert a powerful excitatory effect on the anterior olfactory nucleus, which is a critical hub for odorant processing via extensive bilateral connections with primary olfactory cortices and the olfactory bulb. Acting on the anterior olfactory nucleus, the anterior cingulate may activate the entire primary olfactory cortex to mediate the process of rapid attention to olfactory stimuli. PMID:23797208

  10. Ets homologous factor regulates pathways controlling response to injury in airway epithelial cells.

    PubMed

    Fossum, Sara L; Mutolo, Michael J; Yang, Rui; Dang, Hong; O'Neal, Wanda K; Knowles, Michael R; Leir, Shih-Hsing; Harris, Ann

    2014-12-16

    Ets homologous factor (EHF) is an Ets family transcription factor expressed in many epithelial cell types including those lining the respiratory system. Disruption of the airway epithelium is central to many lung diseases, and a network of transcription factors coordinates its normal function. EHF can act as a transcriptional activator or a repressor, though its targets in lung epithelial cells are largely uncharacterized. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq), showed that the majority of EHF binding sites in lung epithelial cells are intergenic or intronic and coincide with putative enhancers, marked by specific histone modifications. EHF occupies many genomic sites that are close to genes involved in intercellular and cell-matrix adhesion. RNA-seq after EHF depletion or overexpression showed significant alterations in the expression of genes involved in response to wounding. EHF knockdown also targeted genes in pathways of epithelial development and differentiation and locomotory behavior. These changes in gene expression coincided with alterations in cellular phenotype including slowed wound closure and increased transepithelial resistance. Our data suggest that EHF regulates gene pathways critical for epithelial response to injury, including those involved in maintenance of barrier function, inflammation and efficient wound repair. PMID:25414352

  11. The PDK1–Rsk Signaling Pathway Controls Langerhans Cell Proliferation and Patterning

    PubMed Central

    Zaru, Rossana; Matthews, Stephen P.; Edgar, Alexander J.; Prescott, Alan R.; Gomez-Nicola, Diego; Hanauer, André

    2015-01-01

    Langerhans cells (LC), the dendritic cells of the epidermis, are distributed in a distinctive regularly spaced array. In the mouse, the LC array is established in the first few days of life from proliferating local precursors, but the regulating signaling pathways are not fully understood. We found that mice lacking the kinase phosphoinositide-dependent kinase 1 selectively lack LC. Deletion of the phosphoinositide-dependent kinase 1 target kinases, ribosomal S6 kinase 1 (Rsk1) and Rsk2, produced a striking perturbation in the LC network: LC density was reduced 2-fold, but LC size was increased by the same magnitude. Reduced LC numbers in Rsk1/2−/− mice was not due to accelerated emigration from the skin but rather to reduced proliferation at least in adults. Rsk1/2 were required for normal LC patterning in neonates, but not when LC were ablated in adults and replaced by bone marrow–derived cells. Increased LC size was an intrinsic response to reduced LC numbers, reversible on LC emigration, and could be observed in wild type epidermis where LC size also correlated inversely with LC density. Our results identify a key signaling pathway needed to establish a normal LC network and suggest that LC might maintain epidermal surveillance by increasing their “footprint” when their numbers are limited. PMID:26401001

  12. Control of genetic stability by a new heterochromatin compaction pathway involving the Tip60 histone acetyltransferase

    PubMed Central

    Grézy, Aude; Chevillard-Briet, Martine; Trouche, Didier; Escaffit, Fabrice

    2016-01-01

    Pericentric heterochromatin is a highly compacted structure required for accurate chromosome segregation in mitosis. In mammals, it relies on methylation of histone H3K9 by Suv39H enzymes, which provides a docking site for HP1 proteins, therefore mediating heterochromatin compaction. Here we show that, when this normal compaction pathway is defective, the histone acetyltransferase Tip60 is recruited to pericentric heterochromatin, where it mediates acetylation of histone H4K12. Furthermore, in such a context, depletion of Tip60 leads to derepression of satellite transcription, decompaction of pericentric heterochromatin, and defects in chromosome segregation in mitosis. Finally, we show that depletion of BRD2, a double bromodomain–containing protein that binds H4K12ac, phenocopies the Tip60 depletion with respect to heterochromatin decompaction and defects in chromosome segregation. Taking the results together, we identify a new compaction pathway of mammalian pericentric heterochromatin relying on Tip60 that might be dependent on BRD2 recruitment by H4K12 acetylation. We propose that the underexpression of Tip60 observed in many human tumors can promote genetic instability via defective pericentric heterochromatin. PMID:26700317

  13. A new pathway for mitochondrial quality control: mitochondrial-derived vesicles

    PubMed Central

    Sugiura, Ayumu; McLelland, Gian-Luca; Fon, Edward A; McBride, Heidi M

    2014-01-01

    The last decade has been marked by tremendous progress in our understanding of the cell biology of mitochondria, with the identification of molecules and mechanisms that regulate their fusion, fission, motility, and the architectural transitions within the inner membrane. More importantly, the manipulation of these machineries in tissues has provided links between mitochondrial dynamics and physiology. Indeed, just as the proteins required for fusion and fission were identified, they were quickly linked to both rare and common human diseases. This highlighted the critical importance of this emerging field to medicine, with new hopes of finding drugable targets for numerous pathologies, from neurodegenerative diseases to inflammation and cancer. In the midst of these exciting new discoveries, an unexpected new aspect of mitochondrial cell biology has been uncovered; the generation of small vesicular carriers that transport mitochondrial proteins and lipids to other intracellular organelles. These mitochondrial-derived vesicles (MDVs) were first found to transport a mitochondrial outer membrane protein MAPL to a subpopulation of peroxisomes. However, other MDVs did not target peroxisomes and instead fused with the late endosome, or multivesicular body. The Parkinson's disease-associated proteins Vps35, Parkin, and PINK1 are involved in the biogenesis of a subset of these MDVs, linking this novel trafficking pathway to human disease. In this review, we outline what has been learned about the mechanisms and functional importance of MDV transport and speculate on the greater impact of these pathways in cellular physiology. PMID:25107473

  14. WGEF activates Rho in the Wnt–PCP pathway and controls convergent extension in Xenopus gastrulation

    PubMed Central

    Tanegashima, Kosuke; Zhao, Hui; Dawid, Igor B

    2008-01-01

    The Wnt–PCP (planar cell polarity, PCP) pathway regulates cell polarity and convergent extension movements during axis formation in vertebrates by activation of Rho and Rac, leading to the re-organization of the actin cytoskeleton. Rho and Rac activation require guanine nucleotide-exchange factors (GEFs), but the identity of the GEF involved in Wnt–PCP-mediated convergent extension is unknown. Here we report the identification of the weak-similarity GEF (WGEF) gene by a microarray-based screen for notochord enriched genes, and show that WGEF is involved in Wnt-regulated convergent extension. Overexpression of WGEF activated RhoA and rescued the suppression of convergent extension by dominant-negative Wnt-11, whereas depletion of WGEF led to suppression of convergent extension that could be rescued by RhoA or Rho-associated kinase activation. WGEF protein preferentially localized at the plasma membrane, and Frizzled-7 induced colocalization of Dishevelled and WGEF. WGEF protein can bind to Dishevelled and Daam-1, and deletion of the Dishevelled-binding domain generates a hyperactive from of WGEF. These results indicate that WGEF is a component of the Wnt–PCP pathway that connects Dishevelled to Rho activation. PMID:18256687

  15. WGEF activates Rho in the Wnt-PCP pathway and controls convergent extension in Xenopus gastrulation.

    PubMed

    Tanegashima, Kosuke; Zhao, Hui; Dawid, Igor B

    2008-02-20

    The Wnt-PCP (planar cell polarity, PCP) pathway regulates cell polarity and convergent extension movements during axis formation in vertebrates by activation of Rho and Rac, leading to the re-organization of the actin cytoskeleton. Rho and Rac activation require guanine nucleotide-exchange factors (GEFs), but the identity of the GEF involved in Wnt-PCP-mediated convergent extension is unknown. Here we report the identification of the weak-similarity GEF (WGEF) gene by a microarray-based screen for notochord enriched genes, and show that WGEF is involved in Wnt-regulated convergent extension. Overexpression of WGEF activated RhoA and rescued the suppression of convergent extension by dominant-negative Wnt-11, whereas depletion of WGEF led to suppression of convergent extension that could be rescued by RhoA or Rho-associated kinase activation. WGEF protein preferentially localized at the plasma membrane, and Frizzled-7 induced colocalization of Dishevelled and WGEF. WGEF protein can bind to Dishevelled and Daam-1, and deletion of the Dishevelled-binding domain generates a hyperactive from of WGEF. These results indicate that WGEF is a component of the Wnt-PCP pathway that connects Dishevelled to Rho activation. PMID:18256687

  16. An Abscisic Acid-Independent Oxylipin Pathway Controls Stomatal Closure and Immune Defense in Arabidopsis

    PubMed Central

    Mondy, Samuel; Tranchimand, Sylvain; Rumeau, Dominique; Boudsocq, Marie; Garcia, Ana Victoria; Douki, Thierry; Bigeard, Jean; Laurière, Christiane; Chevalier, Anne; Castresana, Carmen; Hirt, Heribert

    2013-01-01

    Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure is conveyed by both LOX1 and the mitogen-activated protein kinases MPK3 and MPK6 and involves salicylic acid whereas the ABA-induced process depends on the protein kinases OST1, MPK9, or MPK12. Finally, we show that the oxylipin and the ABA pathways converge at the level of the anion channel SLAC1 to regulate stomatal closure. Collectively, our results demonstrate that early biotic signaling in guard cells is an ABA-independent process revealing a novel function of LOX1-dependent stomatal pathway in plant immunity. PMID:23526882

  17. Computation of inverse functions in a model of cerebellar and reflex pathways allows to control a mobile mechanical segment.

