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Mps1 promotes rapid centromere accumulation of aurora b

scientific report Mps1 promotes rapid centromere accumulationof Aurora BMaike S. van der Waal1*, Adrian T. Saurin1,2*, Martijn J.M. Vromans1, Mathijs Vleugel1,2,Claudia Wurzenberger3, Daniel W. Gerlich3w, Rene´ H. Medema4, Geert J.P.L. Kops1,2 & Susanne M.A. Lens1+1Department of Medical Oncology, 2Department of Molecular Cancer Research, University Medical Center Utrecht, Utrecht,The Netherlands, 3Institute of Biochemistry, Department of Biology, Swiss Federal Institute of Technology, Zu¨rich, Switzerland,and 4Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands Aurora B localization to mitotic centromeres, which is required The catalytic subunit of the chromosomal passenger complex for proper chromosome alignment during mitosis, relies on (CPC), Aurora B kinase, is a key factor for microtubule attachment Haspin-dependent histone H3 phosphorylation and on Bub1- error correction and impacts on the mitotic checkpoint dependent histone H2A phosphorylation—which interacts with through destabilization of incorrectly attached microtubules, Borealin through a Shugoshin (Sgo) intermediate. We demon- strate that Mps1 stimulates the latter recruitment axis. Mps1 formation of the mitotic checkpoint complex required for APC/C activity enhances H2A-T120ph and is critical for Sgo1 recruit- inhibition Moreover, Aurora B can also directly stimulate the ment to centromeres, thereby promoting Aurora B centromere mitotic checkpoint at the onset of mitosis via kinetochore recruitment in early mitosis. Importantly, chromosome biorienta- recruitment and subsequent activation of the checkpoint kinase tion defects caused by Mps1 inhibition are improved by restoring Mps1 Finally, Aurora B may also function further Aurora B centromere recruitment. As Mps1 kinetochore localiza- downstream in the mitotic checkpoint signalling cascade to tion reciprocally depends on Aurora B, we propose that this maintain the APC/C inhibitory signal Aurora B-Mps1 recruitment circuitry cooperates with the Similar to Aurora B, Mps1 is also a dual function kinase. It is Aurora B-Haspin feedback loop to ensure rapid centromere essential for the mitotic checkpoint and for error correction [ accumulation of Aurora B at the onset of mitosis.
The error correction function of Mps1 has been attributed to the Keywords: Mps1; Aurora B; biorientation; centromere phosphorylation of the CPC subunit Borealin that is required for EMBO reports (2012) 13, 847–854. doi:10.1038/embor.2012.93 Aurora B activation However, Aurora B-independentmechanisms have also been proposed [Therefore it is unclear exactly how Mps1 promotes error correction.
Localization of Aurora B to mitotic centromeres is important for When cells divide, duplicated chromosomes face the challenge ofbiorienting on the mitotic spindle to ensure an exact copy of the efficient error correction. It requires Haspin-dependent phosphor- genome is passed on to the two newly formed daughter cells.
ylation of histone H3 at Thr3 (H3-T3ph) and Bub1-dependentphosphorylation of histone H2A at Thr120 (H2A-T120ph).
Biorientation is assured through destabilization of erroneous H3-T3ph binds the Survivin subunit of the CPC and H2A- kinetochore–microtubule attachments and activation of themitotic checkpoint, which allows time for error correction T120ph interacts with Borealin through a Shugoshin (Sgo)intermediate The signals upstream of these two converging centromere recruitment pathways are now beginning Department of Medical Oncology, 2Department of Molecular Cancer Research, University Medical Center Utrecht, to emerge. Phosphorylation of Borealin by Cdk1 stimulates the Universiteitsweg 100, STR 2.129, 3584 CG Utrecht, The Netherlands interaction between the CPC and Sgo and Aurora B promotes 3Institute of Biochemistry, Department of Biology, Swiss Federal Institute of its own centromere recruitment by reinforcing Haspin activity Technology Zu¨rich (ETHZ), HPM D11.3, Schafmattstrasse 18, 8093 Zu¨rich,Switzerland towards H3-T3ph We show here that Mps1 and Aurora B 4Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, cooperate to stimulate the Bub1/H2A-T120ph/Sgo1 recruitment 1066 CX Amsterdam, The Netherlands axis, which allows rapid centromere accumulation of the CPC at *These authors contributed equally to this work the onset of mitosis.
