imcb symposium 2008 - Institute of Molecular and Cell Biology - A*Star

IMCBSYMPOSIUM2008Wednesday, August 13 th 2008Breakthrough Theatrette,Matrix Building1

CONTENTSProgramme page 3Oral Presentations: Abstracts page 4Poster Presentations: List of Abstracts page 12Poster Presentations: Abstracts page 182

ProgrammeWednesday 13 th August, 200809:30Welcome Address by Professor Neal Copeland;Keynote Address: “Unfinished Genomes” by Professor SydneyBrenner10:20Wip1 phosphatase modulates dendritic morphology and learningand memory processes in hippocampus: Francesca Fernandez,XZC lab, GDD Division.10:50 Coffee Break and 1st Poster Session11:4012:10MRCK-LRAP35a-Myosin 18A Complex Regulates LamellarActomyosin Flow Essential for Cell Migration: Ivan Tan, GSKlab, GDD Division.An intronic Runx1 enhancer marks hematopoietic stem cells:Cherry Ng, RUNX lab, GGD Division.12:40 Lunch14:0014:3015:00Pin1 plays a role in zebrafish neuromasts development bystabilizing NeuroD: Steven Fong, VK lab, CDCBD Division.c-Cbl’s polygamy explained: Jan P. Buschdorf, GG lab, GGDDivisionProbing the genetic networks that regulate the Golgi apparatus:Susanne Ng, FB lab, SBD Division15:30 Coffee Break and 2 nd Poster Session16:3017:0017:30Aph2, a palmitoyl acyltransferase for phospholamban, is anessential regulator of cardiac physiological function and theonset of heart failure: Tielin Zhou, LBJ lab, CDCBD DivisionRegulation of p120ctn in E-cadherin-mediated intercellularadhesion strengthening: Yeh-Shiu Chu, JPT lab, SBD DivisionHappy Hour (sponsored by Invitrogen), including Prize AwardCeremony19:00 Thank You for Coming!!3

ABSTRACTS – ORAL PRESENTATIONSWip1 phosphatase modulates dendritic morphology and learning and memoryprocesses in hippocampusF. Fernandez, Q.H. Ma, S.H. Wei, L. Zeng, A.I. Soon, S.M. Wong, O. Demidov, G.Dawe, D. Bulavin , and Z.C. Xiao.Dendritic spines form the postsynaptic compartment of most excitatory synapses in thebrain, undergoing morphological changes in response to neuronal activity. Thiscorrelation between dendritic morphology and synaptic plasticity may contribute tolearning and memory, but the mechanisms underlying this association remain unclear.The polymerization and/or depolymerization of actin filament plays a key role in thespine motility, growth and shape. Several Protein Kinases (including mitogen-activatedprotein kinase (MAPK)) and protein phosphatases (PP) such as PP1 families, present inthe post synaptic compartment, are essential in the regulation of actin filament and/ordendritic morphology, and also in the synaptic plasticity. The PP wip1, encoded byoncogene ppm1d, participates to the regulation of the protein F-actin. In this study, weinvestigated the role of wip1 in dendritic morphology within the CA1 hippocampalregion and its effect on the synaptic plasticity and learning memory, via behaviouraltesting on mice deficient of wip1 gene. We report a colocation of wip1 and p38 MAPK,main effectors of wip1 protein, at neuronal dendrites. Deficiency of wip1 leads to adecrease of dendritic spine size and of the dendrite density in pyramidal neurons. Thesemorphological dendritic changes are accompanied by an impairment of memory in wip1deficient mice and by a reduction of the Long-Term Potentiation measured in the CA1hippocampal area. We also demonstrated that p38MAPK may contribute to modificationof the synaptic activity observed in the CA1 hippocampal region of wip1 deficient mice,but not to the morphological dendritic changes.4

MRCK-LRAP35a-Myosin 18A Complex Regulates Lamellar Actomyosin FlowEssential for Cell MigrationIvan Tan, Jeffery Yong, Jing Ming Dong, Louis Lim and Thomas Leung(GSK-IMCB Group)Cell migration is important in both development and diseases. It’s a cyclical processinvolving membrane protrusion at leading edge, movement of cell body and retraction ofcell rear. Actomyosin retrograde flow underlies the contraction essential for cell motility.Both lamellipodial and lamellar retrograde flows are essential for membrane protrusionand generation of contractile force by coupling to cell adhesion. We recently found thatRac/Cdc42-binding kinase MRCKa/b, a novel adaptor LRAP35a and myosin II-relatedMYO18A form a functional tripartite complex that is specifically colocalized with andregulates the lamellar retrograde flow required for persistent cell protrusion and asubnuclear actomyosin network. LRAP35a binds independently to MYO18A and MRCK;the binding leads to MRCK activation and its phosphorylation of MYO18A-associatedregulatory myosin light chain on Serine-19, independent of ROK and MLCK. Live cellimaging showed that MRCK-complex moves in concert with the retrograde actomyosinflow, allowing it to regulate the flow continuously. In addition, MRCK-complex wasfound to be functionally linked to specific MYO2A-network known to be responsible forlamellar actomyosin flow and force generation. The promotion of persistent protrusiveactivity and inhibition of cell motility by the respective expression of wild type anddominant negative mutant components of the MRCK-complex show it to be crucial tocell protrusion and migration. Taken together, our findings point to specific roles ofMRCK-complex in actomyosin regulation, and they provide new insights on theregulatory mechanisms underlying the coordination of cellular actomyosin in cellmigration.5

An intronic Runx1 enhancer marks hematopoietic stem cellsCherry Ee Lin Ng, Namiko Yamashita, Giselle Nah, Bindya Jacob, Chelsia Qiuxia Wang,Hao Jin, Zilong Wen, Motomi Osato, Yoshiaki ItoInstitute of Molecular and Cell Biology, Singapore, 138673, Oncology Research Institute,National University of Singapore, Singapore 117597RUNX1 is a transcription factor critical for generation and maintenance of hematopoieticstem cells (HSC) and is frequently mutated in human leukemias. Germline RUNX1mutations cause familial leukemia, named FPD/AML. Despite such widespreadinvolvement in diseases, the precise transcriptional regulation of Runx1 remains largelyunknown due to the extremely large size (approximately 1.0 Mb) of its gene locus. Wefirst took a comparative genomics approach and identified 93 conserved non-codingelements (CNE). In addition, we utilised retroviral integration sites (RIS) information asRIS are potential indicators of active chromatin region. Using this novel approach wehighlighted 10 CNE. These CNE were individually fused with EGFP and injected intozebrafish embryos to assess their function in vivo. We identified a mouse CNE (+24.1mCNE) which drives GFP expression in the intermediate cell mass (ICM) region, whichis a known site of endogenous Runx1 expression and hematopoiesis in zebrafish.Quantitative chromatin-immunoprecipitation (qChIP), using antibodies against modifiedhistones which mark either open or close regions of chromatin, showed a strongcorrelation between state of chromatin structure at the +24.1 mCNE locus and expressionof Runx1. To further characterize this element, +24.1 mCNE-GFP transgenic mouse lineswere generated. Analyses of mouse embryos revealed that this CNE is active in emergingHSC. In adult bone marrow, +24.1 mCNE-driven GFP+ cells were highly enriched forHSC. These results support the role of +24.1 mCNE as a HSC enhancer. +24.1 mCNE-GFP transgenic mice hold the promise to be a powerful tool for research on stem cell andcancer biology.Reference:Ng CEL, Osato M, Ito Y. Retroviral integration sites (RIS) mark cis-regulatoryelements. Critical Reviews in Hematology/Oncology, in press.6

Pin1 plays a role in zebrafish neuromasts development by stabilizing NeuroDSteven Fong 1 , Li-Qun Chao 2 , Vladimir Korzh 1 , Yi-Cherng Liou 21. Institute of Molecular and Cell Biology, Singapore.2. Department of Biological Sciences, National University of Singapore, Singapore.The vertebrate pSer/pThr-Pro specific peptidyl-prolyl-isomerase Pin1 has been shown toplay important roles in cell cycle regulation, apoptosis, oncogenesis, and neuronaldegeneration. However, its role in neuronal development is not clear. We clonedzebrafish Pin1 and investigated its interaction with bHLH factors involved in neuronalspecification. Pin1 is expressed maternally and in a ubiquitous manner early indevelopment, but by 48 hpf it is restricted to the brain and neuromasts. Coimmunoprecipitationassays (CoIP) in cell lines showed that Pin1 interacts with neuroDbut not ngn1 and atoh1a. This binding was reduced when Ser/Thr phosphorylation siteswere mutated. Antisense morpholino oligonucleotide (MO) knockdown of Pin1 led todelay in development. Accounting for the delay, neuroD expression was significantlydiminished in the hindbrain of morphants by 48 hpf equivalent. Morphants in thebackground of Tol2/GFP enhancer trap lines with specific expressions in hair cells (ET4)and mantle cells (ET20) also displayed defects in neuromasts formation. It has beenshown that specification of hair cells in neuromasts is neuroD dependent but ngn1independent. Using siRNA Pin1 knockdown 293T cells and Pin1 knockout MEF cells,we showed that neuroD protein is degraded more rapidly in the absence of Pin1. NeuroDstability was restored when Pin1 is over-expressed. This is the first study to demonstratethe functional regulation of a bHLH factor by post-phosphorylation cis-transisomerization in neuronal specification. In view of the role of Pin1 in neurodegenativediseases, our results may have important pharmaco-therapeutic applications.7

c-Cbl’s polygamy explainedCherlyn Ng, Rebecca A. Jackson, Jan P. Buschdorf, Qingxiang Sun, J. Sivaraman andGraeme R. GuyThe proto-oncogene c-Cbl inhibits several signaling proteins involved in cell growth andthe immune response. c-Cbl keeps in check, via ubiquitination, a number of tyrosinekinases implicated in cancer, including the epidermal growth factor receptor, the c-Metreceptor and c-Src. This requires binding of c-Cbl’s ‘tyrosine kinase-binding domain’(TKB), essentially a derivative SH2 (src homology 2) domain, to specific sequencesaround a phosphotyrosine residue. c-Cbl is unique among the other generally motifspecificphosphotyrosine binding domains in recognizing three unrelated motifs; how thishappens at the molecular level has been enigmatic. To understand the structural basis ofthis feat, we obtained X-ray crystallography structures of c-Cbl bound to five targetpeptide fragments. While the target phosphotyrosine forms several H-bonds with theTKB, only an H-bond from the phosphotyrosine to another residue within the target isconserved. The importance of this H-bond is demonstrated by mutagenesis and byconverting VEGFR1, a receptor not recognized by c-Cbl, into a c-Cbltarget. Surprisingly, one target, c-Met, binds to c-Cbl in the reverse orientation. Thestudy explains how c-Cbl manages to regulate such a diverse set of targets.8

Probing the genetic networks that regulate the Golgi apparatusSusanne San San Ng, Victor Racine and Frederic BardThe Golgi apparatus in mammalian cells is a dynamic and complex organelle regulatedby multiple fluxes of membranes and material. The molecular pathways that orchestratethis complex organization are still poorly understood. We have used a functionalgenomics approach, combining high-throughput RNA interference (RNAi) screeningwith automated microscopy to characterize the genes involved in regulation of the Golgistructure. Hela cells were stained for three different Golgi markers specific for the cis,medial and trans cisternae, and a nuclear dye. Automated image analysis extractsnumerical features from the images. To explore the range of phenotypes that could beobtained after RNAi, we tested a set of known regulators of membrane trafficking. Wefound that Golgi phenotypes can be used to reconstruct functional pathways. For exampletargeting genes implicated in ER to Golgi traffic yields a specific dispersion of the cisGolgi marker. By contrast, the trans Golgi marker is specifically affected when Golgi toplasma membrane traffic genes are targeted. A preliminary systematic screen coveringthe human kinome reveals several classes of Golgi regulating kinases. Some kinasephenotypes suggest a regulation of ER to Golgi traffic while other kinases appear to act atthe trans side of the Golgi. Overall, our preliminary results indicate that up to 70 kinasesare involved in regulating various aspects of Golgi organization. This figure is muchlarger than expected and suggest that a significant number of unknown regulatorynetworks impinge on the secretory pathway and on the Golgi apparatus.9

Aph2, a palmitoyl acyltransferase for phospholamban, is an essential regulator ofcardiac physiological function and the onset of heart failureTielin Zhou (LBJ lab)Reversible protein palmitoylation regulates many aspects of cell function and is largelycarried out by zf-DHHC containing proteins. However, the in vivo physiological functionof protein palmitoylation is largely unknown. To address this question, we generatedAph2-/-(zf-DHHC16) mice, revealing an essential role for Aph2 in postnatal survival andheart development, evidenced by histological/electrophysiological defects and neonatalonset of cardiomyopathy and heart failure. Aph2 is shown to be a palmitoyl acyltransferase for phospholamban (PLN), where palmitoylation on cysteine 36 promotesPLN nucleus envelope localization, its interaction with PKA, and subsequent serine 16phosphorylation. Aph2 deficiency led to PLN hypophosphorylation, defects in PLNpentamer formation and Ca2+ homeostasis. Deletion of PLN in Aph2-/- mice rescuedseveral of the heart phenotypes.These findings establish that Aph2 is a critical in vivo regulator of heart contractilefunction, partially via the modification of PLN, and that Aph2-/- mice represent acongenital heart disease model in which one member of PATs is proven to be involved incardiomyopathy formation, thereby identifying a novel therapeutic target forcardiomyopathy and heart failure.10

Regulation of p120ctn in E-cadherin-mediated intercellular adhesion strengtheningYeh-Shiu Chu, Pierre Nassoy, Alexander Bershadsky, Sylvie Dufour, Jean-Paul Thieryp120ctn is a member of the catenin protein family that controls a wide range of cellularprocesses, including cell adhesion, intracellular signalling, and transcriptional regulation.p120ctn associates with the juxta-membrane domain of E-cadherin via armadillo repeats.The armadillo repeats constitute a core region which indirectly associates with Rho smallGTPases, such as Rac and Cdc42. Although the key roles of p120ctn as a regulator ofcadherin transport from the Golgi complex to the plasma membrane and for cadherinstabilization at the plasma membrane are well-established, the contribution of p120ctn tocadherin-mediated adhesion remains to be investigated.We show here that the strengthening of E-cadherin-mediated cell adhesion is abrogatedby transient expression of an E-cadherin mutant lacking the p120ctn binding site insarcoma S180 cells. In addition, TIRF analysis of the spreading on EcadFC-coatedsurfaces of cells expressing GFP-tagged cadherins indicates that uncoupling of p120ctnfrom cadherin prevents cadherin adhesions and induces increasing lamellipodia formation.The treatment of cells with the Rac inhibitor NSC23766 partially rescues formation ofcadherin adhesions. p120 ctn knockdown experiments shows that reducing p120 ctn incells also weakens E-cadherin-mediated adhesion strengthening. Our data highlight thefact that the association of p120ctn with E-cadherin is important to strengthen thedevelopment of adhesion, possibly by actin cytoskeletal reorganization11