    PubMed

    Ebadzadeh, M; Tondu, B; Darlot, C

    2005-01-01

    The command and control of limb movements by the cerebellar and reflex pathways are modeled by means of a circuit whose structure is deduced from functional constraints. One constraint is that fast limb movements must be accurate although they cannot be continuously controlled in closed loop by use of sensory signals. Thus, the pathways which process the motor orders must contain approximate inverse functions of the bio-mechanical functions of the limb and of the muscles. This can be achieved by means of parallel feedback loops, whose pattern turns out to be comparable to the anatomy of the cerebellar pathways. They contain neural networks able to anticipate the motor consequences of the motor orders, modeled by artificial neural networks whose connectivity is similar to that of the cerebellar cortex. These networks learn the direct biomechanical functions of the limbs and muscles by means of a supervised learning process. Teaching signals calculated from motor errors are sent to the learning sites, as, in the cerebellum, complex spikes issued from the inferior olive are conveyed to the Purkinje cells by climbing fibers. Learning rules are deduced by a differential calculation, as classical gradient rules, and they account for the long term depression which takes place in the dendritic arborizations of the Purkinje cells. Another constraint is that reflexes must not impede voluntary movements while remaining at any instant ready to oppose perturbations. Therefore, efferent copies of the motor orders are sent to the interneurones of the reflexes, where they cancel the sensory-motor consequences of the voluntary movements. After learning, the model is able to drive accurately, both in velocity and position, angular movements of a rod actuated by two pneumatic McKibben muscles. Reflexes comparable to the myotatic and tendinous reflexes, and stabilizing reactions comparable to the cerebellar sensory-motor reactions, reduce efficiently the effects of perturbing torques

  18. Gene Expression Profile of Adult Human Olfactory Bulb and Embryonic Neural Stem Cell Suggests Distinct Signaling Pathways and Epigenetic Control

    PubMed Central

    Marei, Hany E. S.; Ahmed, Abd-Elmaksoud; Michetti, Fabrizio; Pescatori, Mario; Pallini, Roberto; Casalbore, Patricia; Cenciarelli, Carlo; Elhadidy, Mohamed

    2012-01-01

    Global gene expression profiling was performed using RNA from human embryonic neural stem cells (hENSC), and adult human olfactory bulb-derived neural stem cells (OBNSCs), to define a gene expression pattern and signaling pathways that are specific for each cell lineage. We have demonstrated large differences in the gene expression profile of human embryonic NSC, and adult human OBNSCs, but less variability between parallel cultures. Transcripts of genes involved in neural tube development and patterning (ALDH1A2, FOXA2), progenitor marker genes (LMX1a, ALDH1A1, SOX10), proliferation of neural progenitors (WNT1 and WNT3a), neuroplastin (NPTN), POU3F1 (OCT6), neuroligin (NLGN4X), MEIS2, and NPAS1 were up-regulated in both cell populations. By Gene Ontology, 325 out of 3875 investigated gene sets were scientifically different. 41 out of the 307 investigated Cellular Component (CC) categories, 45 out of the 620 investigated Molecular Function (MF) categories, and 239 out of the 2948 investigated Biological Process (BP) categories were significant. KEGG Pathway Class Comparison had revealed that 75 out of 171 investigated gene sets passed the 0.005 significance threshold. Levels of gene expression were explored in three signaling pathways, Notch, Wnt, and mTOR that are known to be involved in NS cell fates determination. The transcriptional signature also deciphers the role of genes involved in epigenetic modifications. SWI/SNF DNA chromatin remodeling complex family, including SMARCC1 and SMARCE1, were found specifically up-regulated in our OBNSC but not in hENSC. Differences in gene expression profile of transcripts controlling epigenetic modifications, and signaling pathways might indicate differences in the therapeutic potential of our examined two cell populations in relation to in cell survival, proliferation, migration, and differentiation following engraftments in different CNS insults. PMID:22485144

  19. Mammalian TOR controls one of two kinase pathways acting upon nPKCdelta and nPKCepsilon.

    PubMed

    Parekh, D; Ziegler, W; Yonezawa, K; Hara, K; Parker, P J

    1999-12-01

    There are three conserved phosphorylation sites in protein kinase C (PKC) isotypes that have been termed priming sites and play an important role in PKC function. The requirements and pathways involved in novel (nPKC) phosphorylation have been investigated here. The evidence presented for nPKCdelta shows that there are two independent kinase pathways that act upon the activation loop (Thr-505) and a C-terminal hydrophobic site (Ser-662) and that the phosphorylation of the Ser-662 site is protected from dephosphorylation by the Thr-505 phosphorylation. Both phosphorylations require C1 domain-dependent allosteric activation of PKC. The third site (Ser-643) appears to be an autophosphorylation site. The serum-dependent phosphorylation of the Thr-505 and Ser-662 sites increases nPKCdelta activity up to 80-fold. Phosphorylation at the Ser-662 site is independently controlled by a pathway involving mammalian TOR (mTOR) because the rapamycin-induced block of its phosphorylation is overcome by co-expression of a rapamycin-resistant mutant of mTOR. Consistent with this role of mTOR, amino acid deprivation selectively inhibits the serum-induced phosphorylation of the Ser-662 site in nPKCdelta. It is established that nPKCepsilon behaves in a manner similar to nPKCdelta with respect to phosphorylation at its C-terminal hydrophobic site, Ser-729. The results define the regulatory inputs to nPKCdelta and nPKCepsilon and establish these PKC isotypes downstream of mTOR and on an amino acid sensing pathway. The multiple signals integrated in PKC are discussed. PMID:10574945

  20. Mitotic Asynchrony Induces Transforming Growth Factor-β1 Secretion from Airway Epithelium

    PubMed Central

    Alcala, Sarah E.; Benton, Angela S.; Watson, Alan M.; Kureshi, Suraiya; Reeves, Erica M. K.; Damsker, Jesse; Wang, Zuyi; Nagaraju, Kanneboyina; Anderson, Julia; Williams, Aaron M.; Lee, Amber J. Y.; Hayes, Kathleen; Rose, Mary C.; Hoffman, Eric P.

    2014-01-01

    We recently proposed that mitotic asynchrony in repairing tissue may underlie chronic inflammation and fibrosis, where immune cell infiltration is secondary to proinflammatory cross-talk among asynchronously repairing adjacent tissues. Building on our previous finding that mitotic asynchrony is associated with proinflammatory/fibrotic cytokine secretion (e.g., transforming growth factor [TGF]-β1), here we provide evidence supporting cause-and-effect. Under normal conditions, primary airway epithelial basal cell populations undergo mitosis synchronously and do not secrete proinflammatory or profibrotic cytokines. However, when pairs of nonasthmatic cultures were mitotically synchronized at 12 hours off-set and then combined, the mixed cell populations secreted elevated levels of TGF-β1. This shows that mitotic asynchrony is not only associated with but is also causative of TGF-β1 secretion. The secreted cytokines and other mediators from asthmatic cells were not the cause of asynchronous regeneration; synchronously mitotic nonasthmatic epithelia exposed to conditioned media from asthmatic cells did not show changes in mitotic synchrony. We also tested if resynchronization of regenerating asthmatic airway epithelia reduces TGF-β1 secretion and found that pulse-dosed dexamethasone, simvastatin, and aphidicolin were all effective. We therefore propose a new model for chronic inflammatory and fibrotic conditions where an underlying factor is mitotic asynchrony. PMID:24669775