Present address: Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria+Corresponding author. Tel: þ 31 88 75 68114; Fax: þ 31 88 75 55430; RESULTS AND DISCUSSION Mps1 is needed to establish Aurora B at centromeresMps1 is required for Bub1 kinetochore recruitment As Received 1 March 2012; revised 4 June 2012; accepted 5 June 2012; publishedonline 26 June 2012 Bub1-dependent H2A-T120ph is involved in CPC centromere &2012 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 13 NO 9 2012 8 4 7




Mps1 promotes centromere accumulation of Aurora B M.S. van der Waal et al scientific report Relative centromere levels Relative centromere levels Relative centromere levels Reversine Mps1-IN-1 Fig 1 Mps1 is required to establish Aurora B centromere localization at the onset of mitosis. All quantifications are signal intensities over CREST(centromere staining). (A) IF and quantifications of Aurora B, Aurora B-T232ph and centromeres (CREST) in HeLa cells. Cells were treated withthe indicated siRNAs (si-Luc ¼ si-Luciferase) for 48 h followed by 30 min treatment with nocodazole and Reversine (1 mM) or DMSO. Early mitoticcells were analysed. Graph represents mean (±s.d.) of 40 cells (Aurora B) or 20 cells (Aurora B-T232ph) per condition, sampled in four or twoindependent experiments, respectively. (B) IF and quantification of Aurora B in U2OS cells treated with nocodazole plus DMSO, Reversine (500 nM) orMps1-IN-1 (10 mM) for 30 min. Early mitotic cells were analysed. Graph represents mean (±s.d.) of 20 cells per condition, sampled in two independentexperiments. (C) IF and quantification of Aurora B in HeLa cells treated with nocodazole and MG132 for 1 h, and DMSO or Reversine (1 mM) foradditional 30 min. Graph represents mean (±s.d.) of 29 cells per condition, sampled in three independent experiments. Scale bars, 5 mm. Forcharacterization of inhibitor concentrations and siRNAs see supplementary Fig S1 online. DMSO, dimethyl sulphoxide; IF, immunofluorescence;siRNAs, short interfering RNAs.
recruitment we determined whether Mps1 was needed for activity was mainly required for the establishment of Aurora B Aurora B centromere localization. We allowed cells to enter localization, because if Mps1 was inhibited during mitosis, when mitosis without Mps1 activity, using the chemical inhibitors Aurora B had already been accumulated at centromeres, then Reversine and Mps1-IN-1 or RNA interference (RNAi)- Aurora B localization was barely affected supplementary mediated protein knockdown supplementary Fig S1A Video S1 online).
online). Cells were treated with nocodazole to exclude effects ofkinetochore–microtubule attachments and, as Mps1 inhibition Mps1 allows rapid Aurora B recruitment to centromeres silences the mitotic checkpoint (supplementary Fig S1C online), As Cdk1 activity is important for proper centromere localization of we only analysed early prometaphase cells recognized by a the CPC we wanted to test if the defects in Aurora B dispersed DNA morphology recruitment upon Mps1 inhibition were related to reduced Cyclin In the absence of Mps1, Aurora B localized to the chromosomal B levels owing to checkpoint inactivation. First, we depleted the arms, instead of accumulating at the inner centromere checkpoint protein BubR1 and found it had no effect on Aurora B supplementary Fig S3C online). The fact that Reversine or localization supplementary Fig S1B,C online). Second, we Mps1-IN-1 phenocopied Mps1 RNAi indicates that Mps1 activity used an assay that allowed Aurora B localization to be measured is required for Aurora B centromere localization The in the absence of Cyclin B degradation. We adapted the protocol reduction of centromeric Aurora B corresponded to a decrease in of Potapova et al and briefly inhibited Cdk1 activity with Aurora B-T232 autophosphorylation Interestingly, Mps1 RO3306 (RO) in cells arrested in mitosis with the Eg5 inhibitor 8 4 8 EMBO reports VOL 13 NO 9 2012 &2012 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION


Mps1 promotes centromere accumulation of Aurora BM.S. van der Waal et al scientific report Washout (into DMSO/MG132) Washout (into Reversine/MG132) t = –15 Centromere levels 0.0 Centromere levels (+/– inhibitors) (YFP-YFP/CFP-YFP) Fig 2 Mps1 inhibition induces a delay in Aurora B centromere accumulation. (A) Schematic depiction of Cdk1 reactivation assay. (B) Time lapseanalysis of Cdk1 reactivation assay in UTR (U2OS stably expressing a Tet repressor) cells expressing wt-INCENP-GFP. Cells were treated as depictedin (A), RO3306 was added after 4 min and washed out after 16 min. Per condition, one representative cell out of eight is shown. (C) IF andquantifications of Aurora B and Aurora B-T232ph in HeLa cells. Cells were treated as depicted in (A), and fixed at indicated time points. Graph showsone representative experiment out of two and represents mean (±s.e.m.) of 10 cells per time point. (D) Time lapse analysis of FRET in U2OS cellsstably expressing a FRET biosensor for Aurora B activity, and treated as depicted in (A). Increased ratio indicates increased FRET sensorphosphorylation. Lines represent means of at least 10 cells (±s.e.m.). Colour-coded images are shown for indicated time points. Scale bars, 5 mm.
