Dicty 2015

Royal Holloway University of London August 9th-13th

London, United Kingdom

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Annual Dictyostelium Conference

2015 August 9-13

Royal Holloway University of London

Organisers Robin Williams Arjan Kortholt

Douwe Veltman

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Dicty2015issponsoredby

AndspecialthankstoJohannesZankerforprovidingtheimagesfortheconferencebag.

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Mapandinformation

FoundersBuilding WindsorBuilding TheHUBReception ‐Diningroom ‐CrosslandsSuite TukeHallAccomodation ‐PictureGallery

AlltalksareheldintheWindsorAuditorium(WindsorBuilding).

Accommodationcheck‐in: Sunday,TheHubReceptionRegistration: Sunday,WindsorBuildingBreakfast: Mon‐Thur7.30‐9am,FoundersDiningRoomLunch: WindsorBuildingDinner: Sun18.00‐19.30,FoundersDiningRoom Mon‐Tue18.30‐20.00,FoundersDiningroomBarService: Sun19.00‐21.00,CrosslandsSuite Mon‐Tue19.30‐23.00CrosslandsSuitePosterSessions: Mon‐Tue20.00‐21.30,WindsorBuildingConferenceBanquet: Wed,Reception18.30‐19.00PictureGallery Banquet19.15‐21.00FoundersDiningRoom EnglishVillageDance21.00‐23.00

Contact: CMS@rhul.ac.ukOrganisersmobile: +44(0)7946548516(emergencyonly)

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Conferenceprogramme

Sunday9th Monday10th

BiomedicalandHost&Pathogen

Tuesday11th

ChemotaxisandDevelopmentI

Wednesday12th

CytokinesisandNuclear

Organization

Thursday13th

ChemotaxisandDevelopmentII

09.30‐10.401stmorningsession

09.00‐10.401stmorningsession

09.00‐10.401stmorningsession

09.30‐10.401stmorningsession

coffeebreak coffeebreak coffeebreak coffeebreak

11.10‐12.302ndmorningsession

11.10‐12.302ndmorningsession

11.10‐12.302ndmorningsession

11.10‐12.302ndmorningsession

12.00‐18.00ArrivalandRegistration

lunchbreak lunchbreak 12.30‐17.30Pickuplunch

pack

ExcursionWindsorCastleorFuller'sGriffinBrewery

12.30‐13.45lunch

14.00‐15.101stafternoon

session

14.00‐15.101stafternoon

session

Departure

coffeebreak coffeebreak

15.40‐17.002ndafternoon

session

15.40‐17.002ndafternoon

session

17.00‐18.00SelectedStudent

Talks

18.00‐19.30Dinner

18.30‐20.00Dinner

18.30‐20.00Dinner

18.30‐23.00ConferenceDinnerand

EnglishVillageDance

20.00‐21.30PosterSessionIoddnumbers

20.00‐21.30PosterSessionIIevennumbers

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Sunday, August 9

12.00 Registrationdeskopenuntil18.0017.00 1 Dicty Jails: a new tool for long‐term single‐cell imaging.AnaTeresa

LópezJiménez17.30 2 Src1 a protein of the innernuclearmembrane is interactingwith the

lamin‐likeproteinNE81inDictyosteliumdiscoideum.PetrosBatsios18.00 Dinner

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Monday, August 10 BIOMEDICAL & HOST PATHOGEN INTERACTIONS

Morningsessionchair:AdrianHarwood09.30 3 Dictyostelium as a biomedical model: Understanding the MCT

ketogenicdiet.RobinWilliams10.00 4 Roco4: A model to investigate LRRK2 inhibitor binding. Bernd

Gilsbach10.20 5 Impedance spectroscopy of Dictyostelium discoideum development:

from collective oscillations to single cell adhesion dynamics.MarcoTarantola

10.40‐11.10 coffeebreak11.10 6 Cell death in Dictyostelium: investigations on causality. Pierre

Golstein11.30 7 HowLigandBreakdownShapesandPotentiatesChemotacticGradients.