  1. Efficient Activation of Apoptotic Signaling during Mitotic Arrest with AK301

    PubMed Central

    Bleiler, Marina; Yeagley, Michelle; Wright, Dennis; Giardina, Charles

    2016-01-01

    Mitotic inhibitors are widely utilized chemotherapeutic agents that take advantage of mitotic defects in cancer cells. We have identified a novel class of piperazine-based mitotic inhibitors, of which AK301 is the most potent derivative identified to date (EC50 < 200 nM). Colon cancer cells arrested in mitosis with AK301 readily underwent a p53-dependent apoptosis following compound withdrawal and arrest release. This apoptotic response was significantly higher for AK301 than for other mitotic inhibitors tested (colchicine, vincristine, and BI 2536). AK301-treated cells exhibited a robust mitosis-associated DNA damage response, including ATM activation, γH2AX phosphorylation and p53 stabilization. The association between mitotic signaling and the DNA damage response was supported by the finding that Aurora B inhibition reduced the level of γH2AX staining. Confocal imaging of AK301-treated cells revealed multiple γ-tubulin microtubule organizing centers attached to microtubules, but with limited centrosome migration, raising the possibility that aberrant microtubule pulling may underlie DNA breakage. AK301 selectively targeted APC-mutant colonocytes and promoted TNF-induced apoptosis in p53-mutant colon cancer cells. Our findings indicate that AK301 induces a mitotic arrest state with a highly active DNA damage response. Together with a reversible arrest state, AK301 is a potent promoter of a mitosis-to-apoptosis transition that can target cancer cells with mitotic defects. PMID:27097159

  2. Taking control over control: use of product sensing in single cells to remove flux control at key enzymes in biosynthesis pathways.

    PubMed

    Schendzielorz, Georg; Dippong, Martin; Grünberger, Alexander; Kohlheyer, Dietrich; Yoshida, Ayako; Binder, Stephan; Nishiyama, Chiharu; Nishiyama, Makoto; Bott, Michael; Eggeling, Lothar

    2014-01-17

    Enzymes initiating the biosynthesis of cellular building blocks are frequently inhibited by the end-product of the respective pathway. Here we present an approach to rapidly generate sets of enzymes overriding this control. It is based on the in vivo detection of the desired end-product in single cells using a genetically encoded sensor. The sensor transmits intracellular product concentrations into a graded optical output, thus enabling ultrahigh-throughput screens by FACS. We randomly mutagenized plasmid-encoded ArgB of Corynebacterium glutamicum and screened the library in a strain carrying the sensor pSenLys-Spc, which detects l-lysine, l-arginine and l-histidine. Six of the resulting N-acetyl-l-glutamate kinase proteins were further developed and characterized and found to be at least 20-fold less sensitive toward l-arginine inhibition than the wild-type enzyme. Overexpression of the mutein ArgB-K47H-V65A in C. glutamicumΔargR led to the accumulation of 34 mM l-arginine in the culture medium. We also screened mutant libraries of lysC-encoded aspartate kinase and hisG-encoded ATP phosphoribosyltransferase. We isolated 11 LysC muteins, enabling up to 45 mM l-lysine accumulation, and 13 HisG muteins, enabling up to 17 mM l-histidine accumulation. These results demonstrate that in vivo screening of enzyme libraries by using metabolite sensors is extremely well suited to identify high-performance muteins required for overproduction. PMID:23829416

  3. Small ubiquitin-related modifier ligase activity of Mms21 is required for maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in Saccharomyces cerevisiae.

    PubMed

    Rai, Ragini; Varma, Satya P M V; Shinde, Nikhil; Ghosh, Shilpa; Kumaran, Srikala P; Skariah, Geena; Laloraya, Shikha

    2011-04-22

    The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast. PMID:21324902

  4. Calbindin and parvalbumin are early markers of non-mitotically regenerating hair cells in the bullfrog vestibular otolith organs

    NASA Technical Reports Server (NTRS)

    Steyger, P. S.; Burton, M.; Hawkins, J. R.; Schuff, N. R.; Baird, R. A.

    1997-01-01

    Earlier studies have demonstrated hair cell regeneration in the absence of cell proliferation, and suggested that supporting cells could phenotypically convert into hair cells following hair cell loss. Because calcium-binding proteins are involved in gene up-regulation, cell growth, and cell differentiation, we wished to determine if these proteins were up-regulated in scar formations and regenerating hair cells following gentamicin treatment. Calbindin and parvalbumin immunolabeling was examined in control or gentamicin-treated (GT) bullfrog saccular and utricular explants cultured for 3 days in amphibian culture medium or amphibian culture medium supplemented with aphidicolin, a blocker of nuclear DNA replication in eukaryotic cells. In control cultures, calbindin and parvalbumin immunolabeled the hair bundles and, less intensely, the cell bodies of mature hair cells. In GT or mitotically-blocked GT (MBGT) cultures, calbindin and parvalbumin immunolabeling was also seen in the hair bundles, cuticular plates, and cell bodies of hair cells with immature hair bundles. Thus, these antigens were useful markers for both normal and regenerating hair cells. Supporting cell immunolabeling was not seen in control cultures nor in the majority of supporting cells in GT cultures. In MBGT cultures, calbindin and parvalbumin immunolabeling was up-regulated in the cytosol of single supporting cells participating in scar formations and in supporting cells with hair cell-like characteristics. These data provide further evidence that non-mitotic hair cell regeneration in cultures can be accomplished by the conversion of supporting cells into hair cells.

  5. The EGFR signaling pathway controls gut progenitor differentiation during planarian regeneration and homeostasis.

    PubMed

    Barberán, Sara; Fraguas, Susanna; Cebrià, Francesc

    2016-06-15

    The planarian Schmidtea mediterranea maintains and regenerates all its adult tissues through the proliferation and differentiation of a single population of pluripotent adult stem cells (ASCs) called neoblasts. Despite recent advances, the mechanisms regulating ASC differentiation into mature cell types are poorly understood. Here, we show that silencing of the planarian EGF receptor egfr-1 by RNA interference (RNAi) impairs gut progenitor differentiation into mature cells, compromising gut regeneration and maintenance. We identify a new putative EGF ligand, nrg-1, the silencing of which phenocopies the defects observed in egfr-1(RNAi) animals. These findings indicate that egfr-1 and nrg-1 promote gut progenitor differentiation, and are thus essential for normal cell turnover and regeneration in the planarian gut. Our study demonstrates that the EGFR signaling pathway is an important regulator of ASC differentiation in planarians. PMID:27122174

  6. PPARs in Regulation of Paraoxonases: Control of Oxidative Stress and Inflammation Pathways

    PubMed Central

    Camps, Jordi; García-Heredia, Anabel; Rull, Anna; Alonso-Villaverde, Carlos; Aragonès, Gerard; Beltrán-Debón, Raúl; Rodríguez-Gallego, Esther; Joven, Jorge

    2012-01-01

    The paraoxonase (PON) group of enzymes, composed of PON1, PON2, and PON3, play an important role in decreasing oxidative stress by degrading lipid peroxides. PON1 synthesis is upregulated by PPAR. Several pharmacological compounds (acting as antioxidants and, hence, atheroprotective) stimulate both PPAR activity and PON1 expression. Recent evidence suggests that PON1 and the monocyte chemoattractant protein-1 (MCP-1) are involved in coordinating the inflammatory response in damaged tissues; PPAR may be central in the regulation of these biochemical pathways. This article reviews the state of knowledge on PON1 biochemistry and function, the influence of genetic variation, and the regulation of PON1 expression by pharmaceutical compounds that increase PPAR activity. We also describe recent lines of evidence suggesting links between PON1 and MCP-1 and how their production may be regulated by PPAR. PMID:22315585

  7. A combinatorial F box protein directed pathway controls TRAF adaptor stability to regulate inflammation.

    PubMed

    Chen, Bill B; Coon, Tiffany A; Glasser, Jennifer R; McVerry, Bryan J; Zhao, Jing; Zhao, Yutong; Zou, Chunbin; Ellis, Bryon; Sciurba, Frank C; Zhang, Yingze; Mallampalli, Rama K

    2013-05-01

    Uncontrolled activation of tumor necrosis factor receptor-associated factor (TRAF) proteins may result in profound tissue injury by linking surface signals to cytokine release. Here we show that a ubiquitin E3 ligase component, Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by destabilizing a sentinel TRAF inhibitor, Fbxl2. Fbxo3 and TRAF protein in circulation positively correlated with cytokine responses in subjects with sepsis, and we identified a polymorphism in human Fbxo3, with one variant being hypofunctional. A small-molecule inhibitor targeting Fbxo3 was sufficient to lessen severity of cytokine-driven inflammation in several mouse disease models. These studies identified a pathway of innate immunity that may be useful to detect subjects with altered immune responses during critical illness or provide a basis for therapeutic intervention targeting TRAF protein abundance. PMID:23542741

  8. Utilising conservative tracers and spatial surveys to identify controls on pathways and DOC exports in an Arctic catchment.