ABSTRACTS CONTENTS – POSTER PRESENTATIONSPosterS/NPresenter Group Title1 Choong‐Tat Keng CAVR2 Ning Sheng Liu HWJ3 Honghui Huang PJR4 Sharon Ling SHW5 Portia G. Loh SHW6 William Go VK7 Igor Kondrychyn VK8 Kar Lai Poon VKGeneration of SARS coronavirus escapemutants by using monoclonal antibodiesthat block membrane fusionTom1L1 as a regulated adaptor for EGFstimulatedendocytosis of EGF receptorMypt1‐Mediated Spatial Positioning ofBmp2‐Producing Cells Is Essential forLiver OrganogenesisCrystal structure of human Edc3 and itsfunctional implicationsStructural basis of translation inhibitionby PDCD4Plasma membrane Ca2+‐ATPase 1 isrequired for lateral line development inzebrafishDevelopmental analysis of the novelzebrafish gene, zic6.Analysis of zebrafish inducible nitricoxide synthase genes duringembryogenesis9 Terk Shin Teng CXM Cytoplasmic role of Stat3 in cell migration10 Alison P Lee FUGE/SB11 Samantha Quah ZQIdentification and characterization ofconserved cis‐regulatory elementsassociated with transcription‐factorencoding genes in the human genomePRL‐3 Initiates Tumor Angiogenesis byRecruiting Endothelial Cells In vitro andIn vivo12 Khay Chun Ang BR Structure of Actin in the Nucleus13 Shalini Nag BR Nuances of Gelsolin Function12

14 Hai Yun See DPLDesigning a synthetic tumour suppressorprotein to validate eIF4E as a potentialanti‐cancer target.15Joanne Zhi HuiChiaFBSNARE genes expression regulation uponvariation of secretory cargo load16 Fa‐Xing Yu LY17 Mei Chin Lee LY18 Jun Yan LY19 Jenn Hui Khong US20 Keng Boon Wee USAdenosine‐containing Molecules AmplifyGlucose Signaling and Enhance TxnipExpressionDissecting a Drosophila melanogasterHistone Transcriptional RegulationPathwayNm23 and Histone H2B gene expressionin HeLa cellsA Mechanism that prevents prematurechromosome segregation when initiationof DNA replication failsRoles of intracellular p53 oscillations incell survival and death21 Kelly Hogue WB Mass Spectrometry in IMCB22 Jasmine Lee ZLH23 Chiaw‐Hwee Lim JYJ24 Ashish Maurya PWI25 Hoe Peng Liew SR26 Hao Zou WYNon‐ribosomal peptides in Pseudomonasaeruginosa quorum sensing regulationLoss of udu function causes activation ofDNA damage checkpointEngrailed gene expression and its controlby hedgehog signalingSpecification of vertebrate slow‐twitchmuscle fiber fate by the transcriptionalregulator Blimp1G‐actin binds to the adenylyl cyclase Cyr1through Cap1 and regulates cAMPsynthesis in C. albicans hyphalmorphogenesis13

27 Yun‐Hua Zhu XZC28 Lihui Goh YXH29 Zhenxing Huang YXHWip1 regulates neural stemcell/progenitor proliferation through p53dependent G2 cell cycle controlMouse embryonic neural stem cells: Amodel for asymmetric cell divisionSearching For Novel Proteins Involved InAsymmetric Cell Division30Joan Siew ChingLeeYXHApim genetically interacts withInscuteable in the dividing Drosophilaneuroblast31 Valentina Migliori EG32 Cheng Chun Wang HWJ33 Cathleen Teh VK34 R.N.Mahalakshmi WH35 Shuo Shen CAVR36 Lei Li AGP37 Jenny Chau LBJIdentification of novel epigeneticregulatorsVAMP8 Is Required for Cell SurfaceDeployment of the Water ChannelAquaporin 2Wnt3 is Essential for Dorsal DiencephalicDifferentiationA regulatory role for Kir/Gem and thenovel muscle‐specific protein CCDC95 inmyogenesisComparing the antibody responsesagainst recombinant hemagglutininproteins of avian influenza A (H5N1)virus expressed in insect cells and bacteriaJNK‐dependent activation of p53 and c‐Jun is required for nitric oxide‐inducedapoptosis of tumor cellsSmad1 provides a link between BMP‐Smad signaling and DNA damageresponse and a target for cancer therapy38 Canhe Chen NNHarnessing piggyBac Transposons forcancer gene discovery in Zebrafish14

39 Bindya Jacob RUNX40 Shreeram Sathya BD41 San San Lee VY42 Permeen Yusoff GG43 Lu Hao CXMStem cell exhaustion due to Runx1deficiency is prevented by Evi5 activationin leukemogenesisCdc25A Serine 123 phosphorylationcouples centrosome duplication withDNA replication and regulatestumorigenesisTrim39 is a MOAP‐1 binding protein thatstabilizes MOAP‐1 through inhibition ofits polyubiquitination processA complex affair between two tumoursuppressors and a famous regulatorGRIM‐19 Is Essential for Maintenance ofMitochondrial Membrane Potential44 Hsiang Ling Teo VT New Controls of the NFkB Pathway45 Xinde Zheng PRK46 Shuhei Kotoshiba PRK47 Haihe Wang ZQ48 Rashmi P Kulkarni FUGE/SB49 Chit Fang Cheok DPLCharacterization of Speedy/RINGO, anew family of cyclin‐like proteinsRb/Cdk2/Cdk4 triple mutant mice elicit anew mechanism for G1/S regulationPRL‐3 Down‐regulates PTEN Expressionand Signals through PI3K to PromoteEpithelial‐Mesenchymal TransitionEvolutionary changes in the regulatoryregion of vertebrate erythropoietin genelocusMolecular therapy : Targeting the p53pathway using small molecules50Christopher JBrownDPLCrystallographic analysis of cap free andcap bound eIF4E with N7 alkylated capderivatives51BalakrishnanKannanBRSpatio‐temporal Mapping of ActinPolymerisation Regulated by Actin‐Binding Proteins15

52 Marten E Larsson BRDirected Evolution of Actin by In VitroCompartmentalization53JayanthaGunaratneWBTracking cellular events using stableisotopelabeling by amino acids in cellculture (SILAC)54 Ya‐Wen He ZLH55 Dimitri Moreau FB56 Xiao Xing Wang LDX57 Tao Zhang US58 Xin Gang Wang PWI59 Shang‐Wei Chong JYJ60 Xuehui Qiu JYJSynergistic regulation of Xanthomonascampestris virulence and adaptation byDSF signaling system and a novel twocomponentsignal transduction systemRavS/RavRProbing the retrograde transport pathwayby RNA interference screeningInhibition of PKR Activation and Upregulationof GADD34 Expression play asynergistic role in the maintenance of denovo Protein Synthesis in Coronavirus‐Infected CellsDNA damage checkpoint maintains Cdh1in quasi‐active state to prevent spindleelongation and the completion ofAnaphaseSearching for novel factors involved inneutrophil chemotaxis by chemicalgeneticsZebrafish nicastrin is required formidbrain and hindbrain developmentTemporal Notch activation throughNotch1a and Notch3 is required formaintaining zebrafish rhombomereboundaries61 Ka Fai William Tse JYJ The Characterization of Novel mib Alleles62 Semil P. Choksi SRIdentifying gene regulatory networks thatcontrol zebrafish regeneration16

63 Xianwen Yu SR64 Yijun Yin YXH65 Chang Run Li WYThe Forkhead domain containingtranscription factor Foxj1 is the masterregulator of the motile ciliogenic programA mouse embryonic stem/progenitor cellsas a model to investigate asymmetric celldivisionCDK regulates cytokinesis byphosphorylation of an IQGAP familyprotein in fungal pathogen Candidaalbicans66YohendranBaskaranGSKRegulation of PAK4, a non‐conventionalPAK17

ABSTRACTS – POSTER PRESENTATIONS(1) Generation of SARS coronavirus escape mutants by using monoclonal antibodiesthat block membrane fusionKeng Choong-Tat (CAVR lab)We have previously shown that a bacterially expressed, denatured spike (S) proteinfragment of the severe acute respiratory syndrome (SARS) coronavirus, containingresidues 1029 to 1192, which include the heptad repeat 2 (HR2) domain, could induceneutralizing polyclonal antibodies (Keng et. al., 2005. J. Virol. 79:3289-3296). We havealso produced monoclonal antibodies (MAbs) against this fragment to identify the linearneutralizing epitopes in this functional domain (Lip et al., 2006. J. Virol. 80:941-950).Eighteen hybridomas secreting the S protein-specific MAbs were obtained. Binding sitesof these MAbs were mapped to 4 linear epitopes. Two of them were located within theHR2 region and two immediately upstream of the HR2 domain. MAbs targeting to theseepitopes show in vitro neutralizing activities and can inhibit cell-cell membrane fusion.These results provide evidence of novel neutralizing epitopes that are located in the HR2domain and the spacer region immediately upstream of it. Using these MAbs, wegenerated SARS coronavirus escape mutants by growing the virus at an antibodyconcentration that reduces the virus concentration by 4 logs. After several passages, wemanaged to isolate viruses that are no longer neutralized by the MAbs. The spike proteinof the escaped viruses was sequenced to identify crucial mutations that enable the virus toescape the inhibition by the MAbs. How such mutations affect the entry ability of theSARS coronavirus will be further investigated using a pseudotyped virus system. Thedynamics of the growth and viability of the escape viruses will also be studied later.18

(2) Tom1L1 as a regulated adaptor for EGF-stimulated endocytosis of EGF receptorNing Sheng Liu (HWJ lab)The molecular mechanism governing ligand-stimulated endocytosis of receptor tyrosinekinases remains elusive. I show here that EGF stimulates transient tyrosinephosphorylationof Tom1L1(Tom-Like 1) by the Src family kinases, resulting in itstransient interaction with the activated EGF receptor (EGFR) bridged by the receptorboundGrb2. Cytosolic Tom1L1 is recruited onto the plasma membrane and subsequentlyredistributes with EGFR into the early endosome. Mutant forms of Tom1L1 defective intyrosine-phosphorylation or interaction with Grb2 is incapable of interaction with EGFRand inhibits endocytosis of EGFR. In addition, siRNA-mediated knockdown of Tom1L1inhibits endocytosis of EGFR. The C-terminal tail of Tom1L1 contains a novel clathrininteractingmotif, which is important for exogenous Tom1L1 to rescue endocytosis ofEGFR in Tom1L1 knocked-down cells. These results suggest that EGF triggers atransient association of EGFR with Tom1L1 to engage the endocytic machinery forendocytosis of the ligand-receptor complex. Moreover, Tom1L1 interacts with ubiquitinand ESCRT family proteins, such as: Hrs, TSG101, STAM1/2, and it is recruited toendosome upon over-expression HA-Hrs. These results suggest that Tom1L1 couldparticipate in the machinery for EGFR sorting and degradation. In addition, Tom1L1negatively regulates Ras activation upon EGF stimulation and A431 colony formation,which indicate that it may play a negative role in Src kinase signaling.19

(3) Mypt1-Mediated Spatial Positioning of Bmp2-Producing Cells Is Essential forLiver OrganogenesisHonghui Huang 1,5 , Hua Ruan 1,5,7 , Meng Yuan Aw 1,7 , Alamgir Hussain 1,7 , Lin Guo 1,5,7 ,Chuan Gao 5 , Feng Qian 1 , Changqing Chang 1 , Thomas Leung 2 , Haiwei Song 3 , DavidKimelman 6 , Zilong Wen 4* and Jinrong Peng 1*1 Laboratory of Functional Genomics, 2 Laboratory of Neural Differentiation andDegeneration, 3 Laboratory of Translation Termination and Messenger RNA Decay, 4Laboratory of Molecular and Developmental Immunology, Institute of Molecular andCell Biology, 61 Biopolis Drive, Proteos, Singapore 138673. 5 Department of BiologicalSciences, National University of Singapore, Singapore 117543. 6 Department ofBiochemistry, University of Washington, Seattle, WA, 98195-7350, USA7 These authors contributed equally to this work.*To whom correspondence should be addressed. E-mail: pengjr@imcb.a-star.edu.sg;zilong@ust.hk;Mesodermal tissues produce various inductive signals essential for morphogenesis ofendodermal organs. However, little is known about how the spatial relationship betweenthe mesodermal signal-producing cells and their target endodermal organs is establishedduring morphogenesis. Here we report that a mutation in the zebrafish myosinphosphatase targeting subunit 1 (mypt1) causes abnormal bundling of actin filaments anddisorganization of lateral plate mesoderm (LPM) cells. As a result, the coordinationbetween mesoderm and endoderm cell movements is disrupted. Consequently, the twostripes of Bmp2a-expressing cells in the LPM fail to align in a V-shaped pocketsandwiching the liver primordium. Mispositioning Bmp2a producing cells with respect tothe liver primordium leads to a reduction of hepatoblast proliferation and final abortion ofhepatoblasts by apoptosis that causes the liverless phenotype. Our results demonstratethat Mypt1 mediates coordination between mesoderm and endoderm cell movements inorder to carefully position the liver primordium such that it receives a Bmp signal that isessential for liver formation in zebrafish.20

(4) Crystal structure of human Edc3 and its functional implicationsSharon Ling (SHW lab)Edc3p is an enhancer of decapping and serves as a scaffold that aggregates mRNAribonucleoproteins (mRNPs) together for P-body formation. It has a modular domainarchitecture consisting of an N-terminal Lsm domain, a central FDF domain and a C-terminal YjeF-N domain, which forms a network of interactions with the components ofthe mRNA decapping machinery. We have determined the crystal structure of the N-terminally truncated human Edc3 at a resolution of 2.2Å. The structure reveals that theYjeF-N domain of Edc3 possesses a divergent Rossmann fold topology that forms adimer, which is supported by sedimentation velocity and sedimentation equilibriumanalysis in solution. The dimerization interface of Edc3 is highly conserved in eukaryotesdespite the overall low sequence homology across species. Structure based site-directedmutagenesis revealed dimerization is required for efficient RNA binding, P-bodyformation and likely for regulating the yeast Rps28B mRNA as well, suggesting that thedimeric form of Edc3 is a structural and functional unit in mRNA degradation.21

(5) STRUCTURAL BASIS OF TRANSLATION INHIBITION BY PDCD4Portia G. Loh 1,2 , and Haiwei Song 1,21 Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673,2 Department of Biological Sciences, National University of Singapore, Lower Kent RidgeRoad, Singapore 117543Pdcd4 is a tumor suppressor protein thought to be an inhibitor of tumor progression andpromoter of apoptosis. The mechanism by which it does so involves interaction with theeukaryotic translation initiation factor eIF4A that functions to unwind secondary structurein mRNA in an ATP-dependent fashion. Loss of Pdcd4 is thought to contribute to celltransformation. Decreased expression of Pdcd4 has been observed in lung, breast,prostate, liver tumors and neuroendocrine carcinomas. To better understand theregulation of eIF4A by Pdcd4, we present here the crystal structures of the MA3 domainsof Pdcd4 at 2.8Å resolution and that of Pdcd4 in complex with eIF4A at 3.5Å resolution.To confirm the Pdcd4 and eIF4A binding interface as seen in the structure, we haveidentified a conserved surface on Pdcd4 extending across both tandem MA3 domains aswell as specific residues on eIF4A for mutagenesis. We analyzed the binding of eIF4A toa series of Pdcd4 mutants in vitro, and vice versa. Mutational analyses have also revealedthat inhibition of ATP hydrolysis induced by interaction with Pdcd4 correlates withconcomitant reduction of RNA unwinding, and that these mutants reduce the ability ofPdcd4 to inhibit the enzymatic activities of eIF4A. These same mutants also reduce theinhibitory effect of Pdcd4 on AP-1 transactivation. Based on our structures and availablestructures of other RNA helicases, we will present a model of the mechanism oftranslation inhibition by Pdcd4.22