CREST, centromere staining; DMSO, dimethyl sulphoxide; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; IF,immunofluorescence; STLC, s-trityl-l-cysteine.
&2012 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 13 NO 9 2012 8 4 9 Mps1 promotes centromere accumulation of Aurora B M.S. van der Waal et al scientific report Fig 3 Mps1 regulates the Bub1/H2A-T120ph/Sgo1 pathway for Aurora B centromere localization. All quantifications are signal intensities over CREST (centromere staining) and early mitotic cells were analysed. (A,B,C) IF and quantifications of Bub1, H2A-T120ph, Sgo1 and centromeres (CREST)in UTR (U2OS cells stably expressing a Tet repressor) cells, and UTR cells expressing CB-INCENP or Mis12-Mps1. Expression was induced withdoxycyclin. Cells were synchronized in G2 with RO3306, released in a medium containing DMSO, Reversine or Hesperadin, and further treated withnocodazole plus MG132 for 30 min. Data represent means (±s.d.) from three independent experiments, each experiment including at least 10 cells percondition. (D) IF of Bub1, Sgo1 and H2A-T120ph in Mis12-Mps1-expressing UTR cells synchronized in G1/S with thymidine and analysed 6 h afterthymidine release (enrichment for G2 cells) in the presence or absence of doxycyclin. (E) IF and quantifications of Aurora B in HeLa cells treated withthe indicated siRNAs for 48 h followed by half an hour treatment with nocodazole and Reversine. Graph shows one representative experiment out oftwo and represents mean (±s.e.m.) of 11 cells per condition. Scale bars, 5 mm. DMSO, dimethyl sulphoxide; IF, immunofluorescence; siRNAs, shortinterfering RNAs; UTR, U2OS cells stably expressing a Tet repressor.
s-trityl-l-cysteine (STLC; in the presence of MG132 to prevent Cyclin intermediate for CPC recruitment was also affected by Mps1 B degradation) and then allowed return to a mitotic state upon inhibition. While Sgo2 levels were only slightly reduced, Sgo1 reactivation of Cdk1 (As expected, Cdk1 inhibition resulted centromere localization was highly sensitive to Mps1 inhibition, in a rapid removal of ectopically expressed green fluorescent protein with Sgo1 localizing over the chromosomal arms instead of to the (GFP)-tagged CPC subunit inner centromere protein (INCEP) from centromere, much like we observed for Aurora B centromeres (supplementary Video S2 online, [This supplementary Fig S3A–C online) effect of Cdk1 inhibition was reversible because B20 min after RO We and others have shown recently that Mps1 localization to washout, the centromere localization of INCENP-GFP was fully kinetochores is Aurora B-dependent We therefore tested recovered ; supplementary Video S2 online). When cells if Aurora B stimulated its own recruitment to centromeres via were released from Cdk1 inhibition in the presence of Reversine the Mps1. Aurora B inhibition with Hesperadin caused a reduction in accumulation of INCENP-GFP at centromeres was significantly Bub1, H2A-T120ph, Sgo1 and H3-T3ph at kinetochores and delayed ; supplementary Video S2 online). We confirmed centromeres, respectively supplementary Fig S3D these results for endogenous Aurora B in fixed cells and observed a online). To determine whether this was a consequence of reduced significant delay in recovery of Aurora B and Aurora B-T232ph when Mps1 activity, we restored Mps1 localization to kinetochores Cdk1 was reactivated in the absence of Mps1 activity Note using Mis12-Mps1 Although, as expected, this did not that Aurora B did relocalize to centromeres, even in the absence of restore H3-T3ph (supplementary Fig S3D online), Bub1 kineto- Mps1 activity, suggesting that Mps1 inhibition merely delays Aurora chore localization and H2A-T120ph were improved, and Sgo1 B recruitment.