    NASA Astrophysics Data System (ADS)

    Lessels, J. S.; Tetzlaff, D.; Dinsmore, K. J.; Street, L. E.; Dean, J.; Washbourne, I. J.; Billett, M. F.; Baxter, R.; Subke, J. A.; Wookey, P. A.

    2014-12-01

    Dissolved organic carbon (DOC) is typically the predominant form of carbon exported from headwater streams, it therefore represents a major carbon export from Arctic catchments. The projected deepening of thaw depth in permafrost regions, due to an increase in air temperature, may have a significant effect on the amount of DOC exported from these systems. However, quantification of the impacts of climate driven changes on DOC export are still highly uncertain. Understanding the processes controlling DOC export is therefore crucial in predicting the potential impact of projected environmental changes. The controls of DOC production and transport are heavily influenced by soil and vegetation, which are highly variable across the landscape. To completely understand these systems information regarding spatial variability of plants, soils and thaw depths must be taken into account. In this study sub-weekly sampling of DOC was undertaken throughout 2014 in a headwater (<1 km2) catchment in the Northwest Territories, Canada. Spatial surveys of soil properties, active thaw depth and normalised difference vegetation index (NDVI) were collected and used in conjunction with conservative stable water isotopes tracers and major ions to understand sources, flow pathways and timing of DOC exports from the catchment. Stable isotope tracers act as fingerprints of water allowing sources and pathways to be assessed. Observations reveal changing DOC concentrations throughout the season as the active layer deepens and the connectivity of the soils to the stream network throughout the catchment increases. Linking the DOC data with the conservative tracer response improves the identification of carbon pathways and fluxes from the soils; preliminary analysis indicates DOC is being delivered via deeper more mineral soils later in the season. The results indicate that the active layer depth has a strong influence on the amount of DOC exported from the system, independent of the amount of

  9. Transformation, products, and pathways of chlorophenols via electro-enzymatic catalysis: How to control toxic intermediate products.

    PubMed

    Du, Penghui; Zhao, He; Li, Haitao; Zhang, Di; Huang, Ching-Hua; Deng, Manfeng; Liu, Chenming; Cao, Hongbin

    2016-02-01

    Chlorophenols can be easily oxidized into chlorobenzoquinones (CBQs), which are highly toxic and have been linked to bladder cancer risk. Herein, we report the transformation, products, and pathways of 2,4-dichlorophenol (DCP) by horseradish peroxidase (HRP) and electro-generated hydrogen peroxide (H2O2) and suggest methods to control the formation of toxic intermediate products. After a 10-min electroenzymatic process, 99.7% DCP removal may be achieved under optimal conditions. A total of 16 reaction products, most of which are subsequently verified as DCP polymers and related quinone derivatives, are identified by using ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOF-MS). A five-step reaction pathway for DCP transformation, including HRP-driven substrate oxidation, substitution and radical coupling, quick redox equilibrium, nucleophilic reaction and precipitation from aqueous solution, is proposed. Current variations and the presence of CO2 could significantly affect these reaction pathways. In particular, higher currents enhance the hydroxylation process by promoting alkaline conditions and abundant H2O2 formation. As both OH(-) and H2O2 are strong nucleophiles, they easily react with CBQ products to form hydroxylated products, which can significantly reduce solution toxicity. An adequate supply of CO2 can provide favorable pH conditions and facilitate enzymatic steps, such as substrate oxidation and radical coupling, to generate precipitable polymerized products. All of the results suggest that toxic intermediate products can be effectively reduced and controlled during the electro-enzymatic process to remove DCP and other phenolic pollutants from wastewaters. PMID:26519798

  10. The Azospirillum brasilense Che1 Chemotaxis Pathway Controls Swimming Velocity, Which Affects Transient Cell-to-Cell Clumping

    PubMed Central

    Bible, Amber; Russell, Matthew H.

    2012-01-01

    The Che1 chemotaxis-like pathway of Azospirillum brasilense contributes to chemotaxis and aerotaxis, and it has also been found to contribute to regulating changes in cell surface adhesive properties that affect the propensity of cells to clump and to flocculate. The exact contribution of Che1 to the control of chemotaxis and flocculation in A. brasilense remains poorly understood. Here, we show that Che1 affects reversible cell-to-cell clumping, a cellular behavior in which motile cells transiently interact by adhering to one another at their nonflagellated poles before swimming apart. Clumping precedes and is required for flocculation, and both processes appear to be independently regulated. The phenotypes of a ΔaerC receptor mutant and of mutant strains lacking cheA1, cheY1, cheB1, or cheR1 (alone or in combination) or with che1 deleted show that Che1 directly mediates changes in the flagellar swimming velocity and that this behavior directly modulates the transient nature of clumping. Our results also suggest that an additional receptor(s) and signaling pathway(s) are implicated in mediating other Che1-independent changes in clumping identified in the present study. Transient clumping precedes the transition to stable clump formation, which involves the production of specific extracellular polysaccharides (EPS); however, production of these clumping-specific EPS is not directly controlled by Che1 activity. Che1-dependent clumping may antagonize motility and prevent chemotaxis, thereby maintaining cells in a metabolically favorable niche. PMID:22522896

  11. Nitrite Control over Dissimilatory Nitrate/Nitrite Reduction Pathways in Shewanella loihica Strain PV-4

    PubMed Central

    Sanford, Robert A.

    2015-01-01

    Shewanella loihica strain PV-4 harbors both a functional denitrification (NO3−→N2) and a respiratory ammonification (NO3−→NH4+) pathway. Batch and chemostat experiments revealed that NO2− affects pathway selection and the formation of reduced products. Strain PV-4 cells grown with NO2− as the sole electron acceptor produced exclusively NH4+. With NO3− as the electron acceptor, denitrification predominated and N2O accounted for ∼90% of reduced products in the presence of acetylene. Chemostat experiments demonstrated that the NO2−:NO3− ratio affected the distribution of reduced products, and respiratory ammonification dominated at high NO2−:NO3− ratios, whereas low NO2−:NO3− ratios favored denitrification. The NO2−:NO3− ratios affected nirK transcript abundance, a measure of denitrification activity, in the chemostat experiments, and cells grown at a NO2−:NO3− ratio of 3 had ∼37-fold fewer nirK transcripts per cell than cells grown with NO3− as the sole electron acceptor. In contrast, the transcription of nrfA, implicated in NO2−-to-NH4+ reduction, remained statistically unchanged under continuous cultivation conditions at NO2−:NO3− ratios below 3. At NO2−:NO3− ratios above 3, both nirK and nrfA transcript numbers decreased and the chemostat culture washed out, presumably due to NO2− toxicity. These findings implicate NO2− as a relevant modulator of NO3− fate in S. loihica strain PV-4, and, by extension, suggest that NO2− is a relevant determinant for N retention (i.e., ammonification) versus N loss and greenhouse gas emission (i.e., denitrification). PMID:25769828

  12. Randomised controlled trial of GP-led in-hospital management of homeless people ('Pathway').

    PubMed

    Hewett, Nigel; Buchman, Peter; Musariri, Jeflyn; Sargeant, Christopher; Johnson, Penny; Abeysekera, Kushala; Grant, Louise; Oliver, Emily A; Eleftheriades, Christopher; McCormick, Barry; Halligan, Aidan; Marlin, Nadine; Kerry, Sally; Foster, Graham R

    2016-06-01

    Homeless people have complex problems. GP enhanced care (Pathway) has shown benefits. We performed a randomised, -parallel arm trial at two large inner city hospitals. Inpatient homeless adults were randomly allocated to either standard care (all management by the hospital-based clinical team) or enhanced care with input from a homeless care team. The hospital data system provided healthcare usage information, and we used questionnaires to assess quality of life. 206 patients were allocated to enhanced care and 204 to usual care. Length of stay (up to 90 days after admission) did not differ between groups (standard care 14.0 days, enhanced care 13.3 days). Average reattendance at the emergency department within a year was 5.8 visits in the standard care group and 4.8 visits with enhanced care, but this decrease was not significant. -Quality of life scores after discharge (in 108 patients) improved with enhanced care (EQ-5D-5L score increased by 0.12 [95% CI 0.032 to 0.22] compared wtih 0.03 [-0.1 to 0.15; p=0.076] with standard care). The proportion of people sleeping on the streets after discharge was 14.6% in the standard care arm and 3.8% in the enhanced care arm (p=0.034). The quality-of-life cost per quality-adjusted life-year was £26,000. The Pathway approach doesn't alter length of stay but improves quality of life and reduces street -homelessness. PMID:27251910