(6) Plasma membrane Ca2+-ATPase 1 is required for lateral line development inzebrafishWilliam Go 1 , Dmitri Bessarab 2 , Vladimir Korzh 11 Institute of Molecular and Cell Biology, Singapore2 Institute of Molecular Biology, SingaporeThe plasma membrane calcium ATPase (PMCA) transports Ca2+ ions out of the cells byATP hydrolysis (Ca2+ pump). It is essential in the control of Ca2+ concentration in thecytosol to maintain a low cytosolic free Ca2+ concentration for the correct functioning ofcells. In mammals, there are four different genes that generate four isoforms (PMCA1,PMCA2, PMCA3, and PMCA4) by alternative splicing. The efforts so far were focusedmainly on isoforms’ physiological significance. Expression of some isoforms started veryearly during embryogenesis but PMCA role in development has not been demonstrated(Sepúlveda et al., 2007).To facilitate developmental studies, enhanced green fluorescent protein (EGFP) geneplaced under control of the keratin8 promoter was used as a reporter in the Tol2transposon based enhancer trap (ET) designed in our laboratory (Parinov et al., 2004). Inline ET4, Tol2 element insertion occurred 100kb upstream of the gene coding forPMCA1. Expression of EGFP faithfully recapitulates the expression of PMCA1 in brain,muscle, inner ear and lateral line neuromasts. During posterior lateral line development,pmca1 transcripts were first detected in the presumptive hair cell precursors locatedwithin the migrating primordium.We used morpholino approach to address the role of PMCA1 in early zebrafishdevelopment and found that abnormalities of development were detected in all organs ortissues expressing pmca1. We focused on the role of PMCA1 in the lateral linedevelopment and our results suggest that PMCA1 is required during development oflateral line from proneuromast deposition to maturation of functional neuromast.References:1. Sepúlveda MR, Hidalgo-Sánchez M, Marcos D, Mata AM. (2007), Developmental distribution of plasmamembrane Ca2+-ATPase isoforms in chick cerebellum, Dev Dyn.;236(5):1227-36.2. Parinov S, Kondrichin I, Korzh V, Emelyanov A. (2004), Tol2 transposon-mediated enhancer trap toidentify developmentally regulated zebrafish genes in vivo. Dev Dyn.;231(2):449-59.23

(7) Developmental analysis of the novel zebrafish gene, zic6.Kondrychyn, Igor and Korzh, VladimirThe zic (zinc finger protein for cerebellum) gene family encoding transcription factors invertebrates is comprised of five members, which play a role in formation of the dorsalneural tube. Developmental analysis of zic genes showed that during the early stage ofneurogenesis they act as pre-pattern genes downstream of BMPs and upstream of Wnts.We isolated and characterized the novel member of the zebrafish zic gene family, zic6,which 3’-untranslated region was tagged by insertion of Tol2 transposon in the ET33enhancer trap transgenic zebrafish line (Parinov et al., 2004). Genomic analysis showedthat in contrast to all other zic’s this gene is present only in teleost phyla. Probably, it waslost during later evolution. During development zic6 mRNA was first detected by RT-PCR at an early blastula stage. By whole mount in situ hybridization zic6 expression wasdetected soon after bud stage at the prospective midbrain-hindbrain boundary and dorsalmidbrain followed by dorsal hindbrain and pretectum. At the later stages, zic6 continuesto be expressed in the dorsal neural tube, in particular, the roof plate. The inhibition ofzic6 translation by antisense morpholino oligonucleotide knockdown revealed that zic6plays a role during morphogenesis of the roof plate cells.24

(8) Analysis of Zebrafish Inducible Nitric Oxide Synthase Genes DuringEmbryogenesisKar Lai Poon (VK lab)Inducible nitric oxide synthase (NOS2) catalyzes the production of nitric oxide (NO), andis one of the players establishing innate immunity. In zebrafish, nos2 is represented bynos2a and nos2b. Here, we report the cloning and expression pattern of the zebrafishnos2b genes, which does not participate in immune defence. nos2b was mapped tozebrafish linkage group 15. The spatial and temporal expression pattern of nos2b inembryonic zebrafish was analyzed by whole mount in situ hybridization. Duringembryogenesis nos2b is expressed constitutively in a highly restricted manner.Expression of zebrafish nos2b appeared at 14 hpf in the forebrain. Later on it was presentin the hypothalamus, where initially it was expressed in the ventral-most cell layer.During late embryogenesis, expression of nos2b was detected immediately posterior tothe adenohypophysis and later on a low level was present in the midline ventral to MHBthat move rostrally. By 72 hpf, the only expression domain of nos2b is associated withthe middle lower jaw.25

(9) Cytoplasmic role of Stat3 in cell migrationTerk Shin Teng (CXM lab)Stat3 is a member of the signal transducer and activator of transcription protein family,which is important for cytokine signaling as well as a number of cellular processesincluding cell proliferation, anti-apoptosis and immune responses. In recent years, thereis emerging evidence that Stat3 also participates in cell invasion and motility. However,how Stat3 regulates these processes still remains poorly understood. In our current study,we use Stat3-deficient and wild-type Stat3-expressing mouse embryonic fibroblasts(MEFs) as a model to investigate the role of Stat3 in cell migration. We find that loss ofStat3 expression leads to an elevation of Rac1 activity, which promotes a random modeof migration by reducing directional persistence. By performing rescued experiments, wedemonstrate that Stat3 can regulate the activation of Rac1 to mediate persisted directionalmigration and this function is not dependent on Stat3 transcriptional activity. We findthat Stat3 binds to -Pix and this interaction could represent a mechanism, whichcytoplasmic Stat3 regulates Rac1 activity to modulate the organization of actincytoskeleton and directional migration.26

(10) Identification and characterization of conserved cis-regulatory elementsassociated with transcription-factor encoding genes in the human genomeAlison P Lee (FUGE/SB lab)Molecular Genetics Laboratory, Institute of Molecular and Cell BiologyComparative genomics is a powerful strategy for identifying conserved gene regulatoryelements in the human genome. We have used this approach for identifying andcharacterizing conserved cis-regulatory elements associated with genes that encodetranscription factors (TFs) in the human genome. TFs represent key nodes in theregulatory networks of important biological processes, and understanding the molecularbasis of regulation of TF-encoding genes is an important step towards decipheringregulatory networks. Human genome is estimated to contain about 2,000 TF-encodinggenes. We created a database of human, mouse and fugu TF-encoding genes andidentified orthologous genes in the three genomes. The genomic sequences of theorthologous loci in the three genomes were aligned using a global alignment program andconserved noncoding elements (CNEs) were predicted using VISTA. These CNEsrepresent regulatory elements under evolutionary selection. A total of 2,843 CNEsassociated with 389 human TF-encoding genes were identified. TF-encoding genes thatare expressed in the nervous system and involved in development contain a significantlyhigher number of CNEs. We predicted four 8-bp motifs that are enriched in CNEs ofcentral nervous system genes. These motifs could be the binding sites of transcriptionfactors important for gene expression in the central nervous system. The functions of aselected set of CNEs are being assayed in transgenic mice. The database of vertebratetranscription factors and the CNEs associated with them, known as TFCONES, is madeavailable in the public domain (http://tfcones.fugu-sg.org/).27

(11) PRL-3 Initiates Tumor Angiogenesis by Recruiting Endothelial Cells In vitroand In vivoSamantha Quah (ZQ lab)Metastasis is the terminal stage of cancer progression. It involves the transformation ofprimary tumors to malignant secondary tumors, and is a multi-step process. Themembers of the PRL (Phosphatase of Regenerating Liver) family belong to a subclass ofprotein tyrosine phosphatases. PRL-3 has been linked to promote cell migration,invasion and metastases. PRL-3 is proposed to be an indicator of poor prognosticoutcome. The molecular mechanisms by which PRL-3 operates in metastasis are onlyjust beginning to be understood. We have investigated the possible functions of PRL-3 inboth physiological and tumor angiogenesis, which is an important step in metastasis.We investigated PRL-3 expressing cells’ ability to crosstalk to human umbilical vascularendothelial cells (HUVEC) using an in vitro co-culture system. HUVEC were grown withfibroblasts, and later overlaid with PRL-3-expressing cells. PRL-3-expressing CHO cellsredirected the migration of HUVEC towards them; while PRL-3-expressing DLD-1 cellsenhanced HUVEC vascular formation. In vivo injection of Matrigel plugs containingPRL-3-expressing CHO cells into nude mice to form local tumors resulted in therecruitment of host endothelial cells (ECs) to the vicinity of the tumor sites and initiationof angiogenesis. Our results suggest that PRL-3 may play a role in initiatingestablishment of microvasculature. We further show that PRL-3 expressing cells reducedIL-4 expression levels, attenuating IL-4 inhibitory effects on the HUVEC vasculature.Our findings provide direct evidence that PRL-3 may be involved in triggeringangiogenesis and may serve as an attractive therapeutic target with respect to bothangiogenesis and cancer metastasis.28

(12) Structure of Actin in the NucleusKhay Chun Ang (BR lab)The existence of actin in the nucleus has been controversial. Traditional cystoplasmicmarkers for actin, such as antibodies and phalloidin, have indicated that conventionalactin filaments do not generally exist in the nucleus. However, over the last few yearsactin has been identified as an integral subunit of the transcription apparatus, DNA andRNA modifying complexes on top of other nuclear binding partners. Hence, actin mostdefinitely is present in the nucleus, however, nothing is known of its conformational state.Actin, as well as actin related proteins (Arps), are found to be integral subunits ofmultisubunit chromatin remodeling complexes (CRC), complexes intimately tasked toactivate genes in eukaryotes by relieving topological constraints exerted by nucleosomes.The role of actin within these complexes is unclear and therefore we believe that a 3-Dstructure approach will not only be vital in clarifying the paradigm but also provideinvaluable structural correlation of actin with existing biochemical and biophysicalobservations.Experimental approaches include purifying targeted nuclear actin binding proteins tohomogeneity, with the final intention of co-crystallization with actin. In addition, atandem affinity purification (TAP)-tag approach is being implemented to harvest native,multisubunit actin complexes and/or Arps containing nuclear complexes forcrystallographic studies. From these structures we expect to clarify the conformationalstate of actin and gain insight into its nuclear roles.29

(13) Nuances of Gelsolin FunctionShalini Nag (BR lab)The gelsolin superfamily of calcium-activated, actin binding proteins regulates actindynamics by severing, capping, nucleating or bundling actin filaments. Expressed in awide range of eukaryotes, they regulate cell motility and phagocytosis, transduce signalsinto dynamic cytoskeletal rearrangements, stimulate apoptosis in some vertebrate cells,and regulate transcription. Due to their varied functions the misregulation of theseproteins results in pathological conditions like inflammation and diseases such as cancerand amyloidosis. Proteins of this family are composed of 100 – 125 residue homologousmodules that are folded into compact domains. Movement between these domains isresponsible for the diverse activities of these proteins. Gelsolin, the founding member ofthis family, has 6 such domains connected by linkers of various lengths. Other membersdiffer in the number of domains, or have residue substitutions that tailor their expression,localization and regulation to the appropriate regions of motile cells. Although 3 decadesof studies have provided numerous insights, the holy grail of the gelsolin field, amolecular movie of this dynamic molecule as it undergoes large conformational changesduring calcium activation and function, has been elusive because of the speed of the actinsevering reaction, multiplicity of steps in the reaction, small size of gelsolin, and theinherently dynamic nature of actin. In an effort to contribute to this molecular movie, Ipresent the structure of human gelsolin domains G1 – G3 bound to actin, along withinsights about gelsolin obtained from various biophysical, biochemical and molecularbiology methods.30

(14) Designing a synthetic tumour suppressor protein to validate eIF4E as apotential anti-cancer target.See Hai Yun, Christopher J.Brown, David Coomber, David P.LaneUpregulation of translation initiation factor eIF4E has been found to selectively increasethe synthesis of oncoproteins, inducing tumourigenesis. Our study aims to design andconstruct a synthetic protein that will bind to and inhibit the tumourigenic activity ofeIF4E.12 amino acids derived from eIF4G, one of eIF4E’s endogenous binding partners, wereinserted between Gly33 and Pro34 of an Escherichia coli protein: thioredoxin (Trx). Thisinsertion disrupts the active site loop of the protein and allows the binding peptide to bepresented at the surface of the aptamer (Trx-eIF4G). Critical contact residues in thebinding amino acid sequence were mutated to alanines to act as the control aptamer (TrxeIF4G-ala).Immunoprecipitations demonstrated the ability of the Trx-eIF4G aptamer to bind toeIF4E, while there was negligible binding of the control aptamer Trx-eIF4G-ala. Theinsertion of additional residues in front of the peptide sequence to allow for increasedflexibility enhanced the binding affinity of the aptamer (Trx-SG-eIF4G). Fluorescentpolarization (FP) experiments using Trx constructs to compete with peptides for bindingfurther confirmed the interaction between the aptamer and its target protein eIF4E. TheFP assays corroborated with the pulldown assays demonstrating enhanced binding of theTrx-SG-eIF4G aptamer to eIF4E as compared to the original Trx-eIF4G aptamer.In vivo transformation assays that involve growing transfected cells on soft agar to testfor independence of contact inhibition (characteristic of tumourigenicity and malignancy)demonstrates the ability of the aptamer to inhibit the tumourigenic effects of eIF4E. Thecapacity of the aptamer to inhibit malignant cell growth in transformation assays wouldprovide strong evidence and support for further research to exploit the therapeuticpotential of eIF4E as an anti-cancer target.31

(15) SNARE genes expression regulation upon variation of secretory cargo loadChia Zhi Hui, Joanne and Frederic BardThe secretory apparatus in higher eukaryotes must be able to adapt to variation in theamount of secretory cargo. For example, terminal B cell differentiation requires anincrease biosynthetic capacity to synthesize massive amounts of antibodies. It remainspoorly understood how the secretory apparatus copes with increase in cargo load.In this study we have used an inducible system in Drosophila S2 cells wherebyexpression of secreted horseradish peroxidase (HRP) is induced in the presence of Cu2+ions. Using quantitative imaging to probe the intracellular HRP (HRPi) levels, we foundthat HRPi increases and decreases over time after Cu2+ induction. This suggests that thesecretory apparatus adapts to the increase in cargo load. To test if genes important forsecretion are being upregulated, we turned to the SNARE family of proteins. The exactSNAREs that regulate Golgi to plasma membrane traffic in Drosophila are currentlyunknown. A systematic survey of SNAREs by RNAi indicated that none of the SNAREcandidates is actually essential for secretion. However, using a co-RNAi screen of allSNAREs in the drosophila genome, we have identified Syntaxin1A, Syntaxin4, VAMP3and possibly VAMP2 as key SNAREs participating in the late secretory pathway.Following activation of secreted HRP production, we found that expression of bothVAMP3 and VAMP2 genes is upregulated 3 to 4 folds. Our results have revealedextensive SNARE functional redundancy in the Golgi to plasma membrane traffic. Theyalso indicate that the secretory apparatus is able to sense an increase of cargo load and toup regulate the expression of secretory genes in response.32