recruitment to centromeres was completely restored To examine the consequences of delayed Aurora B accumula- The incomplete rescue of Bub1 kinetochore localization tion on substrate phosphorylation, we used a chromatin (H2B)- and H2A-T120ph could indicate that Aurora B also affects targeted fluorescence resonance energy transfer (FRET)-based H2A-T120ph via an additional, Mps1-independent pathway.
biosensor for Aurora B activity that is sensitive to Aurora B Indeed, we observed an increase of Bub1 kinetochore localization activity coming from the centromere Treatment of and H2A-T120ph in the absence of Mps1 activity when Aurora B mitotic cells with RO/MG132 resulted in decreased sensor was restored at centromeres by expressing CB-INCENP phosphorylation, and recovery of Aurora B substrate phosphory- The complete rescue of Sgo1 recruitment by lation was delayed when Cdk1 was reactivated in the presence Mis12-Mps1 in cells with inactive Aurora B suggests that Sgo1 is of Reversine/MG132, both in U2OS and HeLa cells under control of Mps1 but only partially via Bub1-dependent H2A- supplementary Fig S2 online). We noticed that Aurora B-T232ph T120ph Indeed, when Bub1 kinetochore localization and sensor phosphorylation were not fully restored in the absence was restored in cells without active Mps1, using Mis12-Bub1, this of active Mps1, which could be owing to the requirement of did not recover Sgo1 localization (supplementary Fig S4A online).
Mps1-dependent Borealin phosphorylation In conclusion, Moreover, expression of Mis12-Mps1 was sufficient to promote Sgo1 we demonstrate in different cell lines, both fixed and live, centromere localization in G2 cells even though H2A-T120 that early in mitosis Mps1 promotes Aurora B centromere phosphorylation was absent . This implies that Mps1 may recruitment and substrate phosphorylation. The relatively small also signal directly to Sgo1 as has been suggested recently by others time window in which Mps1 operates likely explains why the . Importantly, RNAi-mediated Sgo1 depletion resulted in a effect of Mps1 on Aurora B centromere localization was not reduction of centromeric Aurora B to a comparable level as was previously detected observed after inhibition of Mps1 indicating that thereduced Sgo1 levels may indeed be responsible for the impaired Mps1 controls the Bub1/H2A/Sgo CPC recruitment axis Aurora B centromere recruitment after Mps1 inhibition.
Given the importance of the Bub1/H2A-T120ph/Sgo pathway forCPC centromere localization we rationalized that the defects Mps1 recruits Aurora B to stimulate error correction in this pathway could explain the delay in Aurora B centromere We next tested the significance of the Mps1-Aurora B recruitment accumulation following Mps1 inhibition. While Mps1 activity circuitry for error correction, a process that relies on Aurora B and was dispensable for Haspin-dependent H3-T3ph (supplementary Mps1 Chromosome alignment was examined upon Fig S3A,D online), it was indeed required for Bub1 kinetochore inhibition of Mps1 in stable cell lines with inducible expression of localization, and H2A-T120ph supplementary Fig S3A CB-INCENP, which renders Aurora B localization insensitive to online) in mitotic cells We next determined if the Sgo Mps1 regulation Cells that had entered mitosis with 8 5 0 EMBO reports VOL 13 NO 9 2012 &2012 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION


Mps1 promotes centromere accumulation of Aurora BM.S. van der Waal et al scientific report Relative centromere levels Relative centromere levels Reversine Hesperadin Reversine Hesperadin Relative centromere levels Reversine Hesperadin Relative centromere levels Mis12-Mps1 H2A-T120ph Merge &2012 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 13 NO 9 2012 8 5 1



Mps1 promotes centromere accumulation of Aurora B M.S. van der Waal et al scientific report % Of mitotic cells Severe misalignment Mild misalignment Complete alignment Fig 4 Mps1-dependent Aurora B centromere recruitment promotes efficient chromosome biorientation. (A) IF of Aurora B and centromeres (CREST)in UTR cells with inducible expression of wt-INCENP-GFP or CB-INCENP-GFP. Cells were treated as in (B) Cells were synchronized in G2 withRO3306 and released to enter mitosis in the presence or absence of Reversine for 60 min. MG132 was added for an additional 30 min to accumulatecells in metaphase. Low expression levels of CB-INCENP at the onset of mitosis were accomplished by a relative short protein induction time (6 h).