  13. The Novel Smad Protein Expansion Regulates Receptor Tyrosine Kinase Pathway to Control Drosophila Tracheal Tube Size

    PubMed Central

    Iordanou, Ekaterini; Chandran, Rachana R.; Yang, Yonghua; Essak, Mina; Blackstone, Nicholas; Jiang, Lan

    2014-01-01

    Tubes with distinct shapes and sizes are critical for the proper function of many tubular organs. Here we describe a unique phenotype caused by the loss of a novel, evolutionarily-conserved, Drosophila Smad-like protein, Expansion. In expansion mutants, unicellular and intracellular tracheal branches develop bubble-like cysts with enlarged apical membranes. Cysts in unicellular tubes are enlargements of the apical lumen, whereas cysts in intracellular tubes are cytoplasmic vacuole-like compartments. The cyst phenotype in expansion mutants is similar to, but weaker than, that observed in double mutants of Drosophila type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. Ptp4E and Ptp10D negatively regulate the receptor tyrosine kinase (RTK) pathways, especially epithelial growth factor receptor (EGFR) and fibroblast growth factor receptor/breathless (FGFR, Btl) signaling to maintain the proper size of unicellular and intracellular tubes. We show Exp genetically interacts with RTK signaling, the downstream targets of RPTPs. Cyst size and number in expansion mutants is enhanced by increased RTK signaling and suppressed by reduced RTK signaling. Genetic interaction studies strongly suggest that Exp negatively regulates RTK (EGFR, Btl) signaling to ensure proper tube sizes. Smad proteins generally function as intermediate components of the transforming growth factor-β (TGF-β, DPP) signaling pathway. However, no obvious genetic interaction between expansion and TGF-β (DPP) signaling was observed. Therefore, Expansion does not function as a typical Smad protein. The expansion phenotype demonstrates a novel role for Smad-like proteins in epithelial tube formation. PMID:24973580

  14. Functional convergence of oxylipin and abscisic acid pathways controls stomatal closure in response to drought.

    PubMed

    Savchenko, Tatyana; Kolla, Venkat A; Wang, Chang-Quan; Nasafi, Zainab; Hicks, Derrick R; Phadungchob, Bpantamars; Chehab, Wassim E; Brandizzi, Federica; Froehlich, John; Dehesh, Katayoon

    2014-03-01

    Membranes are primary sites of perception of environmental stimuli. Polyunsaturated fatty acids are major structural constituents of membranes that also function as modulators of a multitude of signal transduction pathways evoked by environmental stimuli. Different stresses induce production of a distinct blend of oxygenated polyunsaturated fatty acids, "oxylipins." We employed three Arabidopsis (Arabidopsis thaliana) ecotypes to examine the oxylipin signature in response to specific stresses and determined that wounding and drought differentially alter oxylipin profiles, particularly the allene oxide synthase branch of the oxylipin pathway, responsible for production of jasmonic acid (JA) and its precursor 12-oxo-phytodienoic acid (12-OPDA). Specifically, wounding induced both 12-OPDA and JA levels, whereas drought induced only the precursor 12-OPDA. Levels of the classical stress phytohormone abscisic acid (ABA) were also mainly enhanced by drought and little by wounding. To explore the role of 12-OPDA in plant drought responses, we generated a range of transgenic lines and exploited the existing mutant plants that differ in their levels of stress-inducible 12-OPDA but display similar ABA levels. The plants producing higher 12-OPDA levels exhibited enhanced drought tolerance and reduced stomatal aperture. Furthermore, exogenously applied ABA and 12-OPDA, individually or combined, promote stomatal closure of ABA and allene oxide synthase biosynthetic mutants, albeit most effectively when combined. Using tomato (Solanum lycopersicum) and Brassica napus verified the potency of this combination in inducing stomatal closure in plants other than Arabidopsis. These data have identified drought as a stress signal that uncouples the conversion of 12-OPDA to JA and have revealed 12-OPDA as a drought-responsive regulator of stomatal closure functioning most effectively together with ABA. PMID:24429214

  15. Nitrite Control over Dissimilatory Nitrate/Nitrite Reduction Pathways in Shewanella loihica Strain PV-4.

    PubMed

    Yoon, Sukhwan; Sanford, Robert A; Löffler, Frank E

    2015-05-15

    Shewanella loihica strain PV-4 harbors both a functional denitrification (NO3 (-)→N2) and a respiratory ammonification (NO3 (-)→NH4 (+)) pathway. Batch and chemostat experiments revealed that NO2 (-) affects pathway selection and the formation of reduced products. Strain PV-4 cells grown with NO2 (-) as the sole electron acceptor produced exclusively NH4 (+). With NO3 (-) as the electron acceptor, denitrification predominated and N2O accounted for ∼90% of reduced products in the presence of acetylene. Chemostat experiments demonstrated that the NO2 (-):NO3 (-) ratio affected the distribution of reduced products, and respiratory ammonification dominated at high NO2 (-):NO3 (-) ratios, whereas low NO2 (-):NO3 (-) ratios favored denitrification. The NO2 (-):NO3 (-) ratios affected nirK transcript abundance, a measure of denitrification activity, in the chemostat experiments, and cells grown at a NO2 (-):NO3 (-) ratio of 3 had ∼37-fold fewer nirK transcripts per cell than cells grown with NO3 (-) as the sole electron acceptor. In contrast, the transcription of nrfA, implicated in NO2 (-)-to-NH4 (+) reduction, remained statistically unchanged under continuous cultivation conditions at NO2 (-):NO3 (-) ratios below 3. At NO2 (-):NO3 (-) ratios above 3, both nirK and nrfA transcript numbers decreased and the chemostat culture washed out, presumably due to NO2 (-) toxicity. These findings implicate NO2 (-) as a relevant modulator of NO3 (-) fate in S. loihica strain PV-4, and, by extension, suggest that NO2 (-) is a relevant determinant for N retention (i.e., ammonification) versus N loss and greenhouse gas emission (i.e., denitrification). PMID:25769828

  16. Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance

    PubMed Central

    Malo, Mackenzie E.; Postnikoff, Spike D.L.; Arnason, Terra G.; Harkness, Troy A.A.

    2016-01-01

    The Saccharomyces cerevisiae Forkhead Box (Fox) orthologs, Forkheads (Fkh) 1 and 2, are conserved transcription factors required for stress response, cell cycle progression and longevity. These yeast proteins play a key role in mitotic progression through activation of the ubiquitin E3 ligase Anaphase Promoting Complex (APC) via transcriptional control. Here, we used genetic and molecular analyses to demonstrate that the APC E3 activity is necessary for mitotic Fkh1 protein degradation and subsequent cell cycle progression. We report that Fkh1 protein degradation occurs specifically during mitosis, requires APCCdc20 and proteasome activity, and that a stable Fkh1 mutant reduces normal chronological lifespan, increases genomic instability, and increases sensitivity to stress. Our data supports a model whereby cell cycle progression through mitosis and G1 requires the targeted degradation of Fkh1 by the APC. This is significant to many fields as these results impact our understanding of the mechanisms underpinning the control of aging and cancer. PMID:27099939

  17. TSC1 controls IL-1β expression in macrophages via mTORC1-dependent C/EBPβ pathway.

    PubMed

    Yang, Tao; Zhu, Linnan; Zhai, Yanhua; Zhao, Qingjie; Peng, Jianxia; Zhang, Hongbing; Yang, Zhongzhou; Zhang, Lianfeng; Ding, Wenjun; Zhao, Yong

    2016-09-01

    The tuberous sclerosis complex 1 (TSC1) is a tumor suppressor that inhibits the mammalian target of rapamycin (mTOR), which serves as a key regulator of inflammatory responses after bacterial stimulation in monocytes, macrophages, and primary dendritic cells. Previous studies have shown that TSC1 knockout (KO) macrophages produced increased inflammatory responses including tumor necrosis factor-α (TNF-α) and IL-12 to pro-inflammatory stimuli, but whether and how TSC1 regulates pro-IL-1β expression remains unclear. Here using a mouse model in which myeloid lineage-specific deletion of TSC1 leads to constitutive mTORC1 activation, we found that TSC1 deficiency resulted in impaired expression of pro-IL-1β in macrophages following lipopolysaccharide stimulation. Such decreased pro-IL-1β expression in TSC1 KO macrophages was rescued by reducing mTORC1 activity with rapamycin or deletion of mTOR. Rictor deficiency has no detectable effect on pro-IL-1β synthesis, suggesting that TSC1 positively controls pro-IL-1β expression through mTORC1 pathway. Moreover, mechanism studies suggest that mTORC1-mediated downregulation of the CCAAT enhancer-binding protein (C/EBPβ) critically contributes to the defective pro-IL-1β expression. Overall, these findings highlight a critical role of TSC1 in regulating innate immunity by control of the mTOR1-C/EBPβ pathway. PMID:27593484