(16) Adenosine-containing Molecules Amplify Glucose Signaling and EnhanceTxnip ExpressionFa-Xing YU and Yan LUOGlucose uptake and utilization are tightly regulated and fundamental for cellularfunctions. Eukaryotic cells have developed different mechanisms that sense the glucoseand regulate the expression of certain genes involved in metabolism. The thioredoxininteractionprotein (Txnip) has been identified as a glucose sensor, for the expression ofwhich is tightly correlated with extracellular glucose concentrations. Here, we presentdata showing that, at a transcriptional level, the Txnip expression is induced by a novelclass of adenosine-containing molecules such as NAD(H), NADP(H), FAD, ATP, ADP,AMP and adenosine, and that an intact adenosine moiety is necessary and sufficient forthis induction. The induction is abolished in presence of glucose transporter inhibitors orupon glucose withdrawal, thus is critically dependent on glucose. This is consistent withthat the Txnip promoter contains a cis-regulatory element, carbohydrate response element(ChoRE), which mediates the induction. MondoA and MLX have been recentlydocumented to be ChoRE-binding transcription factor partners involved in the glucosestimulatedTxnip expression, and are shown here to be required to transmit stimulatorysignals from extracellular adenosine-containing molecules to the Txnip promoter. Ourstudies suggest that adenosine-containing molecules can amplify the glucose-mediatedsignaling and further stimulate the Txnip expression, and that the revealed function ofthese molecules might set the stage for developing novel pharmacological interventionsor modulations of certain cellular processes in which glucose plays fundamental roles.33

(17) Dissecting a Drosophila melanogaster Histone Transcriptional RegulationPathwayMei Chin LEE, Ling Ling TOH and Yan LUOMammalian OCA-S acts as an Oct-1 Co-Activator in S-phase that stimulates thetranscription of the histone 2B (H2B) gene. Oct-1 binds the essential octamer element inthe H2B promoter, whilst OCA-S occupies the promoter via an interaction with Oct-1.As a multi-component protein complex, OCA-S comprises of p38/GAPDH, p36/LDHand other components that are essential for OCA-S function. This exemplifiesmoonlighting nuclear gene-switching activities of certain metabolic enzymes, thusexpands the one-protein-multi-function paradigm.A similar regulatory pattern may be conserved in Drosophila melanogaster (dm). Wethus studied expression patterns of the dmH2B gene. The octamer elements in thedmH2B promoter are bound by Pdm-1, a counterpart of mammalian Oct-1; this binding isessential for dmH2B expression. We have developed an effective RNAi approach toanalyze functions of Pdm-1 and the components of the fly OCA-S (dmOCA-S) in the dmSchneider-2 cell line. Each dmOCA-S component is essential for H2B expression, andthe H2B promoter occupancy by dmOCA-S is critically dependent upon Pdm-1. This isin line with an OCA-S-Oct-1 interaction in mammalian cells, and suggests a conservedhistone expression program in higher eukaryotes. We are testing the functions of Pdm-1and dmOCA-S at an organism level--in embryos--and are using a genome-wide RNAiapproach to search for other potential transcription (co)-activators involved in regulatingthe expression of the dmH2B gene as well as other dm histone genes.34

(18) Nm23 and Histone H2B gene expression in HeLa cellsJun Yan (LY lab)OCA-S is a key coactivator that enables transcription factor Oct 1 to regulate histoneH2B transcription. OCA-S is composed of seven components including nm23 H1 andnm23 H2, two subunits of nucleotide diphosphate (NDP) kinase. To investigate the roleof nm23 H1/H2 in OCA-S coactivation of H2B transcription, we knocked down the H1and/or H2 transiently with siRNA; and created 3 types of stable knockdown cell lines forlong term effects by using shRNA. We found that the combination of siRNA against bothH1 and H2 decreased H2B expression by 30% at 72 hours post-transfection. In stableknockdown cells, H1 and double knockdown lines grew at a faster rate than parentalHeLa cells whereas H2 knockdown lines grew at a slower rate. Compared with HeLacells by real time PCR, all stable knockdown lines didn’t produce significant differencein overall H2B mRNA level. However, double knockdown lines showed a reduced H2Btranscription initiation by 70%-80% using luciferase reporter assay, suggesting theexistence of compensatory mechanisms at posttranscriptional level. Here we report themRNA stability increase by 50% in double knockdown lines at late S phase (6 hours afterrelease from thymidine); and stable knockdown lines had lower NDP kinase activities.These findings indicate that nm 23 H1 and H2 positively contributed to OCA-S mediatedH2B expression at transcription initiation and we further suggest that nm23 mightfunction through its NDP kinase activity.35

(19) A Mechanism that prevents premature chromosome segregation wheninitiation of DNA replication failsJenn Hui KhongUS lab, Systems Biology Division, Institute of Molecular and Cell BiologyCdc6, an evolutionarily conserved protein, is required for the initiation of DNAreplication and is essential for cell viability. Yeast cells deficient in Cdc6 fail to initiateDNA replication but proceed to elongate their spindles and segregate the un-replicatedchromosomes leading to a “reductional” anaphase. Therefore, Cdc6 has been defined asan important component of the G1-M checkpoint that prevents untimely onset of mitosiswhen cells fail to initiate DNA replication. Our results suggest that untimelychromosome segregation in the absence of Cdc6 function is not due to premature entryinto mitosis but is a result of the deregulation of spindle dynamics. This dramaticderegulation is a result of the interplay of three cellular events as cells proceed to DNAreplication: activation of the E3 ubiquitin ligase SCF, destruction of Cdk inhibitorSic1and inactivation of another ubiquitin ligase APCCdh1 (Anapahase PromotingComplex activated by Cdh1). Surprisingly, we find that premature chromosomesegregation is a not a property specifically associated with the loss of Cdc6 function but itis a common characteristic of cells (such as cdc7 or cdc45 mutants) that execute the threeevents but fail to initiate DNA replication. These results suggest that initiation of DNAreplication saves the cells from potential chromosome-segregation catastrophe which theconvergence of the three regulatory elements APC, SCF and Sic1 can cause.36

(20) Roles of intracellular p53 oscillations in cell survival and deathKeng Boon WeeUS lab, Systems Biology Division, Institute of Molecular and Cell Biology,NUS Graduate School for Integrative Sciences & Engineering, National University ofSingaporeIntracellular levels of p53 and MDM2 proteins have been shown to oscillate in responseto ionizing radiation (IR). The p53-MDM2 negative feedback loop – the putative causeof the oscillations – is embedded in a larger network involving a mutual antagonismbetween p53 and AKT. We have shown earlier that this p53-AKT network predicts anall-or-none switching behavior from a pro-survival cellular state (low p53 and high AKTsteady state) to a pro-apoptotic state (high p53 and low AKT steady state). Here, weshow that upon IR, this p53-AKT network model can also reproduce experimentallyobserved oscillations, and that these oscillations are possible only at pro-survival states.Notably, p53 oscillations markedly decrease the IR intensity threshold at which switchingto a pro-apoptotic state occurs. Several biological advantages conferred by suchoscillations are demonstrated, including the increased ability of p53 as a transcriptionfactor to induce expression of DNA repair and pro-apoptotic target genes at higher levels,though no advantage of p53 oscillations in non-proliferating cell-types is deduced.Taking the analyses of p53-AKT regulation of the BCL2 family proteins andexperimental evidence together, p53 oscillations appear to confer proliferative cells withthe ability to strike the critical balance between rapid responses to DNA damage andtimely apoptosis after irreparable damage but yet prevent premature cell death.37

(21) Mass Spectrometry in IMCBKelly Hogue and Walter BlackstockIn his 2007 essay in Cell “Is Proteomics the New Genomics?” Mann proposed that “theexpression levels of all proteins would arguably provide the most relevant single data setcharacterizing a biological system.” Realizing this goal is the aim of quantitativeproteomics. Of late there have been major advances in mass spectrometry, mostnoticeably in the manipulation and storage of ions and image-current detection in thefrequency domain. With stable isotope labeling this makes a potent combination forlarge-scale protein quantification, the protein analogue of the micro-array. Thetechnology is expensive and at the moment its use far from routine. Mass spectrometerskills are scarce as there is little opportunity to learn mass spectrometry in graduateschools. There is also a need to improve understanding of what is possible and ensure thatinstrument time is productively used.To improve mass spectrometry in IMCB specifically and A*Star in general we arebuilding a small team of biologists, mass spectroscopists, and software engineers workingcollaboratively in the area of quantitative biology using stable isotope labeling Weemploy two state-of-the art Orbitrap mass spectrometers, an investment of over S$ 3.5million, in work-flows optimized for the analysis of protein digests. Data analysis is aprocess of inference as the protein information has to be recovered from the peptideinformation. This can be computationally intensive particularly for post-translationalmodifications and is being actively developed.38

(22) Non-ribosomal peptides in Pseudomonas aeruginosa quorum sensingregulationJasmine Lee (ZLH lab)Quorum Sensing (QS) bacteria sense and communicate with one another through therelease of chemical signals known as autoinducers. These signals are recognized by theircognate receptors, which then act as transcriptional regulators to activate the expressionof downstream target genes. There are currently three known QS systems inPseudomonas aeruginosa, i.e., the LasI/R, RhlI/R, and Pseudomonas quinolone signal(PQS) systems. Random mutagenesis revealed an operon, consisting of 4 genesdesignated as qrpABCD, implicated in regulation of PQS biosynthesis. They arepredicted to be a non-ribosomal peptide synthetase gene cluster. When individuallydeleted from the genome, a dramatic reduction in transcriptional expression of PQSbiosynthesis genes is observed. This is translated into an actual physiological reduction inPQS level, as well as corresponding decrease in production of PQS-controlled virulencefactors, including pyocyanin and elastase. In trans expression of wild type qrp genes incorresponding mutants reversed the trends and caused increased production of PQS andits related virulence factors. These evidences attest to the role of QrpABCD in positiveregulation of the PQS quorum sensing system in P. aeruginosa. Work has started onunderstanding the mechanism behind the control. Recent data suggests that theqrpABCD-dependent products are secreted into the extracellular environment and can bechemically extracted from the bacterial culture supernatant. When the extracts are addedto the qrpABCD-null mutants, PQS production is restored to wild type level. Furtherexperiments are underway to purify the peptide for structural analysis in a bid to identifyhow PQS is regulated.39

(23) Loss of udu function causes activation of DNA damage checkpointChiaw-Hwee Lim, Shang-Wei Chong and Yun-Jin JiangLaboratory of Developmental Signalling and Patterning, Genes and DevelopmentDivision, Institute of Molecular and Cell Biology, A*STAR (Agency for Science,Technology and Research), 61 Biopolis Drive, Singapore 138673Although Udu has been showed to play an essential role during blood cell development,its roles in other cellular processes remain largely unexplored. We used a zebrafishmutant; ugly duckling (udutu24) isolated in the 1996 Tübingen genetic screen andshowed that its extensive p53-dependent apoptosis is a consequence of activation of theAtm-Chk2 pathway. The DNA damage response pathway is a cellular surveillancesystem that senses the presence of damaged DNA and elicits an appropriate response tothe damage. Our FACS analysis showed that the loss of udu function resulted in defectivecell cycle progression and comet assay indicated the presence of increased DNA damagein udutu24 mutants. Immunofluoresence staining of transfected COS7 cells demonstratesa dynamic association between PAH-L (Paired Amphipathic α-Helix like), SANT-L(SW13, ADA2, N-Cor and TFIIIB like) domains and replicating pericentromericheterochromatin, which is known to serve functions from gene regulation to protection ofthe integrity of chromosomes. Taken together, these data suggest a possible role of Uduin protecting the integrity of the genome and the loss of Udu function, particularly PAH-L and SANT-L domains can contribute to DNA damage, leading to the activation ofDNA damage pathway and subsequently, cell cycle arrest and apoptosis.40

(24) Engrailed gene expression and its control by hedgehog signalingAshish Maurya (PWI lab)In the zebrafish myotome several muscle cell types are specified by varying levels andtiming of hedgehog signaling. Muscle pioneer cells that are specified by the highestamount of hedgehog signaling express high levels of engrailed homeeobox genes. In thepresent study, I am studying regulation of engrailed genes in the muscle pioneers andhow it is controlled by hedgehog signaling?To isolate engrailed cis-elements I have tested various conserved elements around theengrailed genes for activity in an enhancer assay in transgenic zebrafish. I have alsotested various lengths of the En2a promoter and mapped a muscle pioneer enhancerelement within 4 to 8kb region upstream of the gene. Currently I am trying to identify theminimal region necessary for this muscle pioneer activity. Within this 4kb region no Glibinding sites (Gli transcription factors mediate hedgehog responsive gene expression) arepredicted. This suggests that hedgehog signaling is indirectly regulating engrailed. Inagreement with this the -4 to -8kb region does contain binding sites for transcriptionfactors that are positively and strongly regulated by hedgehog signaling like MyoD andPrdm1.To identify the trans-acting factors that bind to this element I plan to screen a medakacDNA library in a cell culture based assay. In this assay a reporter gene will be driven bythe engrailed regulatory elements and the cDNA clones will be individually tested fortheir ability to either enhance or suppress the reporter.41

(25) Specification of vertebrate slow-twitch muscle fiber fate by the transcriptionalregulator Blimp1Hoe Peng Liew, Semil P. Choksi, Kangli Noel Wong, Sudipto RoyInstitute of Molecular and Cell BiologyVertebrate skeletal muscles are typically composed of slow and fast-twitch fibers thatdiffer in their morphology, gene expression profiles, contraction speeds, metabolicproperties and patterns of innervation. During myogenesis, how muscle precursors areinduced to mature into distinct slow or fast-twitch fiber-types remains inadequatelyunderstood. We have previously shown that within the somites of the zebrafish embryo,activity of the zinc finger and SET domain containing transcriptional regulator Blimp1 isessential for the specification of slow muscle fibers. Here, we have investigated themechanism by which Blimp1 programs myoblasts to adopt the slow-twitch fiber fate. Inslow myoblasts, expression of the Blimp1 protein is transient, and precedes the onset ofexpression of slow muscle-specific differentiation genes. We demonstrate that thecompetence of somitic myoblasts to commit to the slow lineage in response to Blimp1changes as a function of developmental time. Furthermore, we provide evidence thatmammalian Blimp1 can fully recapitulate the slow myogenic program in the zebrafish,suggesting that zebrafish Blimp1 can bind to the same consensus DNA sequence that hasbeen ascribed to the mammalian protein. In line with this, zebrafish Blimp1 can target therepression of fast muscle-specific myosin light chain through direct binding near thepromoter of this gene, indicating that an important aspect of the transcriptional activity ofBlimp1 in slow muscle development is the suppression of fast-muscle-specific geneexpression. Taken together, all of these findings provide new insights into the molecularbasis of vertebrate muscle fiber-type specification, and underscore Blimp1 as the centraldeterminant of this process.42