Graph shows one representative experiment out of two. The number (n) of cells analysed per condition is indicated. Scale bars, 5 mm. (C) Model forthe recruitment circuitries that operate between Mps1-Sgo1-Aurora B (black arrows) and Mps1-Bub1-Sgo1-Aurora B (grey arrows) at the beginningof mitosis to ensure robust establishment of the mitotic checkpoint and error correction machineries. DAPI, 4,6-diamidino-2-phenylindole; DMSO,dimethyl sulphoxide; GFP, green fluorescent protein; IF, immunofluorescence; UTR, U2OS cells stably expressing a Tet repressor.
active or inactive Mps1 were scored for chromosome alignment onset of mitosis via centromere recruitment of Sgo1, and we after 30 min treatment with MG132 to allow sufficient time for suggest that this recruitment circuitry acts together with the Aurora alignment in control cells supplementary Fig S4B online).
B-Haspin feedback loop We propose that the mutual As expected, inhibition of Mps1 reduced the ability of cells to dependence of Aurora B and Mps1 for the rapid establishment align their chromosomes. Whereas expression of wt-INCENP did of Aurora B centromere activity and Mps1 kinetochore activity not improve alignment under these conditions, we observed a allows for a fast and coordinated start-up of the error correction and striking improvement when Aurora B localization to centromeres mitotic checkpoint machineries so that both processes are fully was restored by CB-INCENP expression supplementary functional at the point of nuclear envelope breakdown ).
Fig S4B online). Only mild chromosome misalignments persisted,which might reflect the Aurora B-independent role of Mps1 in chromosome biorientation Cell lines and cell culture. Inducible cell lines expressing LAP-Mis12-Mps1D200, wt-INCENP-GFP and CB-INCENP-GFP werepreviously described All inducible cell lines were derived from U2OS cells stably expressing a Tet repressor (UTR) and Our data show that an Aurora B-Mps1 regulatory circuitry cultured in Tet-free medium. Protein expression was induced with promotes timely and robust Aurora B centromere activity at the 1 mg/ml doxycycline (Sigma-Aldrich) for at least 6 h. HeLa and 8 5 2 EMBO reports VOL 13 NO 9 2012 &2012 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION Mps1 promotes centromere accumulation of Aurora BM.S. van der Waal et al scientific report U2OS cells were transfected with a pIRESpuro2b vector Ruchaud S, Carmena M, Earnshaw WC (2007) Chromosomal passengers: containing H2B-targeted FRET sensor for Aurora B activity conducting cell division. Nat Rev Mol Cell Biol 8: 798–812 Tanaka TU, Rachidi N, Janke C, Pereira G, Galova M, Schiebel E, and selected with 1 mg/ml puromycin (Sigma-Aldrich) for stable Stark MJ, Nasmyth K (2002) Evidence that the Ipl1-Sli15 (Aurora expression. HeLa and U2OS cells were grown in DMEM kinase-INCENP) complex promotes chromosome bi-orientation by supplemented with 6% fetal bovine serum, pen/strep and altering kinetochore-spindle pole connections. Cell 108: 317–329 l-glutamine (2 mM).