  18. The complex STATes of astrocyte reactivity: How are they controlled by the JAK-STAT3 pathway?

    PubMed

    Ceyzériat, Kelly; Abjean, Laurene; Carrillo-de Sauvage, María-Angeles; Ben Haim, Lucile; Escartin, Carole

    2016-08-25

    Astrocytes play multiple important roles in brain physiology. In pathological conditions, they become reactive, which is characterized by morphological changes and upregulation of intermediate filament proteins. Besides these descriptive hallmarks, astrocyte reactivity involves significant transcriptional and functional changes that are far from being fully understood. Most importantly, astrocyte reactivity seems to encompass multiple states, each having a specific influence on surrounding cells and disease progression. These diverse functional states of reactivity must be regulated by subtle signaling networks. Many signaling cascades have been associated with astrocyte reactivity, but among them, the JAK-STAT3 pathway is emerging as a central regulator. In this review, we aim (i) to show that the JAK-STAT3 pathway plays a key role in the control of astrocyte reactivity, (ii) to illustrate that STAT3 is a pleiotropic molecule operating multiple functions in reactive astrocytes, and (iii) to suggest that each specific functional state of reactivity is governed by complex molecular interactions within astrocytes, which converge on STAT3. More research is needed to precisely identify the signaling networks controlling the diverse states of astrocyte reactivity. Only then, we will be able to precisely delineate the therapeutic potential of reactive astrocytes in each neurological disease context. PMID:27241943

  19. The insulator protein BEAF-32 is required for Hippo pathway activity in the terminal differentiation of neuronal subtypes.

    PubMed

    Jukam, David; Viets, Kayla; Anderson, Caitlin; Zhou, Cyrus; DeFord, Peter; Yan, Jenny; Cao, Jinshuai; Johnston, Robert J

    2016-07-01

    The Hippo pathway is crucial for not only normal growth and apoptosis but also cell fate specification during development. What controls Hippo pathway activity during cell fate specification is incompletely understood. In this article, we identify the insulator protein BEAF-32 as a regulator of Hippo pathway activity in Drosophila photoreceptor differentiation. Though morphologically uniform, the fly eye is composed of two subtypes of R8 photoreceptor neurons defined by expression of light-detecting Rhodopsin proteins. In one R8 subtype, active Hippo signaling induces Rhodopsin 6 (Rh6) and represses Rhodopsin 5 (Rh5), whereas in the other subtype, inactive Hippo signaling induces Rh5 and represses Rh6. The activity state of the Hippo pathway in R8 cells is determined by the expression of warts, a core pathway kinase, which interacts with the growth regulator melted in a double-negative feedback loop. We show that BEAF-32 is required for expression of warts and repression of melted Furthermore, BEAF-32 plays a second role downstream of Warts to induce Rh6 and prevent Rh5 fate. BEAF-32 is dispensable for Warts feedback, indicating that BEAF-32 differentially regulates warts and Rhodopsins. Loss of BEAF-32 does not noticeably impair the functions of the Hippo pathway in eye growth regulation. Our study identifies a context-specific regulator of Hippo pathway activity in post-mitotic neuronal fate, and reveals a developmentally specific role for a broadly expressed insulator protein. PMID:27226322

  20. Integration of Light Signals by the Retinoblastoma Pathway in the Control of S Phase Entry in the Picophytoplanktonic Cell Ostreococcus

    PubMed Central

    Moulager, Mickael; Corellou, Florence; Vergé, Valérie; Escande, Marie-Line; Bouget, François-Yves

    2010-01-01

    Although the decision to proceed through cell division depends largely on the metabolic status or the size of the cell, the timing of cell division is often set by internal clocks such as the circadian clock. Light is a major cue for circadian clock entrainment, and for photosynthetic organisms it is also the main source of energy supporting cell growth prior to cell division. Little is known about how light signals are integrated in the control of S phase entry. Here, we present an integrated study of light-dependent regulation of cell division in the marine green alga Ostreococcus. During early G1, the main genes of cell division were transcribed independently of the amount of light, and the timing of S phase did not occur prior to 6 hours after dawn. In contrast S phase commitment and the translation of a G1 A-type cyclin were dependent on the amount of light in a cAMP–dependent manner. CyclinA was shown to interact with the Retinoblastoma (Rb) protein during S phase. Down-regulating Rb bypassed the requirement for CyclinA and cAMP without altering the timing of S phase. Overexpression of CyclinA overrode the cAMP–dependent control of S phase entry and led to early cell division. Therefore, the Rb pathway appears to integrate light signals in the control of S phase entry in Ostreococcus, though differential transcriptional and posttranscriptional regulations of a G1 A-type cyclin. Furthermore, commitment to S phase depends on a cAMP pathway, which regulates the synthesis of CyclinA. We discuss the relative involvements of the metabolic and time/clock signals in the photoperiodic control of cell division. PMID:20502677

  1. A sperm-activating peptide controls a cGMP-signaling pathway in starfish sperm.

    PubMed

    Matsumoto, Midori; Solzin, Johannes; Helbig, Annika; Hagen, Volker; Ueno, Sei-ichi; Kawase, Osamu; Maruyama, Yoshinori; Ogiso, Manabu; Godde, Matthias; Minakata, Hiroyuki; Kaupp, U Benjamin; Hoshi, Motonori; Weyand, Ingo

    2003-08-15

    Peptides released from eggs of marine invertebrates play a central role in fertilization. About 80 different peptides from various phyla have been isolated, however, with one exception, their respective receptors on the sperm surface have not been unequivocally identified and the pertinent signaling pathways remain ill defined. Using rapid mixing techniques and novel membrane-permeable caged compounds of cyclic nucleotides, we show that the sperm-activating peptide asterosap evokes a fast and transient increase of the cGMP concentration in sperm of the starfish Asterias amurensis, followed by a transient cGMP-stimulated increase in the Ca(2+) concentration. In contrast, cAMP levels did not change significantly and the Ca(2+) response evoked by photolysis of caged cAMP was significantly smaller than that using caged cGMP. By cloning of cDNA and chemical crosslinking, we identified a receptor-type guanylyl cyclase in the sperm flagellum as the asterosap-binding protein. Sperm respond exquisitely sensitive to picomolar concentrations of asterosap, suggesting that the peptide serves a chemosensory function like resact, a peptide involved in chemotaxis of sperm of the sea urchin Arbacia punctulata. A unifying principle emerges that chemosensory transduction in sperm of marine invertebrates uses cGMP as the primary messenger, although there may be variations in the detail. PMID:12921734

  2. Calorie restriction and methionine restriction in control of endogenous hydrogen sulfide production by the transsulfuration pathway.

    PubMed

    Hine, Christopher; Mitchell, James R

    2015-08-01

    H2S is a gas easily identified by its distinctive odor. Although environmental exposure to H2S has been viewed alternately as therapeutic or toxic through the centuries, H2S has recently regained recognition for its numerous beneficial biological effects. Most experiments documenting such benefits, including improved glucose tolerance, increased stress resistance, and even lifespan extension, are based on exposure of experimental organisms to exogenous sources of H2S. However, appreciation is growing for the importance of H2S produced endogenously by the evolutionary conserved transsulfuration pathway (TSP) in health and longevity. Recent data implicate H2S produced by the TSP in pleiotropic benefits of dietary restriction (DR), or reduced nutrient/energy intake without malnutrition. DR, best known as the most reliable way to extend lifespan in a wide range of experimental organisms, includes various regimens aimed at either reducing overall calorie intake (calorie restriction, intermittent/every-other-day fasting) or reducing particular nutrients such as protein or the essential amino acid, methionine (methionine restriction), with overlapping functional benefits on stress resistance, metabolic fitness and lifespan. Here we will review the small but growing body of literature linking the TSP to the functional benefits of DR in part through the production of endogenous H2S, with an emphasis on regulation of the TSP and H2S production by diet and mechanisms of beneficial H2S action. PMID:25523462

  3. Calorie restriction and methionine restriction in control of endogenous hydrogen sulfide production by the transsulfuration pathway

    PubMed Central

    Hine, Christopher; Mitchell, James R.