(26) G-actin binds to the adenylyl cyclase Cyr1 through Cap1 and regulates cAMPsynthesis in C. albicans hyphal morphogenesisHao Zou and Yue WangCandida albicans Molecular & Cell Biology Laboratory, Genes and DevelopmentDivision, Institute of Molecular and Cell BiologyThe yeast-to-hyphal growth switch is a key virulence trait of C. albicans. It is induced bycertain environmental signals and primarily mediated by the cAMP/PKA pathway. Actincytoskeleton plays a central role in the hyphal growth. However, the mechanisms ofinteraction between the cAMP/PKA pathway and the actin cytoskeleton remain unclear.The aim of this study is to test the hypothesis that the Cyclase Associated Protein, Cap1of C. albicans, which has the capacity to interact with both actin and the adenylyl cyclaseCyr1, may play an important role in linking the actin cytoskeleton and the cAMP/PKApathway. We have found that Cyr1, Cap1 and actin can indeed form a ternary complex inwhich Cap1 acts as a bridge, and this complex is sufficient for producing cAMP in vitrounder hyphal-inducing conditions. Since the C-terminus of Cap1 contains the G-actinbinding site and is required for the formation of the ternary complex, it is reasonable tospeculate that G-actin might be able to influence Cyr1 activity through its interactionwith Cap1. Consistent with this view, cells expressing a Cap1 mutant lacking the G-actinbinding site exhibited delayed and reduced cAMP production and defects in hyphalmorphogenesis. Consistently, while wild-type cells exhibited suppressed adenylyl cyclaseactivity when treated with actin depolymerization drugs, the mutant cells were resistant.These results for the first time demonstrate that G-actin plays an active role in directlyregulating cAMP synthesis by forming a ternary complex with Cyr1 and Cap1, revealinga mechanistic link for regulation of the cAMP/PKA pathway by cellular actin status.43

(27) Wip1 regulates neural stem cell/progenitor proliferation through p53dependent G2 cell cycle controlYun-Hua. Zhu, Lu Li, Dmitry V. Bulavin and Zhi-Cheng XiaoContinuing new neuron formation in adult mammalian brain has been realized as animportant process. However, the intrinsic regulation of Neural Stem/progenitor Cells(NSC) remains to be elucidated. Here we report an essential function of theserine/thronine phosphatase wip1 in NSC proliferation and new neuron formation. wip1knockout (ko) resulted in a 90% decrease in new neuron formation in the Olfactory Bulb(OB), without obvious defects in migration and apoptosis. wip1 ko mice show a over twofold decrease in proliferating NSC number in the Sub-Ventricular Zone (SVZ). Similar toin vivo data, in vitro findings indicated the defect of wip1 ko NSC in proliferation, selfrenewal and new neuron formation. Cell cycle analysis by CSFE pulse labeling proved adirect evidence of prolonged cell cycle in wip1 ko NSC, and that could be a result fromrepressed G2/M transition. Molecular analysis revealed that wip1 ko NSC have increasedp21 and reprimo levels in a p53 dependent manner, which might be responsible for therepression in G2 progression. Most interestingly, wip1 ko could not trigger G2/Maccumulation without p53. Finally, we confirmed that NSC regulation by wip1 is p53dependent in vitro and in vivo by the finding that ko of p53, but not other targets of wip1including atm or p38, diminishes the effect of wip1 ko on NSC proliferation. Thus, ourfinding established wip1 as an essential regulator in maintaining NSC proliferation andnew cell formation during through p53 dependent cell cycle control at G2/M transition.44

(28) Mouse embryonic neural stem cells: A model for asymmetric cell divisionGoh Lihui (YXH lab)A stem cell typically divides asymmetrically to give two different daughter cells, one ofwhich remains as an undifferentiated stem cell, the other which would differentiate intospecialized cells. The mechanism of asymmetric cell division is well studied inDrosophila Melanogaster neuroblasts and sensory organ precursor cells. The mechanismdirecting asymmetric cell division in mammals, however, is largely unknown.Polarity genes like Partioning Defective (Par) 3, Par 6, Lethal Giant Larvae (Lgl), whichare known to be involved in asymmetric cell division in Caenorhabditis elegans andDrosophila Melanogaster, are highly conserved in mammals. Studies have found thatthese genes and their gene product are required for the establishment of polarity inmammalian epithelial cells. This brings us to wonder if they also play a role in theprocess of asymmetric cell division in mammals.Using embryonic neural stem cells (NSC) from mouse embryonic cerebral cortex as amodel, we aim to investigate if these different polarity genes are involved in asymmetriccell division. By knocking down these genes in NSC using lentiviral or retroviralparticles carrying the gene specific shRNA constructs, we seek to determine the functionsof the genes in asymmetric cell division using various real time cell markers like Hes-1promoter and Notch Receptors.45

(29) Searching For Novel Proteins Involved In Asymmetric Cell DivisionZhenxing Huang, Siu Kwan Sze and Xiaohang YangAsymmetric cell division is a fundamental mechanism of generating cell diversity duringdevelopment. Drosophila Neuroblast is a model system for studying cell polarity,asymmetric cell division as well as stem cell self-renewal. The apical proteins, such asPar6, aPKC and Pins, control and regulate various aspects of neuroblast asymmetricdivision, such as basal cell fate determinant localization, spindle orientation as well assibling cell size. Despite the fact that large numbers of proteins have been identified asplayers in this mechanism in the recent decade, the complicated network of proteininteractions during asymmetric cell division is still poorly understood. This suggests thatthere are still a large number of proteins contributing in this event awaiting to bediscovered. In this study, we generated transgenic flies expressing TAP (tandem affinitypurification)-tagged Par6 fusion protein specifically in neuronal cells. The fusion proteintogether with its interacting partners was co-pulled down from fly embryos extracts. Thecomponents of the protein complex were subsequently separated and identified byLC/MS/MS. Some known interacting partners, such as aPKC and Lgl, together withdozens of novel interacting proteins have been identified. Base on the expression patterns,we chose six candidate genes for further investigation.46

(30) Apim genetically interacts with Inscuteable in the dividing DrosophilaneuroblastJoan Siew Ching Lee (YXH lab)Asymmetric cell division is an evolutionary conserved mechanism used by progenitorcells to generate cellular diversity during development. Apim (Apical Miranda), a coiledcoilprotein, was isolated from a genetic screen. Embryos lacking Apim and Inscuteable(Insc) exhibit delayed development. Mislocalization of proteins normally segregated tothe basal cortex of dividing neuroblasts such as Miranda and Prospero (Pros) is observedin double mutant embryos. This mislocalization occurs progressively from the cortex ofthe dividing neuroblast to the mitotic spindle. Neuroblasts in double mutant embryos areeventually stalled in metaphase. Our findings show that in the absence of Inscuteable,Apim is involved in embryonic development, regulates the basal localization of the keycell fate determinant Pros, and plays a role in cell cycle progression in Drosophilaembryos.47

(31) Identification of Novel Epigenetic RegulatorsMigliori, V 1 , Casadio F 2 , Bassi C 2 , Amati B 2 , Gozani O 3 and Guccione E 1 .1IMCB, Singapore 2 Ifom-Ieo-Campus, Dpt of Experimental Oncology, Milan Italy 3 Stanford University,Dept. of Biological Sciences, USAIn recent years several groups have generated genome wide epigenetic maps of differentmouse and human cell types. One of the next challenging questions in biological researchis to understand how this epigenetic information is then transduced by chromatinassociatedproteins to downstream effects.The project aims at identifying the network of interaction between protein-motifs that arecommonly found among chromatin-associated proteins and methyl-histone modifications(HMs).The experimental strategy involved the cloning of a library of protein domains into avector, which allows in vitro transcription and translation. The library includes the entireset of human protein domains potentially associated with modified histone tails: Chromo-,Tudor-, MBT-, Plant Agenet-, PWWP-, all belonging to the Tudor domain "Royalfamily", as well as BRK domains and selected PHD fingers. The second step of theproject consisted in hybridizing the library onto an array of Histone H3 and H4 modifiedpeptides.This has resulted in the identification of new histone binders, like the chromodomain ofCDYL1 and CDYL2 that recognize the trimethylation of lysine 9 of histone H3(H3K9me3), and the WD40 domain of WDR5 that binds to the symmetric dimethylationof histone H3 (H3R2me2s).We have further validated the newly discovered interactions by alternative methods suchas standard peptide immunoprecipitations from bacterial lysates, immunofluorescence,BIAcore and crystalization. The project, which is still ongoing, as allowed so far toincrease the information available on how proteins "read" the histone epigeneticinformation and to further investigate the function of some of these interactions indownstream signaling events.48

(32) VAMP8 Is Required for Cell Surface Deployment of the Water ChannelAquaporin 2Wang Cheng Chun (HWJ lab)Water is the major constituent of living cells and water homeostasis is fundamentallyimportant for cell metabolism. AQP2 is a vasopressin-regulated water channel that playsa critical role in maintaining body water balance. Despite the major progress indeciphering the structure and function of AQP2, the SNARE proteins mediating AQP2exocytosis remain elusive. Here we show that VAMP8 is required for regulated surfaceexpression of AQP2. Ablation of VAMP8 in mice resulted in hydronephrosis associatedwith a three to five folds up-regulation of AQP2. Forskolin and [desamino-Cys1, D-Arg8]-vasopressin (DDAVP)-induced AQP2 exocytosis was impaired in VAMP8-nullcollecting duct cells. VAMP8 was revealed to co-localize with AQP2 on intracellularvesicles and to interact with the plasma membrane t-SNARE proteins syntaxin4 andsyntaxin3, suggesting that VAMP8 may mediate regulated fusion of AQP2-positivevesicles with the plasma membrane.49

(33) Wnt3 is Essential for Dorsal Diencephalic DifferentiationCathleen Teh, Michael Richardson, Yin-Ming Lim, Lee-Thean Chu, Igor Kondrychynand Vladimir KorzhThe Wnt signaling pathway has been implicated in dorsal diencephalic differentiation.However, the Wnt involved has not been defined. In zebrafish, wnt3 is persistentlyexpressed in the dorsal diencephalon, specifically, the zona limitans intrathalamica (ZLI),ventral epithalamus and thalamus. We developed a zebrafish stable transgenic line,Tg(wnt3:GFP) whose GFP expression during development faithfully recapitulates theexpression pattern of endogenous wnt3 at the mRNA level. We showed that during neuraldevelopment Wnt3 acts within the context of the canonical Wnt signaling pathway.Morpholino-mediated Wnt3 knockdown results in morphants characterized by atruncated ZLI and lack of neurogenesis in the ventral epithalamus and thalamus, whichparallels the deficient GFP expression in Tg(wnt3:GFP) embryos. In vivo electroporationof either human or zebrafish Wnt3 expression constructs into morphant backgroundrescues these neural developmental defects. Taken together, our results demonstrate thatWnt3 plays an evolutionarily conserved role during cell differentiation in the dorsaldiencephalon.50

(34) A regulatory role for Kir/Gem and the novel muscle-specific protein CCDC95in myogenesisR.N.Mahalakshmi (WH lab)Kir/Gem, a member of the RGK family of Ras related small G proteins, is implicated inregulation of voltage gate calcium channel activity and cytoskeleton remodeling. Thefunctions of Kir/Gem are modulated by its subcellular localization, which in turn is underregulation by calmodulin, 14.3.3, importins and phosphorylation.In our present study, we identify CCDC95, a novel heart and muscle specific protein, asan interacting partner of Kir/Gem. CCDC95 modulates the subcellular localization ofKir/Gem by effectively mediating its nuclear translocation, independent of the importinα5 pathway previously reported by us. It thereby affects the role of Kir/Gem incytoskeleton remodeling and cell shape changes. In an invitro model of muscledifferentiation, Kir/Gem relocalizes from nucleus to cytosol in the initial stages ofdifferentiation. Followed by the induction of CCDC95 expression in the late stages,Kir/Gem and CCDC95 are found to be present in apoptotic nuclei of myotubes.The process of muscle development is a series of well orchestrated events- withdrawal ofmyoblasts from cell cycle, expression of myogenic regulatory factors (MRFs) andformation of multinucleated mytoubes by fusion. Interestingly, overexpression ofKir/Gem interferes with the differentiation of myoblasts to myotubes. This effect ofKir/Gem on myotube formation is overcome by co-expression of CCDC95 and Kir/Gem.Thus our study unravels a novel function of Kir/Gem, in co-ordination with CCDC95, inmuscle development or myogenesis.51

(35) Comparing the antibody responses against recombinant hemagglutinin proteinsof avian influenza A (H5N1) virus expressed in insect cells and bacteriaShen Shuo (CAVR lab)The hemagglutinin (HA) of influenza A virus plays an essential role in mediating theentry of the virus into host cells. Here, recombinant full-length HA5 protein from a H5N1isolate (A/chicken/hatay/2004(H5N1)) was expressed and purified from the baculovirusinsectcell system. As expected, full-length HA5 elicits strong neutralizing antibodies, asevaluated in micro-neutralization tests using HA5 pseudotyped lentiviral particles. Inaddition, we have expressed two fragments of HA5 in bacteria and found that the N-terminal fragment, covering the ectodomain before the HA1/HA2 polybasic cleavage site,can also elicit neutralizing antibodies. But the C-terminal fragment, which covers theremaining portion of the ectodomain, did not. Neutralizing titer of the anti-serum againstthe N-terminal fragment is only four times lower than the anti-serum against the fulllengthHA5 protein. Using a novel membrane fusion assay, we further showed that theabilities of these antibodies to block membrane fusion correlate well with theneutralization activities.52

(36) JNK-dependent activation of p53 and c-Jun is required for nitric oxide-inducedapoptosis of tumor cellsLei Li, S.H. Lim, G. Aw and Alan G PorterCell Death & Human Disease Group, Cancer & Developmental Biology Division,Institute of Molecular and Cell Biology, #07-03B, 61 Biopolis Drive, Proteos,Singapore, e-mail: mcblil@imcb.a-star.edu.sgExcessive nitric oxide (NO) contributes to cell death in trauma, strokes andneurodegenerative diseases. Here we investigate the mechanisms of NO-inducedapoptosis in human neuronal and other cell lines. We show that NO-induced, JNKdependentactivation of c-Jun by phosphorylation on Ser-63 promotes caspase-3-dependent apoptosis in human SH-Sy5y cells; and that p53 is also an essential mediatorof NO-induced apoptosis in these cells. With published evidence that JNK canphosphorylate various amino acids on p53, we addressed if NO-activated JNK is requiredfor p53 Ser-15 phosphorylation and p53 activation. Time course and dose responseexperiments in neuroblastoma and colon carcinoma cells treated with two different NOdonors demonstrated activation of JNK concomitant with or prior to phosphorylation ofp53 on Ser-15 and p53 up-regulation. A JNK chemical inhibitor substantially decreasedJNK activation, p53 Ser-15 phosphorylation, p53 up-regulation, apoptosis, andexpression of the p53 transcriptional targets of NO we recently identified - viz. maspinand plasminogen activator inhibitor-1 (PAI-1). Neuroblastoma cells expressingdominant-negative JNK2 were relatively resistant to NO-induced apoptosis, exhibitedreduced p53 activation, and showed decreases in maspin and PAI-1 at both thetranscriptional and translational levels. Conversely, in p53-deficient cells JNK was fullyactivated by NO even though the cells were resistant to apoptosis. Altogether our resultssuggest that JNK-dependent activation of c-Jun and p53 leads to NO-induced apoptosis indifferent tumor cell types.53