Santaguida S, Vernieri C, Villa F, Ciliberto A, Musacchio A (2011)Evidence that Aurora B is implicated in spindle checkpoint signalling Cdk1 reactivation assay. Cells were incubated for 1.5 h with independently of error correction. EMBO J 30: 1508–1519 STLC (20 mM, Tocris Bioscience) to arrest cells in mitosis with Saurin AT, van der Waal MS, Medema RH, Lens SM, Kops GJ (2011) monopolar spindles. MG132 was added 30 min before addition of Aurora B potentiates Mps1 activation to ensure rapid checkpoint RO3306 (10 mM, Enzo Life Sciences). After 10 min, the drug was establishment at the onset of mitosis. Nat Commun 2: 316 removed by washing the cells with STLC, MG132 (10 mM, Sigma- Maldonado M, Kapoor TM (2011) Constitutive Mad1 targeting tokinetochores uncouples checkpoint signalling from chromosome Aldrich) and either dimethyl sulphoxide (DMSO), Hesperadin biorientation. Nat Cell Biol 13: 475–482 (125 nM, Selleck Chemicals), Mps1-IN-1 (10 uM, gift of Dr N.
Abrieu A, Magnaghi-Jaulin L, Kahana JA, Peter M, Castro A, Vigneron S, Grey or Reversine (Sigma-Aldrich, 500 nM for U2OS cells or Lorca T, Cleveland DW, Labbe JC (2001) Mps1 is a kinetochore- 1 mM for HeLa and UTR cells, supplementary Fig S1A online)- associated kinase essential for the vertebrate mitotic checkpoint.
containing medium Cells were either fixed at different time Cell 106: 83–93 points or recovery was imaged live. Details of live and fixed cell 10. Jelluma N, Brenkman AB, van den Broek NJ, Cruijsen CW, van Osch MH, Lens SM, Medema RH, Kops GJ (2008) Mps1 imaging procedures (including FRET sensor experiments), indirect phosphorylates Borealin to control Aurora B activity and chromosome immunofluorescence, antibodies, expression vectors and short alignment. Cell 132: 233–246 interfering RNAs (siRNAs) are available as Supplementary 11. Maure JF, Kitamura E, Tanaka TU (2007) Mps1 kinase promotes information online.
sister-kinetochore bi-orientation by a tension-dependent mechanism.
Curr Biol 17: 2175–2182 Chromosome alignment assay. Protein induction was started 3 h 12. Stucke VM, Sillje HH, Arnaud L, Nigg EA (2002) Human Mps1 kinase is before cells were arrested in late G2 by treatment with RO3306 required for the spindle assembly checkpoint but not for centrosome (7.5 mM) for 2.5 h. The relative short protein induction time is duplication. EMBO J 21: 1723–1732 important to ensure that the levels of CB-INCENP are still low 13. Hewitt L, Tighe A, Santaguida S, White AM, Jones CD, Musacchio A, when cells enter mitosis. Synchronized cells were released to Green S, Taylor SS (2010) Sustained Mps1 activity is required in mitosisto recruit O-Mad2 to the Mad1-C-Mad2 core complex. J Cell Biol 190: enter mitosis in the presence of Reversine or DMSO. When cells entered mitosis (about 1 h after release) they were treated for 14. Maciejowski J, George KA, Terret ME, Zhang C, Shokat KM, Jallepalli PV 30 min with MG132 before fixation.
(2010) Mps1 directs the assembly of Cdc20 inhibitory complexes during Supplementary information is available at EMBO reports online interphase and mitosis to control M phase timing and spindle checkpoint signaling. J Cell Biol 190: 89–100 15. Santaguida S, Tighe A, D'Alise AM, Taylor SS, Musacchio A (2010) Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine. J Cell Biol We thank A. Janssen for manuscript reading, W. Bruinsma for analyses help and Dr. Losada and Dr. Grey for reagents. This work was supported 16. Kelly AE, Ghenoiu C, Xue JZ, Zierhut C, Kimura H, Funabiki H (2010) by the Netherlands Organization for Scientific Research (Vidi 91766332 Survivin reads phosphorylated histone H3 threonine 3 to activate the to S.M.A.L. and Vidi 91776336 to G.J.P.L.K.), the Dutch Cancer Society mitotic kinase Aurora B. Science 330: 235–239 (UU 2009-4311 to S.M.A.L.), the European Research Council (ERC-Stg 17. Wang F, Dai J, Daum JR, Niedzialkowska E, Banerjee B, Stukenberg PT, KINSIGN to G.J.P.L.K.), the European Communities 7th Framework Gorbsky GJ, Higgins JM (2010) Histone H3 Thr-3 phosphorylation by Programme (FP7/2007-2013; n1 241548/MitoSys and n1 258068/Systems Haspin positions Aurora B at centromeres in mitosis. Science 303: 231–235 Microscopy to D.W.G.) and Boehringer Ingelheim Fonds (C.W.).