    2015-01-01

    H2S is a gas easily identified by its distinctive odor. Although environmental exposure to H2S has been viewed alternately as therapeutic or toxic through the centuries, H2S has recently regained recognition for its numerous beneficial biological effects. Most experiments documenting such benefits, including improved glucose tolerance, increased stress resistance, and even lifespan extension, are based on exposure of experimental organisms to exogenous sources of H2S. However, appreciation is growing for the importance of H2S produced endogenously by the evolutionary conserved transsulfuration pathway (TSP) in health and longevity. Recent data implicate H2S produced by the TSP in pleiotropic benefits of dietary restriction (DR), or reduced nutrient/energy intake without malnutrition. DR, best known as the most reliable way to extend lifespan in a wide range of experimental organisms, includes various regimens aimed at either reducing overall calorie intake (calorie restriction, intermittent/ every-other-day fasting) or reducing particular nutrients such as protein or the essential amino acid, methionine (methionine restriction), with overlapping functional benefits on stress resistance, metabolic fitness and lifespan. Here we will review the small but growing body of literature linking the TSP to the functional benefits of DR in part through the production of endogenous H2S, with an emphasis on regulation of the TSP and H2S production by diet and mechanisms of beneficial H2S action. PMID:25523462

  4. A new pathway in the control of the initiation of puberty: the MKRN3 gene

    PubMed Central

    Abreu, Ana Paula; Macedo, Delanie B.; Brito, Vinicius N.; Kaiser, Ursula B.; Latronico, Ana Claudia

    2015-01-01

    Pubertal timing is influenced by complex interactions among genetic, nutritional, environmental, and socioeconomic factors. The role of MKRN3, an imprinted gene located in the Prader-Willi syndrome critical region (chromosome 15q11-q13), in pubertal initiation was first described in 2013 after the identification of deleterious MKRN3 mutations in five families with central precocious puberty (CPP) using whole-exome sequencing analysis. Since then, additional loss-of-function mutations of MKRN3 have been associated with the inherited premature sexual development phenotype in girls and boys from different ethnic groups. In all of these families, segregation analysis clearly demonstrated autosomal dominant inheritance with complete penetrance, but with exclusive paternal transmission, consistent with the monoallelic expression of MKRN3 (a maternally imprinted gene). Interestingly, the hypothalamic Mkrn3 mRNA expression pattern in mice correlated with a putative inhibitory input on puberty initiation. Indeed, the initiation of puberty depends on a decrease in factors that inhibit the release of GnRH combined with an increase in stimulatory factors. These recent human and animal findings suggest that MKRN3 has an inhibitory role in the reproductive axis to represent a new pathway in pubertal regulation. PMID:25957321

  5. Using the stress response to monitor process control: pathways to more effective bioremediation.

    PubMed

    Hazen, Terry C; Stahl, David A

    2006-06-01

    Environmental contamination with a variety of pollutants has prompted the development of effective bioremediation strategies. But how can these processes be best monitored and controlled? One avenue under investigation is the development of stress response systems as tools for effective and general process control. Although the microbial stress response has been the subject of intensive laboratory investigation, the environmental reflection of the laboratory response to specific stresses has been little explored. However, it is only within an environmental context, in which microorganisms are constantly exposed to multiple changing environmental stresses, that there will be full understanding of microbial adaptive resiliency. Knowledge of the stress response in the environment will facilitate the control of bioremediation and other processes mediated by complex microbial communities. PMID:16616486

  6. PTEN regulates EG5 to control spindle architecture and chromosome congression during mitosis.

    PubMed

    He, Jinxue; Zhang, Zhong; Ouyang, Meng; Yang, Fan; Hao, Hongbo; Lamb, Kristy L; Yang, Jingyi; Yin, Yuxin; Shen, Wen H

    2016-01-01

    Architectural integrity of the mitotic spindle is required for efficient chromosome congression and accurate chromosome segregation to ensure mitotic fidelity. Tumour suppressor PTEN has multiple functions in maintaining genome stability. Here we report an essential role of PTEN in mitosis through regulation of the mitotic kinesin motor EG5 for proper spindle architecture and chromosome congression. PTEN depletion results in chromosome misalignment in metaphase, often leading to catastrophic mitotic failure. In addition, metaphase cells lacking PTEN exhibit defects of spindle geometry, manifested prominently by shorter spindles. PTEN is associated and co-localized with EG5 during mitosis. PTEN deficiency induces aberrant EG5 phosphorylation and abrogates EG5 recruitment to the mitotic spindle apparatus, leading to spindle disorganization. These data demonstrate the functional interplay between PTEN and EG5 in controlling mitotic spindle structure and chromosome behaviour during mitosis. We propose that PTEN functions to equilibrate mitotic phosphorylation for proper spindle formation and faithful genomic transmission. PMID:27492783

  7. PTEN regulates EG5 to control spindle architecture and chromosome congression during mitosis

    PubMed Central

    He, Jinxue; Zhang, Zhong; Ouyang, Meng; Yang, Fan; Hao, Hongbo; Lamb, Kristy L.; Yang, Jingyi; Yin, Yuxin; Shen, Wen H.

    2016-01-01

    Architectural integrity of the mitotic spindle is required for efficient chromosome congression and accurate chromosome segregation to ensure mitotic fidelity. Tumour suppressor PTEN has multiple functions in maintaining genome stability. Here we report an essential role of PTEN in mitosis through regulation of the mitotic kinesin motor EG5 for proper spindle architecture and chromosome congression. PTEN depletion results in chromosome misalignment in metaphase, often leading to catastrophic mitotic failure. In addition, metaphase cells lacking PTEN exhibit defects of spindle geometry, manifested prominently by shorter spindles. PTEN is associated and co-localized with EG5 during mitosis. PTEN deficiency induces aberrant EG5 phosphorylation and abrogates EG5 recruitment to the mitotic spindle apparatus, leading to spindle disorganization. These data demonstrate the functional interplay between PTEN and EG5 in controlling mitotic spindle structure and chromosome behaviour during mitosis. We propose that PTEN functions to equilibrate mitotic phosphorylation for proper spindle formation and faithful genomic transmission. PMID:27492783

  8. Mitotic noncoding RNA processing promotes kinetochore and spindle assembly in Xenopus.

    PubMed

    Grenfell, Andrew W; Heald, Rebecca; Strzelecka, Magdalena

    2016-07-18

    Transcription at the centromere of chromosomes plays an important role in kinetochore assembly in many eukaryotes, and noncoding RNAs contribute to activation of the mitotic kinase Aurora B. However, little is known about how mitotic RNA processing contributes to spindle assembly. We found that inhibition of transcription initiation or RNA splicing, but not translation, leads to spindle defects in Xenopus egg extracts. Spliceosome inhibition resulted in the accumulation of high molecular weight centromeric transcripts, concomitant with decreased recruitment of the centromere and kinetochore proteins CENP-A, CENP-C, and NDC80 to mitotic chromosomes. In addition, blocking transcript synthesis or processing during mitosis caused accumulation of MCAK, a microtubule depolymerase, on the spindle, indicating misregulation of Aurora B. These findings suggest that co-transcriptional recruitment of the RNA processing machinery to nascent mitotic transcripts is an important step in kinetochore and spindle assembly and challenge the idea that RNA processing is globally repressed during mitosis. PMID:27402954

  9. Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibres without a 30-nm chromatin structure

    PubMed Central

    Nishino, Yoshinori; Eltsov, Mikhail; Joti, Yasumasa; Ito, Kazuki; Takata, Hideaki; Takahashi, Yukio; Hihara, Saera; Frangakis, Achilleas S; Imamoto, Naoko; Ishikawa, Tetsuya; Maeshima, Kazuhiro

    2012-01-01

    How a long strand of genomic DNA is compacted into a mitotic chromosome remains one of the basic questions in biology. The nucleosome fibre, in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fibre and further hierarchical regular structures to form mitotic chromosomes, although the actual existence of these regular structures is controversial. Here, we show that human mitotic HeLa chromosomes are mainly composed of irregularly folded nucleosome fibres rather than 30-nm chromatin fibres. Our comprehensive and quantitative study using cryo-electron microscopy and synchrotron X-ray scattering resolved the long-standing contradictions regarding the existence of 30-nm chromatin structures and detected no regular structure >11 nm. Our finding suggests that the mitotic chromosome consists of irregularly arranged nucleosome fibres, with a fractal nature, which permits a more dynamic and flexible genome organization than would be allowed by static regular structures. PMID:22343941

  10. Phosphorylation of the proapoptotic BH3-only protein bid primes mitochondria for apoptosis during mitotic arrest.

    PubMed

    Wang, Pengbo; Lindsay, Jennefer; Owens, Thomas W; Mularczyk, Ewa J; Warwood, Stacey; Foster, Fiona; Streuli, Charles H; Brennan, Keith; Gilmore, Andrew P

    2014-05-01

    Mitosis is a moment of exquisite vulnerability for a metazoan cell. Failure to complete mitosis accurately can lead to aneuploidy and cancer initiation. Therefore, if the exit from mitosis is delayed, normal cells are usually removed by apoptosis. However, how failure to complete mitosis activates apoptosis is still unclear. Here, we demonstrate that a phosphorylated form of the BH3-only protein Bid regulates apoptosis if mitotic exit is delayed. Bid is phosphorylated on serine 66 as cells enter mitosis, and this phosphorylation is lost during the metaphase-to-anaphase transition. Cells expressing a nonphosphorylatable version of Bid or a BH3-domain mutant were resistant to mitotic-arrest-induced apoptosis. Thus, we show that Bid phosphorylation primes cells to undergo mitochondrial apoptosis if mitotic exit is delayed. Avoidance of this mechanism may explain the selective pressure for cancer cells to undergo mitotic slippage. PMID:24767991

  11. Association of Chromosome Loss with Centromere-Adjacent Mitotic Recombination in a Yeast Disomic Haploid

    PubMed Central

    Campbell, D. A.; Fogel, S.