(37) Smad1 provides a link between BMP-Smad signaling and DNA damageresponse and a target for cancer therapyJenny Chau, Yuanyu Hu, Jianhe Wang, and Baojie LiThe Institute of Molecular and Cell BiologyA*STAR (Agency for Science, Technology and Research)SingaporeGenome instability is a major driving force of tumorigenesis and DNA damage activatedAtm-p53 pathway acts as an anti-cancer barrier by limiting cell growth. Here we reportthat the BMP-Smad pathway acts downstream of Atm in DNA damage response, as a p53alternative pathway. Atm phosphorylates Smad1 on Ser240 in the linker region, whichinhibits Smad1 dephosphorylation by PPM1A, leading to Smad1 activation, nuclearretention and stabilization, accompanied by transactivation of some BMP target genes.Furthermore, when signaling from Atm/Atr to p53 is disrupted in p53 deficient cells,Smad1 is up-regulated, as a backup mechanism. Elevation of Smad1 enhances DNAdamage induced apoptosis and diminishes cell transformation and tumorigenesis, whiledeficiency of Smad1 shows opposite effects. Moreover, up-regulation of Smad1 is amechanism by which many of the non-genotoxic chemotherapeutic drugs kill tumor cells.These results suggest that the BMP-Smad1 pathway is an integral part of DNA damageresponse that suppresses oncogenesis, and that Smad1 is a target for cancer therapy.54

(38) Harnessing piggyBac Transposons for cancer gene discovery in ZebrafishCanhe Chen 1 , Jolene Y.Z. Ooi 1 , Lifang Li 1 , Sook Yee Wong 1 , Sergey Parinov 2 ,Alexander Emelyanov 2 , Nancy A. Jenkins 1 and Neal G. Copeland 11 Institute of Molecular and Cell Biology, Singapore 138673; 2 Temasek Life SciencesInstitute, Singapore 117604Transposon-based insertional mutagenesis offers enormous potential for systematicallyidentifying the genes that cause cancer, which is a prerequisite for developing new targetbasedtherapeutics for treating cancer. While such screens have recently been developedin mice, their cost is a major limitation for most labs. The development of such screensin zebrafish would dramatically reduce their cost and make it possible to performsaturation screens for new cancer genes in fish. In my experiments, I am making use ofpiggyBac (PB), a novel class II transposon, which can accept large inserts and transposein both dividing and non-dividing cells. I have shown that PB can transpose in zebrafishat very high frequencies where it preferentially inserts into the coding-region of genes,similar to what has been reported for mammalian cells. I have also constructedmutagenic versions of the SB transposon that are capable of insertionally activatingoncogenes as well as insertionally inactivating tumor suppressor genes and generatedzebrafish lines that harbor multiple copies of these mutagenic transposons. In addition, Ihave generated a number of zebrafish transgenic lines that carry a codon-optimizedversion of the PB transposase under the control of a chemically inducible promoter.Since the site of integration affects PB transposase expression, it has been possible toselect zebrafish transposase lines that vary in their intensity and pattern of transposaseexpression. I have also made polyclonal and monoclonal PB transposase antibodies andused them to characterize these zebrafish transposase lines. Finally, I have generatedlarge numbers of zebrafish that carry the mutagenic transposase and a chemically inducedubiquitously expressed transposase and I am currently monitoring these zebrafish fortumor development. If my experiments are successful they have the potential todramatically accelerate cancer gene discovery in zebrafish and open up a whole new areaof zebrafish genetics.55

(39) Stem cell exhaustion due to Runx1 deficiency is prevented by Evi5 activation inleukemogenesisBindya Jacob *1 , Motomi Osato 1, 2 , Namiko Yamashita 1 , Ichiro Taniuchi 3 , Dan Littman 4 ,Norio Asou 5 , Yoshiaki Ito 1, 2*Postdoctoral fellow, Runx lab1 Institute of Molecular and Cell Biology, Singapore, 2 Oncology Research Institute,National University of Singapore, Singapore 117456, 3 RIKEN, Research Center forAllergy and Immunology, Yokohama, Japan, 4 New York University, New York, U.S.A,5 Department of Hematology, Kumamoto University School of Medicine, Kumamoto,Japan.The balance between the self-renewal and differentiation of hematopoietic stem cells(HSC) is perturbed in acute leukemia. Quiescence is an alternative state for HSC, evendominant in adult. However, its involvement in leukemogenesis remains poorlyunderstood. We show that conditional deletion of Runx1, the most frequently targetedgene in human leukemia, results in an increase of HSC due to downregulation of CXCR4,a key factor for niche interaction. The weakened niche interaction of Runx1-/- HSC failedto sustain quiescence and instead facilitated cell cycle entry. The decreased dormancy inturn progressively resulted in stem cell loss, called HSC exhaustion. In the leukemiadevelopment, the stem cell exhaustion was rescued by additional genetic changes, such asoverexpression of Evi5. These results reiterate the importance of interaction of leukemiainitiating cells with niche in leukemogenesis. Our results also emphasize the usefulnessof retroviral insertional mutagenesis to precisely dissect the multiple steps ofleukemogenesis: subsequent genetic hits compensate negative aspects given by precedinggenetic alterations. Elucidation of these combinatorial mechanisms, in particular stem cellquiescence related mechanism, may provide novel direction for therapeutic applications.56

(40) Cdc25A Serine 123 phosphorylation couples centrosome duplication with DNAreplication and regulates tumorigenesisShreeram Sathya (BD lab)The cell division cycle 25A (Cdc25A) phosphatase is a critical regulator of cell cycleprogression under normal conditions and after stress. Stress-induced degradation ofCdc25A has been proposed as a major way of delaying cell cycle progression. In vitrostudies pointed towards serine 123 as a key site in regulation of Cdc25A stability afterexposure to ionizing radiation (IR). To address the role of this phosphorylation site invivo, we generated a knock-in mouse in which serine 123 was substituted for alanine.The Cdc25 Ser123A knock-in mice appeared normal,and, unexpectedly, cells derived from them exhibited unperturbed cell cycle and DNAdamage responses. In turn, we found that Cdc25A was present in centrosomes and thatCdc25A levels were not reduced after IR in knock-in cells. This resulted in centrosomeamplification due to lack of induction of Cdk2 inhibitory phosphorylation after IRspecifically in centrosomes. Further, Cdc25A knock-in animals appeared sensitive to IRinducedcarcinogenesis. Our findings indicate that Cdc25A Ser123 phosphorylation iscrucial for coupling centrosome duplication to DNA replication cycles after DNAdamage, and therefore is likely to play a role in the regulation of tumorigenesis.57

(41) TRIM39 is a MOAP-1 Binding Protein That Stabilizes MOAP-1 ThroughInhibition of its Polyubiquitination ProcessSan San Lee, Nai Yang Fu, Sunil K. Sukumaran, Kah Fei Wan, Qian Wan, and Victor YuBax, a multi-domain pro-apoptotic member of the Bcl-2 family, is a key regulator for therelease of apoptogenic factors from mitochondria. MOAP-1, which was first isolatedfrom a screen for Bax-associating proteins, interacts with Bax upon apoptotic induction.Gain- and loss-of-function analyses suggest that MOAP-1 may play an effector role infacilitating the function of Bax in mitochondria. MOAP-1 is a short-lived protein that isconstitutively degraded by the ubiquitin-proteasome system. Apoptotic stimuli upregulateMOAP-1 rapidly through inhibition of its poly-ubiquitination process. However,cellular factors that play a role in regulating the stability of MOAP-1 have not yet beenidentified. In this study, we report the identification of TRIM39 as a MOAP-1-associatingprotein. TRIM39 belongs to a family of proteins characterized by a Tripartite Motif(TRIM), consisting of a RING domain, a B-box and a coiled-coil domain. Severalmembers of the TRIM family are known to demonstrate E3 ubiquitin ligase activity.Surprisingly, TRIM39 significantly extends the half-life of MOAP-1 by inhibiting itspoly-ubiquitination process. TRIM39 localizes predominantly in the cytosol but a portionof TRIM39 co-localizes with MOAP-1 in the mitochondria. In agreement with its effecton enhancing MOAP-1 stability, TRIM39 sensitizes cells to etoposide-induced apoptosis.Furthermore, TRIM39 elevates the level of MOAP-1 in mitochondria and promotescytochrome c release from isolated mitochondria stimulated by recombinant Bax. Takentogether, these data suggest that TRIM39 is a regulator of MOAP-1 stability that canpromote Bax-dependent apoptotic function in mitochondria.58

(42) A complex affair between two tumour suppressors and a famous regulatorPermeen Yusoff, Yim GR Daniel and Graeme R. GuySprouty (Spry) proteins are inhibitors of the central, cancer-implicated Ras/ERKsignaling pathway and have been implicated as likely tumour suppressors. Spry proteinsshare a common C-terminal, cysteine-rich domain and two short sequences in the N-terminus The function of Spry2, the most profound inhibitor of the Ras/ERK pathway,appears to be governed by a controlled level of phosphorylation on key tyrosine andserine residues. Tyr 55 is situated within a conserved, canonical c-Cbl TKB domainbinding site and upon receptor tyrosine kinase (RTK) activation c-Cbl binds with highaffinity to this sequence. c-Cbl is a well-characterised docker protein that also hasubiquitin E3 ligase activity and appears to be central in the downregulation of manyRTKs and non-RTKs of the Src family. The second conserved sequence consists mainlyof Ser and Thr residues, some or all of which are dephosphorylated upon RTK activation,despite the whole protein showing an increased phosphorylation. Evidence indicates thatthe phosphatase responsible for the specific RTK-induced dephosphorylation is theubiquitous PP2A, which also plays an important role in controlling the activity of variousproteins within and those controlling the Ras/ERK pathway. PP2A, consisting of threevariable scaffold, catalytic and activator subunits, has also been implicated as playing arole as a tumour suppressor and is a major target of tumourigenic viruses. PP2A-A, thescaffold subunit binds directly to Spry2, coincidently at the same location as c-Cbl. Thecellular pools of Spry2 binding c-Cbl and to PP2A are mutually exclusive and we arecurrently working on the physiological implication of this complex relationship.59

(43) GRIM-19 Is Essential for Maintenance of Mitochondrial Membrane PotentialLu Hao (CXM lab)GRIM-19 was found to copurify with complex I of mitochondrial respiratory chain andsubsequently was demonstrated to be involved in complex I assembly and activity. Tofurther understand its function in complex I, we dissected its functional domains bygenerating a number of deletion, truncation, and point mutants. The mitochondriallocalization sequences were located at the N-terminus. Strikingly, deletion of residues70–80, 90–100, or the whole C-terminal region (70–144) led to a loss of mitochondrialtransmembrane potential (m). However, similar deletions of another two complex Isubunits, NDUFA9 and NDUFS3, did not show such effect. We also found that deletionof the last 10 residues affected GRIM-19's ability to be assembled to complex I. Weconstructed a dominant-negative mutant containing the N-terminal 60 and the last C-terminal 10 residues, which could be assembled into complex I, but failed to maintainnormal m. Cells overexpressing this mutant did not spontaneously undergo cell death, butwere sensitized to apoptosis induced by cell death agents. Our results demonstrate thatGRIM-19 is required for electron transfer activity of complex I, and disruption of m byGRIM-19 mutants enhances the cells' sensitivity to apoptotic stimuli.60

(44) New Controls of the NFkB PathwayHsiang Ling Teo (VT lab)The transcription factor NFkB is critical for several cellular and developmental events inmetazoans. Given that over 200 physiological stimuli activate NFkB, which in turnregulates an equally large number of genes, understanding how specificity is generated insuch a pleiotropic pathway is also a major challenge. Using a large-scale functionalgenomics approach, parallel interrogation of approximately 20,000 sequence-annotatedgenes was carried out and several novel modifiers of NFkB activity were identified. I willdescribe the characterization of one of these modulators and the mechanisms by which itregulates NFkB signalling.61

(45) Characterization of Speedy/RINGO, a new family of cyclin-like proteinsXinde Zheng and Philipp KaldisCyclin-dependent kinases (Cdk) are master regulators of cell cycle progression. Thefunction of Cdk is largely decided by the bound cyclins, which determine the substratespecificity. Recently, a new family of cyclin-like proteins, Speedy/RINGO, has beenidentified. Similar to classical cyclins, Speedy/RINGO family members can bind andactivate Cdk1, Cdk2, and Cdk3. Through phylogenetic analysis, Speedys can only befound in the species of Deuterostome, including sea squirt, bird, fish, mouse, and primate.Mouse and human have four Speedy genes. Several cell culture studies indicated thatSpeedys may have functions in cell cycle and DNA damage control. In Xenopus, Speedycan promote the meiotic maturation. However, their roles on meiosis and somatic cellcycle have not been firmly established. We use mouse models to explore their in vivofunctions. Conditional knockout mice of Speedy A, B1, B2 and B3 are being made. Todifferentiate these highly homologous speedy genes, monoclonal antibody and peptidepolyclonal antibody are being developed. Testis-specific expression of Speedy A, B1 andB2 suggest that mammalian Speedys may be key players in meiosis. Ubiquitousexpression of Speedy B3 indicates that Speedy may also have functions in the somaticcell cycle. The potential binding partners found in yeast two hybrid screens suggest thatSpeedys have close relation with Cdks, and may contribute to the unique chromosomebehavior during meiosis. Interestingly, the only phenotype of Cdk2-/- male mice isspermatogenesis block. We hypothesize that at least one of the Speedys may be themeiotic partner of Cdk2.62