18. Yamagishi Y, Honda T, Tanno Y, Watanabe Y (2010) Two histone marks Authors contributions: M.S.v., A.T.S., G.J.P.L.K. and S.M.A.L. designed establish the inner centromere and chromosome bi-orientation. Science the research. M.S.v. and S.M.A.L. wrote the paper with the input of A.T.S.
and G.J.P.L.K. M.S.v. performed most of the experiments and data 19. Tsukahara T, Tanno Y, Watanabe Y (2010) Phosphorylation of the CPC analysis, with contribution of A.T.S. and M.V. C.W. and D.W.G.
by Cdk1 promotes chromosome bi-orientation. Nature 467: 719–723 generated the FRET sensor vector and the FRET-HeLa cell line.
20. Wang F, Ulyanova NP, van der Waal MS, Patnaik D, Lens SM, Higgins M.V. generated Mis12-Bub1. R.H.M. contributed financially.
JM (2011b) A positive feedback loop involving Haspin and Aurora Bpromotes CPC accumulation at centromeres in mitosis. Curr Biol 21: CONFLICT OF INTEREST The authors declare that they have no conflict of interest.
21. Sliedrecht T, Zhang C, Shokat KM, Kops GJ (2010) Chemical genetic inhibition of Mps1 in stable human cell lines reveals novel aspects of Mps1 function in mitosis. PloS One 5: e10251 Santaguida S, Musacchio A (2009) The life and miracles of kinetochores.
22. Kwiatkowski N, Jelluma N, Filippakopoulos P, Soundararajan M, EMBO J 28: 2511–2531 Manak MS, Kwon M, Choi HG, Sim T, Deveraux QL, Rottmann S et al Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T, (2010) Small-molecule kinase inhibitors provide insight into Mps1 cell Mortlock A, Keen N, Taylor SS (2003) Aurora B couples chromosome cycle function. Nat Chem Biol 6: 359–368 alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to 23. Potapova TA, Daum JR, Pittman BD, Hudson JR, Jones TN, Satinover DL, kinetochores. J Cell Biol 161: 267–280 Stukenberg PT, Gorbsky GJ (2006) The reversibility of mitotic exit in Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, Heckel A, vertebrate cells. Nature 440: 954–958 van Meel J, Rieder CL, Peters JM (2003) The small molecule Hesperadin 24. Fuller BG, Lampson MA, Foley EA, Rosasco-Nitcher S, Le KV, reveals a role for Aurora B in correcting kinetochore-microtubule Tobelmann P, Brautigan DL, Stukenberg PT, Kapoor TM (2008) attachment and in maintaining the spindle assembly checkpoint.
Midzone activation of Aurora B in anaphase produces an intracellular J Cell Biol 161: 281–294 phosphorylation gradient. Nature 453: 1132–1136 &2012 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 13 NO 9 2012 8 5 3 Mps1 promotes centromere accumulation of Aurora B M.S. van der Waal et al scientific report 25. Wang E, Ballister ER, Lampson MA (2011a) Aurora B dynamics at 27. Liu D, Vader G, Vromans MJ, Lampson MA, Lens SM (2009) Sensing centromeres create a diffusion-based phosphorylation gradient.
chromosome bi-orientation by spatial separation of Aurora B kinase from J Cell Biol 194: 539–549 kinetochore substrates. Science 323: 1350–1353 26. Vazquez-Novelle MD, Petronczki M (2010) Relocation of the 28. Storchova Z, Becker JS, Talarek N, Kogelsberger S, Pellman D (2011) chromosomal passenger complex prevents mitotic checkpoint Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome engagement at anaphase. Curr Biol 20: 1402–1407 biorientation in budding yeast. Mol Biol Cell 22: 1473–1485 8 5 4 EMBO reports VOL 13 NO 9 2012 &2012 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION

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