    1977-01-01

    Experiments designed to characterize the association between disomic chromosome loss and centromere-adjacent mitotic recombination were performed. Mitotic gene convertants were selected at two heteroallelic sites on the left arm of disomic chromosome III and tested for coincident chromosome loss. The principal results are: (1) Disomic chromosome loss is markedly enhanced (nearly 40-fold) over basal levels among mitotic gene convertants selected to arise close to the centromere; no such enhancement is observed among convertants selected to arise relatively far from the centromere. (2) Chromosome loss is primarily associated with proximal allele conversion at the centromere-adjacent site, and many of these convertants are reciprocally recombined in the adjacent proximal interval. (3) Partial aneuploid exceptions provisionally identified as carrying left arm telocentrics have been found. A testable model is proposed suggesting that centromere involvement in genetic recombination may precipitate segregational disfunction leading to mitotic chromosome loss. PMID:324869

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

    PubMed

    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. PMID:26247711

  13. Inhibition of a Mitotic Motor Protein: Where, How, and Conformational Consequences

    SciTech Connect

    Yan, Youwei; Sardana, Vinod; Xu, Bei; Homnick, Carl; Halczenko, Wasyl; Buser, Carolyn A.; Schaber, Michael; Hartman, George D.; Huber, Hans E.; Kuo, Lawrence C.

    2010-11-16

    We report here the first inhibitor-bound structure of a mitotic motor protein. The 1.9 {angstrom} resolution structure of the motor domain of KSP, bound with the small molecule monastrol and Mg{sup 2+} {center_dot} ADP, reveals that monastrol confers inhibition by 'induced-fitting' onto the protein some 12 {angstrom} away from the catalytic center of the enzyme, resulting in the creation of a previously non-existing binding pocket. The structure provides new insights into the biochemical and mechanical mechanisms of the mitotic motor domain. Inhibition of KSP provides a novel mechanism to arrest mitotic spindle formation, a target of several approved and investigative anti-cancer agents. The structural information gleaned from this novel pocket offers a new angle for the design of anti-mitotic agents.

  14. Centromeric barrier disruption leads to mitotic defects in Schizosaccharomyces pombe.

    PubMed

    Gaither, Terilyn L; Merrett, Stephanie L; Pun, Matthew J; Scott, Kristin C

    2014-04-01

    Centromeres are cis-acting chromosomal domains that direct kinetochore formation, enabling faithful chromosome segregation and preserving genome stability. The centromeres of most eukaryotic organisms are structurally complex, composed of nonoverlapping, structurally and functionally distinct chromatin subdomains, including the specialized core chromatin that underlies the kinetochore and pericentromeric heterochromatin. The genomic and epigenetic features that specify and preserve the adjacent chromatin subdomains critical to centromere identity are currently unknown. Here we demonstrate that chromatin barriers regulate this process in Schizosaccharomyces pombe. Reduced fitness and mitotic chromosome segregation defects occur in strains that carry exogenous DNA inserted at centromere 1 chromatin barriers. Abnormal phenotypes are accompanied by changes in the structural integrity of both the centromeric core chromatin domain, containing the conserved CENP-A(Cnp1) protein, and the flanking pericentric heterochromatin domain. Barrier mutant cells can revert to wild-type growth and centromere structure at a high frequency after the spontaneous excision of integrated exogenous DNA. Our results reveal a previously undemonstrated role for chromatin barriers in chromosome segregation and in the prevention of genome instability. PMID:24531725

  15. Centrin: Another target of monastrol, an inhibitor of mitotic spindle

    NASA Astrophysics Data System (ADS)

    Duan, Lian; Wang, Tong-Qing; Bian, Wei; Liu, Wen; Sun, Yue; Yang, Bin-Sheng

    2015-02-01

    Monastrol, a cell-permeable inhibitor, considered to specifically inhibit kinesin Eg5, can cause mitotic arrest and monopolar spindle formation, thus exhibiting antitumor properties. Centrin, a ubiquitous protein associated with centrosome, plays a critical role in centrosome duplication. Moreover, a correlation between centrosome amplification and cancer has been reported. In this study, it is proposed for the first time that centrin may be another target of the anticancer drug monastrol since monastrol can effectively inhibit not only the growth of the transformed Escherichia coli cells in vivo, but also the Lu3+-dependent self-assembly of EoCen in vitro. The two closely related compounds (Compounds 1 and 2) could not take the same effect. Fluorescence titration experiments suggest that four monastrols per protein is the optimum binding pattern, and the binding constants at different temperatures were obtained. Detailed thermodynamic analysis indicates that hydrophobic force is the main acting force between monastrol and centrin, and the extent of monastrol inhibition of centrin self-assembly is highly dependent upon the hydrophobic region of the protein, which is largely exposed by the binding of metal ions.

  16. Pathways to Problem Behaviors: Chaotic Homes, Parent and Child Effortful Control, and Parenting

    ERIC Educational Resources Information Center

    Valiente, Carlos; Lemery-Chalfant, Kathryn; Reiser, Mark

    2007-01-01

    Guided by Belsky's and Eisenberg, Cumberland, and Spinrad's heuristic models, we tested a process model with hypothesized paths from parents' effortful control (EC) and family chaos to indices of parenting to children's EC, and finally children's externalizing problem behavior. Parents reported on all constructs and children (N = 188; M age = 9.55…

  17. Recent advances in understanding of meiosis initiation and the apomictic pathway in plants.

    PubMed

    Wang, Chung-Ju R; Tseng, Ching-Chih

    2014-01-01

    Meiosis, a specialized cell division to produce haploid cells, marks the transition from a sporophytic to a gametophytic generation in the life cycle of plants. In angiosperms, meiosis takes place in sporogenous cells that develop de novo from somatic cells in anthers or ovules. A successful transition from the mitotic cycle to the meiotic program in sporogenous cells is crucial for sexual reproduction. By contrast, when meiosis is bypassed or a mitosis-like division occurs to produce unreduced cells, followed by the development of an embryo sac, clonal seeds can be produced by apomixis, an asexual reproduction pathway found in 400 species of flowering plants. An understanding of the regulation of entry into meiosis and molecular mechanisms of apomictic pathway will provide vital insight into reproduction for plant breeding. Recent findings suggest that AM1/SWI1 may be the key gene for entry into meiosis, and increasing evidence has shown that the apomictic pathway is epigenetically controlled. However, the mechanism for the initiation of meiosis during sexual reproduction or for its omission in the apomictic pathway still remains largely unknown. Here we review the current understanding of meiosis initiation and the apomictic pathway and raised several questions that are awaiting further investigation. PMID:25295051

  18. Recent advances in understanding of meiosis initiation and the apomictic pathway in plants

    PubMed Central

    Wang, Chung-Ju R.; Tseng, Ching-Chih

    2014-01-01

    Meiosis, a specialized cell division to produce haploid cells, marks the transition from a sporophytic to a gametophytic generation in the life cycle of plants. In angiosperms, meiosis takes place in sporogenous cells that develop de novo from somatic cells in anthers or ovules. A successful transition from the mitotic cycle to the meiotic program in sporogenous cells is crucial for sexual reproduction. By contrast, when meiosis is bypassed or a mitosis-like division occurs to produce unreduced cells, followed by the development of an embryo sac, clonal seeds can be produced by apomixis, an asexual reproduction pathway found in 400 species of flowering plants. An understanding of the regulation of entry into meiosis and molecular mechanisms of apomictic pathway will provide vital insight into reproduc