(46) Rb/Cdk2/Cdk4 triple mutant mice elicit a new mechanism for G1/S regulationShuhei Kotoshiba 1 , Weimin Li 2 , and Philipp Kaldis 11 Institute of Molecular and Cell Biology (IMCB), Cell Division and CancerLaboratory (PRK), 2 University of Wisconsin-Madison, Department of PharmacologyThe G1/S transition is a well-toned switch in the mammalian mitotic cell cycle. The cellcycle kinases, Cdk2 and Cdk4, and the rate-limiting tumor suppressor Retinoblastomaprotein (Rb) have been studied in separate animal models leaving an unattendedinteraction between the kinases and Rb in vivo. To further dissect the regulation of theG1 to S phase progression, we generated Cdk2-/-Cdk4-/-Rb-/- (TKO) mutant mice. Themice died in mid-gestation with major defects in the circulatory systems and displayedcombined phenotypes of Rb-/- and Cdk2-/-Cdk4-/- mutants. However, the TKO MEFswere not only resistant to senescence and became immortal but displayed enhanced Sphase entry as well as proliferation rates similar to wild type. The effects were moreremarkable in hypoxia compared to normoxia. Interestingly, depletion of the pocketproteins by HPV-E7 in the TKO background elicited a second mechanism for the G1/Sregulation, where p27Kip1 bound to Cdk1 and cyclin E complex due to disrupted Skp2-p27Kip1 interactions. Our work indicates that the G1/S transition can be controlled indifferent ways depending on the situation, resembling a regulatory network63

(47) PRL-3 Down-regulates PTEN Expression and Signals through PI3K toPromote Epithelial-Mesenchymal TransitionHaihe Wang, Samantha Yiling Quah, Jing Ming Dong, Edward Manser, Jing Ping Tang,and Qi ZengPRL-3, a metastasis-associated phosphatase has been demonstrated to increases cellmotility and invasiveness with its overexpression. These phenotypic changes arereminiscent of the epithelial-mesenchymal transition (EMT) that occurs duringembryonic development and oncogenesis and is a complex process that converts epitheliainto migratory mesenchymal cells. We here attempt to unravel the underlyingmechanistic basis of these phenomena. HeLa cells transiently expressing EGFP-PRL-3(HeLa-PRL-3) exhibit reduced levels of paxillin. Similarly, CHO cells stably expressingmyc-PRL-3 (CHO-PRL-3) also show marked reductions in paxillin, phospho-paxillin-Tyr31, and vinculin at focal adhesion complexes, and notable reductions in the levels ofRhoA-GTP, Rac1-GTP, and F-actin filaments. DLD-1 human colorectal cancer cellsengineered to express EGFP-PRL-3 (DLD-1-PRL-3) underwent changes consistent withEMT. In these cells, PRL-3 activates Akt and inactivates GSK-3β as assessed byphosphospesific antibodies. PRL-3 upregulates mesenchymal markers: Fibronectin andSnail, and downregulates epithelial markers: E-cadherin, γ-catenin (plakoglobin), andIntegrin β3, which are major effectors in the EMT pathway. The changes in these EMTcharacteristics brought about by PRL-3 can be abrogated by the phosphoinositide 3kinase (PI3K) inhibitor LY294002, implying that PRL-3 acts upstream of PI3K and couldplay an initiating role to trigger the EMT switch during cancer metastasis. In addition,PRL-3 can downregulate PTEN which is an important antagonist of PI3K, furtherreinforcing PI3K/Akt function in PRL-3-triggered EMT. Catalytically inactive PRL-3(C104S) was impaired in the above PRL-3-mediated events, indicating that theseproperties require phosphatase activity. Targeting PRL-3 may thus be a useful strategy toimpede cancer cell invasion and metastasis.64

(48) Evolutionary changes in the regulatory region of vertebrate erythropoietin genelocusRashmi P Kulkarni (FUGE/SB lab)Erythropoietin (Epo) is the principal regulator of red blood cell production. Decreasedoxygen levels induce a dramatic increase in the expression levels of Epo gene. Inmammals, Epo is expressed mainly in the fetal liver and adult kidney. A highly induciblehypoxia-response element (HRE) has been identified in the 3’-flanking sequences ofmammalian Epo genes. Interestingly, cloning of Epo gene from a teleost fish (fugu)showed that in fishes, the major site of Epo expression is the adult heart. Furthermore, 3’-flanking region of fugu Epo does not contain a HRE. Thus, the regulatory regions of Epolocus seem to have undergone significant changes during the evolution of vertebrates. Totrace these evolutionary changes and to understand better the regulation of Epo gene, wehave characterized tissue-specific enhancers and HRE in the fugu Epo locus. In vitrostudies in human Hep3B cells demonstrated that fugu Epo locus contains a HRE 2.3 kbupstream of the transcription initiation site, on the opposite strand of DNA. Using azebrafish reporter-assay system we were able to identify the heart-specific enhancerlocated 1.8 kb upstream of the transcription initiation site. To elucidate the molecularchanges that have led to the replacement of this heart-specific enhancer by a kidneyspecificenhancer in mammals, we are testing the fugu heart-specific enhancer intransgenic mice. These studies should provide insights into the architecture and evolutionof tissue-specific enhancers in vertebrates.65

(49) Molecular therapy : Targeting the p53 pathway using small moleculesChris Brown, Chit Fang Cheok, David P LaneThe tumor suppressor p53 is frequently inactivated in a wide variety of cancers. At least50% of all tumours carry either point mutations or deletions of the p53 gene and amongmany of the remaining tumours, the signaling pathways of p53 is/are defective. One ofthe common lesions in tumours carrying inactivated p53 is the amplification of murinedouble minute 2 (MDM2) status, a negative regulator of p53. Disruption of the p53-MDM2 complex therefore provides an attractive strategy for reactivating p53 functions.Detailed analysis of the p53-MDM2 interaction interface provides clues to better designin small molecules targeting this interaction. We use peptide aptamer molecules as leadstructures for drug design to develop more potent and specific small molecule inhibitors.Fluorescence polarization-based assays are used to screen and validate potential ligandsthat bind to the target used for selection.As proof-of-concept that inhibitors of p53-MDM2 complex have therapeutic value, weWe demonstrate the correlation between p53 activation and the induction of apoptosis,using an inhibitor of p53-MDM2 complex (nutlin-3a). Given that p53 activity is tightlycontrolled by multiple levels of regulation, simultaneously targeting these multi-pathwaysmay provide new therapeutic regimes. We explored the concept of drug synergism in theactivation of p53 responses. The antitumor effect of low doses of CDK inhibitors issynergistically enhanced by nutlin-3a, resulting in p53-induced apoptosis in varioustumor-derived cell lines. Importantly, the combination is nongenotoxic and may be usefulas a therapeutic strategy to circumvent the problems of using genotoxic drugs inchemotherapy.66

(50) Crystallographic analysis of cap free and cap bound eIF4E with N7 alkylatedcap derivativesChristopher J Brown (DPL lab)We present here the first reported crystal structure of cap-free human eIF4E. Thisstructure reveals that in the absence of m7GTP the cap-binding site undergoes significantstructural changes with the loop containing residue W56 becoming disordered and theside-chain of W102 rotating out of the cap-binding site. The eIF4E complex crystalstructures with Bn7GMP and FBn7GMP show that the eIF4E cap-binding pocket isformed by an induced fit mechanism, with the W102 side-chain rotating into analternative position compared to the m7GTP bound structure. The phosphate binding siteremains invariant between the cap-free and cap-analogue bound structures which implieselectrostatic steering of the cap-analogue into the cap-binding site followed by orderingof the W56 loop and rotation of W102 into the cap binding site. Mass spectrometry wasalso used to determine apparent gas-phase Kd constants of 0.15 M, 13.6 M, and 55.7M for eIF4E with m7GTP, GTP, and GMP, respectively. These measurements showthat for tight and specific binding to eIF4E there seems to be a clear requirement forguanosine derivatives to possess both the delocalised positive charge of the N7-methylated guanine system and at least one phosphate group. We also showed that theN7benzylated monophosphates Bn7GMP and FBn7GMP bind eIF4E substantially moretightly than non-N7-alkylated guanosine derivatives (Kd values of 7.0 M and 2.0 M,respectively). The eIF4E complex crystal structures with Bn7GMP and FBn7GMP showthat additional favourable contacts of the benzyl groups with eIF4E and the localstructured water network contribute binding energy that compensates for the loss of thebeta and gamma-phosphates. The structural observations made here will be useful in thedesign and discovery of new families of eIF4E inhibitors, which may have potentialtherapeutic applications in cancer. The cap free structure also offers a new pose forvirtual drug docking studies67

(51) Spatio-temporal Mapping of Actin Polymerisation Regulated by Actin-BindingProteinsKannan Balakrishnan (BR lab)Actin filaments not only provide mechanical support to the cell but also participate in cellmovement. Assembly and disassembly of actin filaments are regulated by a variety ofproteins called the actin-binding proteins. To understand the mechanism of cellmovement in health and disease, a detailed understanding of the self-assembly of actinmonomers into filaments and its regulation by the actin-binding proteins is required.Conventional bulk measurements yield ensemble averaged data which does not allow anunderstanding of the process at the single molecule level. Total internal reflectionfluorescence (TIRF) microscopy coupled with fluorescence anisotropy and spectroscopymeasurements provide a means to follow the actin dynamics with single moleculesensitivity. In the present work, in vitro real-time TIRF assays of actin polymerization inthe presence and absence of actin-binding proteins in combination with anisotropy andspectroscopy analyses will be presented.68

(52) Directed Evolution of Actin by In Vitro CompartmentalizationMarten E Larsson (BR lab)Actin is a protein involved in many important cellular functions, including musclecontraction, cell motility, cytokinesis and is a central protein of the cytoskeleton. Actinmonomers (G-actin) rapidly polymerize into heterogeneous filaments (F-actin) in thepresence of even low concentration of salt. This feature has been a major sumbling blockin elucidating the structure of F-actin.Expression of soluble, functional actin in E. coli has been difficult partly due to the lackof chaperonins needed for the correct folding, and perhaps, by the lack ofposttranslational modifications. Overexpression in other systems like yeast,Dictyostelium discoideum or baculovirus/insect cells have either been lethal, yielded verylow expression or insoluble aggregates. Expression of actin with the option of addingdifferent purification tags, or functional mutations would be highly desirable forstructural studies of F-actin and its complexes with actin-binding proteins.Using in vitro transcription/translation of randomly mutated biotin-labelled actin cDNAscoupled to streptavidin beads located within the aqueous compartment of an oil-in-wateremulsion (in vitro compartmentalization) we are trying to evolve a soluble yet functionalactin. Correctly folded actin will polymerize within the compartment and the producedpolymer can be captured by biotin-labelled phalloidin coupled to the beads. Selection ofhighly polymerizable actin is done by breaking open the emulsion, staining withfluorescein-labelled phalloidin and subsequent FACS sorting. cDNAs encodingpolymerizable actin can be amplified by PCR and used for further rounds of selection orfor expression in E. coli and functional analyzes.69

(53) Tracking cellular events using stable-isotope labeling by amino acids in cellculture (SILAC)Jayantha Gunaratne, PhD (WB lab)Recent progress in mass spectrometry (MS) technology offers exciting new strategies foradvances in virtually all areas of biology. Among the MS strategies that focus onquantitative proteomics, stable (non-radio active) isotope labeling by amino acids in cellculture (SILAC) has emerged as a simple and robust approach to solve a wide range ofbiological problems. SILAC eliminates false positives in protein-interaction studies, andreveals large-scale dynamics of proteomes and protein turnover kinetics by adding a timedimension to proteomics. It has become an important assay for measuring changes in thephospho-proteome, ranging from the yeast pheromone pathway to stem-celldifferentiation. As a major research focus in our laboratory we initially established thistechnology by successfully labeling whole proteomes of commonly used cancer cell linesfor the study of p53, PTEN and Evi1. Currently we are examining SILAC’s capability toquantify both the proteome and its modifications in response to stimuli and perturbationsreflecting the research interests of collaborators. In a study to measure gene translation ofp53 hot-spot mutant R175H transfected into p53 null cells (H1299) we have quantifiedmore than 6000 proteins in the whole proteome using SILAC under basic fractionationconditions. SILAC-based interactomic study of tagged PTEN has revealed newinteracting partners with competent removal of false positives. In this presentation I willdiscuss the principles, practical stratagems, applications, limitations and improvedformats of SILAC.70

(54) Synergistic regulation of Xanthomonas campestris virulence and adaptationby DSF signaling system and a novel two-component signal transduction systemRavS/RavR.Ya-Wen HE, Calvin Boon, Lian Zhou, and Lian-Hui ZhangThe ability of Xanthomonas campestris pv. campestris (Xcc) to incite disease in crucifersdepends upon a number of virulence factors including extracellular enzymes andextracellular polysaccharides (EPS). Previous work shows that production of thesevirulence factors is positively regulated by a DSF quorum sensing system, which acts bymodulating the intercellular content of a novel second messenger cyclic di-GMP (c-di-GMP). In this study, the roles of 18 genes encoding proteins with HD or EAL domains,which are implicated in cyclic di-GMP degradation, were examined in EPS productionand extracellular enzyme activity in Xcc. We found that deletion of Xcc1958 (ravR),which encodes a response regulator with GGDEF-EAL domains, led to decreased EPSproduction and extracellular enzyme activity. Transcriptional analysis showed that ravRis co-transcribed with Xcc1960 (ravS), which encodes a sensor kinase with PAS-PACdomains. Deletion analysis confirmed that the null mutant of ravS displayed similarphenotypes as the ravR mutant, suggesting that RavS and RavR constitute a twocomponentregulatory system. Microarray analysis revealed that RavS/RavR systemregulates diverse functions. Bioinformatic and genetic evidence suggest that RavR mightfunction as a c-di-GMP phosphodiesterase. Further studies suggest that RavS/RavRsystem sense low oxygen conditions and regulated EPS production and extracellularenzyme activity through the Clp transcriptional regulatory network. Furthermore, geneticand biochemical analysis revealed that disruption of RavS and RavR system did notaffect the functionality of DSF signaling system and both systems synergisticallyregulated virulence factor production and virulence. These results outline a dualsignaling system that may allow Xcc to integrate population density and environmentalcues to modulate virulence factor production and adaptation.71

(55) Probing the retrograde transport pathway by RNA interference screeningDimitri Moreau, Shin Yi Chew and Frederic BardThe cellular secretory route involves an anterograde membrane flow that is compensatedby retrograde transport of proteins and lipids to maintain organelle homeostasis and torecycle components of the transport machineries. The retrograde pathway is activebetween the endosomes, the Golgi apparatus and the endoplasmic reticulum. Today, thisendogenous process appears to be of special importance for the targeted delivery of drugsto the cytosol of cells. Indeed, the retrograde traffic pathway can provide a route todeliver drugs bypassing the hydrolytic environment of the lysosome. However, relativelyfew molecular players in this pathway have been identified. Pseudomonas Exotoxin A(PE) and Ricin are two toxins able to travel from the cell surface through the Golgiapparatus and the endoplasmic reticulum before translocation to the cytosol where theyblock protein synthesis. Therefore, we have developed a biological assay based on theinhibition of protein synthesis induced by the PE and Ricin toxins. We used this assay toperform a pilot RNAi screen with the goal to identify missing key genes. We foundseveral genes already known to be involved in the retrograde pathway, like the clathrinheavy chain, the KDEL receptor and Rab6A. We also found several potential new genesin this pathway, including MAPK8IP1 (JIP1), which has been involved in neuronalvesicular trafficking. JIP1 is able to link vesicular cargo with the molecular motor kinesin.Various signal transduction pathways regulate JIP1 through phosphorylation, raising theintriguing possibility that retrograde traffic could also be a target of regulation byextracellular signals.72

(56) Inhibition of PKR Activation and Up-regulation of GADD34 Expression play asynergistic role in the maintenance of de novo Protein Synthesis in Coronavirus-Infected CellsXiaoxingWang, Ying Liao, Kim J Png, Pei Ling Yap, and D.X. LiuA diversity of strategies is evolved by RNA viruses to manipulate the host translationmachinery in order to create an optimal environment for viral replication and progenyproduction. One of the common targets of the translation machinery is the eukaryoticinitiation factor 2a (eIF2a). In this report, we showed that phosphorylation of eIF2α wasseverely suppressed during IBV infection. To understand if this suppression is throughinhibition of PKR, the double-stranded RNA dependent kinase that is responsible forphosphorylation of eIF2α, cells infected with IBV were analyzed by Western blot. Theresults showed that phosphorylated PKR was greatly reduced in IBV-infected cells.Over-expression of a number of IBV-encoded RNA-binding proteins results in theinhibition of PKR activation, suggesting the involvement of these proteins in viralevasion of host-antiviral response. Furthermore, GADD34, a component of the proteinphosphatase 1 complex that dephosphorylates eIF2α, was significantly induced in IBVinfectedcells. Over-expression of GADD34 showed significant enhancement of IBVinfection in a dose-dependent manner. Taken together, these results confirm that IBVmay have exploited one combination of two mechanisms, i.e. by blocking PKR activationand inducing GADD34, to maintain a minimal level of de novo protein synthesis duringthe infection cycles.73

(57) DNA damage checkpoint maintains Cdh1 in quasi-active state to preventspindle elongation and the completion of AnaphaseTao ZhangUS lab, Systems Biology Division, Institute of Molecular and Cell BiologyDNA damage checkpoint imposes cell cycle arrest to prevent segregation of damagedchromosomes and allows sufficient time for the damaged chromosomes to be repaired.In yeast, Mec1, Chk1 and Rad53 (homologous to human ATM/ATR, Chk1 and Chk2kinase, respectively) are among the main effectors of this surveillance pathway. Whileactivated Chk1 phosphorylates securin to prevent cohesin cleavage, thus inhibitingchromosome segregation, Rad53 branch inhibits the mitotic exit network (MEN),preventing cells from exiting mitosis. Rad53 also inhibits Cdc5 polo kinase, an essentialcomponents of MEN. Restraining cohesin cleavage by stabilizing Pds1 (securin) isconsidered to be the predominant mechanism by which DNA damage checkpointprevents segregation of damaged chromosomes. We have uncovered a new regulatorynetwork that prevents segregation of damaged chromosomes by suppressing spindleelongation, even after sister-chromatid cohesion is lost. This control circuit involvesRad53, polo kinase, anaphase promoting complex (APC) activator Cdh1 and the bimCkinesin family proteins Cin8 (homologous to human Eg5) and Kip1. Hence, mitoticspindle emerges as a novel target of the DNA damage checkpoint.74

(58) Searching for novel factors involved in neutrophil chemotaxis by chemicalgeneticsXingang Wang and Philip W. InghamDevelopmental and Biomedical Genetics, IMCBNeutrophil chemotaxis, directed cell movement in response to a gradient ofchemoattractant, plays an important role in wound healing and immune response. Thoughsome factors involved in cell migration have been identified in vitro using primaryneutrophils, and HL-60 cells as well as through studies in Dictyostelium discoideum,there is a need to verify and extend these finding in an in vivo context. In this study,mpo:GFP transgenic zebrafish - that heave neutrophils labeled with green fluorescenceprotein – have been used as a model to screen for compounds that can affect theirbehavior in vivo. Two compounds have been found that completely inhibit the migrationof neutrophils towards an injury site after screening a drug library containing about onethousand natural extracts isolated from different organisms. Time-lapse imaging analysisshowed that in animals treated with these compounds neutrophils failed to polarize, wereunable to form pseudopod or formed pseudopods in random directions. Further analysisis under way to determine whether these compounds affect the subcellular localization ofproteins such as PI3K, Rac and Rho which are important in establishing the cell polarityand controlling the F-actin polymerization and myosin contraction. These compounds arealso being subfractioned and further analyzed to determine their molecular structure. Theprotein targets of these molecules are to be determined in the future.75

(59) Zebrafish nicastrin is required for midbrain and hindbrain developmentShang-Wei Chong 1 , Adam Amsterdam 2 , Haw Siang Ang 3 , He Yang 3 , Nancy Hopkins 2and Yun-Jin Jiang 11 Laboratory of Developmental Signalling and Patterning, Institute of Molecular and CellBiology, 61 Biopolis Dr., Proteos, 138673, Singapore. 2 Center for Cancer Research andDepartment of Biology, Massachusetts Institute of Technology, 77 Massachusetts AvenueE17-340, Cambridge, MA 02139, USA. 3 Genome and Gene Expression Data AnalysisDivision, Bioinformatics Institute, 30 Biopolis Street, Matrix, 138671, SingaporeWe carried out a zebrafish mind bomb (mib) genetic interaction screen and discoveredthat one such cross between heterozygous mibta52b X hi1384 produced a low percentageof phenotype (

(60) Temporal Notch activation through Notch1a and Notch3 is required formaintaining zebrafish rhombomere boundariesXuehui Qiu, Chiaw-Hwee Lim, Kian-Hong Lee and Yun-Jin JiangLaboratory of Developmental Signalling and Patterning, Genes and DevelopmentDivision, Institute of Molecular and Cell Biology, A*STAR (Agency for Science,Technology and Research), 61 Biopolis Drive, Singapore 138673In vertebrates, hindbrain is subdivided into seven segments termed rhombomeres and theinterface between each rhombomere forms the boundary. Similar to the D/V boundaryformation in Drosophila, Notch activation has been shown to regulate the segregation ofrhombomere boundary cells. Here we further explored the function of Notch signaling inthe formation of rhombomere boundaries. By using bodipy-ceramide cell labelingtechnique, we found that the hindbrain boundary is formed initially in mib mutants butlost after 24 hpf. This phenotype was more severe in mibta52b allele than in mibtfi91allele. Similarly, injection of su(h)-MO led to boundary defects in a dosage-dependentmanner. Though cell proliferation was decreased in mibta52b mutants, inhibition of cellproliferation by aphidicolin did not lead to the loss of boundary cells. Boundary cellsdifferentiated into neurons at later stages and were recovered in mibta52b mutants afterinhibition of neurogenesis by injecting hdac1-MO. Furthermore, boundary cells lostsensitivity to reduced Notch activation from 15 somite onwards. We also showed thatknockdown of notch3 function in notch1a mutants leads to the loss of rhombomereboundary cells and causes neuronal hyperplasia, indicating Notch1a and Notch3 play aredundant role in the maintenance of rhombomere boundary.77

(61) The Characterization of Novel mib AllelesKa Fai William TSE*, Qimei NG and Yun-Jin JIANGLaboratory of Developmental Signalling and Patterning, Genes and DevelopmentDivision, Institute of Molecular and Cell Biology, A*STAR (Agency for Science,Technology and Research), 61 Biopolis Drive, Singapore 138673The Notch signaling pathway is an evolutionarily conserved intercellular signaling pathway thatplays essential roles in a wide range of developmental processes, such as pattern formation andcell fate determination. Three E3 ubiquitin ligases, Neuralized (Neur), Mind bomb (Mib), andMind bomb 2 (Mib2) are essential in Notch activation by targeting Notch ligand, Delta and/orSerrate/Jagged for endocytosis. Mib protein contains mib/her2 domain, zz zinc finger domain,and Mib repeat in the N-terminus, ankyrin repeats in the middle, and three Ring Fingers (RFs) inthe C-terminus. Different mib alleles (mibta52b, mibm132, and mibtfi91) in Danio renio had beenidentified and characterized in our lab. In brief, the mib mutants display curly-up tail anddifferent severity of delayed somite development and pigmentation. In addition, decreased her4expression and increased huC expression were found with different degree. Recently, three newmib mutant alleles, mibSJI-1006, mibSJB-1017, and mibSJT-1207 were discovered in our lab bynon-complementation screen. Both mibSJI-1006 and mibSJB-1017, showed typical mibphenotypes. Point mutation in mibSJI-1006 generates a premature stop codon (C898stop) in RF2,resulting in a truncated protein without RF3 that is required for Mib E3 ligase activity. FormibSJB-1017, preliminary analysis suggested a point mutation in intron between exon 8 and 9generating a splicing variant, which leads to a truncated protein of 968 amino acids that deletesRF3. Interestingly, mibSJT-1207 was found to have different morphological phenotypes whencompared to the typical mib alleles, though it did not complement with the existing mib alleles.The mibSJT-1207 embryos, besides its delayed somite development, started early cell death(apoptosis) at 18s stage. In addition, huC expression level was higher. Its stronger neurogenicphenotype and the lethal development are different from other mib alleles, which raised aquestion of its mutation nature: a new mib allele or a mutation in another gene that can interactwith Mib and/or its homolog. By mapping, using the SSLP in LG2, we have found that there is ahigh potential linkage between the mibSJT-1207 and a SSLP marker Z13620 (3.32cm away frommib). This result suggested that the new mibSJT-1207 mutant is another mib allele. We are nowworking on the mutation nature of this new mib allele.78

(62) Identifying gene regulatory networks that control zebrafish regenerationSemil P. Choksi, Leong Wan Ying, Sudipto RoyInstitute of Molecular and Cell Biology, SingaporeOne of the major obstacles in regenerative medicine is directing progenitor cells torestore damaged or diseased tissues. In order to overcome this hurdle, we can look tonature: endogenous regeneration occurs in several complex tissues of the zebrafish,Danio rerio. To understand the molecular mechanisms underlying this process, we haveset out to identify networks of genes that regulate tissue regeneration in the adultzebrafish.We have performed a candidate-based quantitative reverse transcription and PCR screento find transcriptional regulators that are induced during both fin and heart regeneration.Using RNA in situ hybridization, we have tracked the expression domains of several ofthese genes within the regenerating tissue. Through this analysis we have identified fourgenes which are induced during both fin regeneration and heart regeneration.One of these genes, LIM domain only 4a (lmo4a), is highly expressed in the underlyingmesenchyme of the regenerating fin. Morpholino-based gene knockdown indicates thatlmo4a is critical for fin regeneration. Currently we are performing overexpression andgenetic interaction studies to further dissect the function of this gene. Finally, we will usechromatin immunopreciptiation to identify the targets of Lmo4a, placing it into aregeneration-specific transcriptional network.79

(63) The Forkhead domain containing transcription factor Foxj1 is the masterregulator of the motile ciliogenic programXianwen Yu, Chee Peng Ng, Hermann Habacher, Sudipto RoyInstitute of Molecular and Cell Biology, SingaporeIn many tissues, motile cilia induce fluid movement through their rhythmic beatingactivity. Dysfunctional motile cilia are the hallmark of human genetic abnormalities likePrimary Ciliary Dyskinesia or Kartagener syndrome. In mice, the transcription factorFoxj1 has been implicated in motile cilia formation. However, the regulation of Foxj1expression in ciliated tissues and its precise role in ciliogenesis have remained unclear.Here, we show that in zebrafish embryos, foxj1 is a novel target of Hedgehog signaling inthe floor-plate. Loss of Foxj1 severely compromised assembly of motile cilia whichdecorate floor-plate cells. Besides the floor-plate, foxj1 is expressed in the embryonicnode and pronephric ducts, and its function is also necessary for ciliary differentiation inthese tissues. We provide evidence that Foxj1 activates expression of a constellation ofgenes essential for motile cilia formation and function, and that its activity is sufficientfor motile cilia formation. These findings identify a pivotal master regulatory role forFoxj1 in the transcriptional program that controls the production of motile cilia.80

(64) A mouse embryonic stem/progenitor cells as a model to investigate asymmetriccell divisionYin Yijun and Yang XiaohangCellular diversity in every organism is generated from asymmetric cell divisions. Ourunderstanding of the molecular mechanism of asymmetric cell divisions in animaldevelopment comes mainly from studies in C. elegans and Drosophila. However,knowledge of asymmetric division in mammal is less studied. We use in vitro culturedmouse embryonic neural stem/progenitor cells as a model to investigate the asymmetriccell division in mammals. Time-lapse imaging demonstrated that 64% cell divisions (55division in 26 cells) were asymmetric based on several parameters in control, but thefrequency of asymmetric division decreased to 48% (148 division in 62 cells) whenmouse Inscuteable was knockdown (mInsc-kd). Data from pair-assay using Ki67 as amarker to label the proliferating cells further showed that in control, the percentages of p-n (p=positive, n=negative) were 24% (16 pairs), p-p 32% (22 pairs), and n-n 44% (30pairs), as compared with p-n 18% (12 pairs), p-p 58% (32 pairs), and n-n 31% (20 pairs)in mInsc-kd, respectively. The immunocytochemical staining of numb (cell fatedeterminant) at anaphase further showed that the angle between numb crescent stainingand spindle axis was changed. From these data we conclude that asymmetric divisionoccurs in cultured mouse embryonic neural stem/progenitor cells, and there is a shift fromasymmetric to symmetric division in mInsc-kd cells.81

(65) CDK regulates cytokinesis by phosphorylation of an IQGAP family protein infungal pathogen Candida albicansChang Run Li, Yan Ming Wang and Yue WangCandida albicans Molecular & Cell Biology Laboratory, Genes and DevelopmentDivision, Institute of Molecular and Cell BiologyCyclin-dependent kinases (CDKs) drive and coordinate multiple cell-cycle events,including construction and contraction of the actomyosin ring during cytokinesis.However, it remains unclear whether CDKs regulate cytokinesis by directly targetingcomponents of the ring. Here we show that Iqg1, an IQGAP family protein, is directlyphosphorylated by CDK1 in vivo and in vitro. This phosphorylation event mediates theassembly and disassembly of the actomyosin ring, in part through the regulation of theinteraction between Iqg1 and actin-nucleating formins. Furthermore, the phosphorylationof Iqg1 regulates the stability of Iqg1. These results suggest that CDK1 regulatescytokinesis by directly targeting the actomyosin ring component Iqg1 in Candida albicans.82

(66) Regulation of PAK4, a non-conventional PAKYohendran Baskaran (GSK lab)The p21-activated kinase (PAK) family of proteins are critical regulators of cell signalingdownstream of the Cdc42 and Rac subfamilies of Rho GTPases. The group II of PAKs(mammalian PAK4, 5 and 6) is less understood both in terms of their targets and kinaseregulation. Here we define novel mechanisms of regulation, not seen in the group IPAKs.The ubiquitous PAK4 can affect the actin cytoskeleton and is over-expressed in a numberof tumours, however the basis for its role in adhesion-independent growth and survival isnot understood. We have mapped and functionally characterized all the conservedregulatory domains in PAK4 that determine both localization and activity of the kinase.This includes 14-3-3 binding sites that have profound effects on the function of the kinase.Using zebrafish as a model system to study kinase function, we show that PAK4operates via kinase-independent mechanisms. This includes PAK4 nuclear-cytoplasmicshuttling which assists in relocalization of important PAK4-associated proteins (PAPs).83