RobertInsall11.50 8 Towards the Application of CRISPR for Whole Gene and Nucleotide‐

SpecificMutagenesis.AlanKimmel12.10 9 Amoeba Tastes Bitter: A Novel Non Animal Model for Bitterness

Research.MarcoCocorocchio12.30‐14.00 lunch

Afternoonsessionchair:AnnetteMüller‐Taubenberger14.00 10 BacteriacaptureandkillingbyNOX‐dependentDNAextracellulartraps

predatestheevolutionofanimals.ThierrySoldati14.30 11 Identificationofmacrame‐aNovelregulatorofmicrotubuledynamics

andlysosomaltrafficking.JasonKing14.50 12 NutritionalimmunityandironhomeostasisinDictyostelium.Salvatore

Bozzaro15.10‐15.40 coffeebreak15.40 13 Interactionsbetweenamoebaeandnon‐pathogenicbacteria:agenetic

approach.PierreCosson16.00 14 Is the TOR pathway controlled by Mycobacterium marinum? Elena

Cardenal‐Muñoz16.20 15 TheCrop Sows theFarmer:Burkholderia bacteria infectiously induce

the proto‐farming symbiosis of Dictyostelium amoebae and foodbacteria.SusanneDiSalvo

16.40 Flashtalks17.00‐18.30 Break18.30‐20.00 Dinner20.00‐21.30 PosterSessionI‐oddnumbers

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Tuesday, August 11 CHEMOTAXIS AND DEVELOPMENT I

Morningsessionchair:PetervanHaastert09.00 16 Systems biology of cellular rhythms: from microorganisms to

mammaliancells.AlbertGoldbeter09.30 17 Identification of a novel chemoattractant GPCR that regulates both

chemotaxisandphagocytosis.TianJin10.00 18 A pseudopod‐centred model for cell migration also replicates

phagocytosis(plusabitofamplification).MatthewNeilson10.20 19 Understanding cell movement in the back of traveling waves of

chemoattractant.MonicaSkoge10.40‐11.10 coffeebreak11.10 20 Investigating the effects of inositol depletion in Dictyostelium.Anna

Frej11.30 21 FunctionofRhoGTPaseinGradientSensing.HiroshiSenoo11.50 22 Regulation of Population Density in Dictyostelium by Extracellular

Polyphosphate.PatrickSuess12.10 23 Why does Dictyostelium discoideum have so many identical actin

genes?EdwardTunnacliffe12.30‐14.00 lunch

Afternoonsessionchair:RichardGomer14.00 24 Noise dampening is required for robust cell fate oscillator dynamics.

ChrisThompson14.30 25 Gene regulation by c‐di‐GMP in Dictyostelid social amoebas. Zhi‐hui

Chen14.50 26 Anovelubiquitin likedomaincontainingprotein is required forearly

developmentandcAMPpropagationinD.discoideum.SatoshiKuwana15.10‐15.40 coffeebreak15.40 27 ThenewfaceofdictyBase.DavidJimenez‐Morales16.00 28 Characterization of Dictyostelium genes by parallel phenotyping of

barcodedmutants.AdamKuspa16.20 Flashtalks16.40 29 Introducing a new dictyExpress:from raw reads to gene expression

browsing.MihaStajdoharandBlazZupan17.00‐18.30 Break18.30‐20.00 Dinner20.00‐21.30 PosterSessionII‐evennumbers

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Wednesday, August 12 CYTOKINESIS AND NUCLEAR ORGANIZATION

Morningsessionchair:JonathanChubb09.00 30 Cellshapethroughthecellcycle.BuzzBaum09.30 31 TheSTEgroupkinaseSepAcontrolscellmigrationandcleavagefurrow

formation.AnnetteMüller‐Taubenberger10.00 32 ADP‐ribosylationofhistoneH2BinrepairofDNAdoublestrandbreaks.

CatherinePears10.20 33 Measurement and modelling of stochastic transcription dynamics.

AdamCorrigan10.40‐11.10 coffeebreak11.10 34 Spacing Remodelers in Dictyostelium Control Transcription Through

DistinctEffectsonNucleosomeSize,PositioningandOccupancy.MarkRobinson

11.30 35 Proteolytic regulation in Dictoystelium mitochondria. ElinorThompson

11.50 36 C‐module‐binding factor supports retrotransposition of TRE5‐A bysuppressinganRNAipathway.ThomasWinckler

12.10 37 REMI‐seq – generation of a genome‐wide mutant resource forDictyosteliumfunctionalgenomics.AmyBaldwin

12.30 Pickuplunchpack13.00 DepartureforWindsorCastleandFuller'sGriffinBrewery18.30 Reception,PictureGallery, Banquet,FoundersDiningRoom EnglishVillageDance

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Thursday, August 10 CHEMOTAXIS AND DEVELOPMENT II

Morningsessionchair:ElinorThompson09.30 38 ForminAfunctionduringmigrationinconfinedenvironments.JanFaix10.00 39 Polychromatic‘greenbeard’genesdeterminepatternsofaggregationin

asocialamoeba.NicoleGruenheit10.20 40 Locally Regulated Ras Signaling Reveals Inhibitory Process in GPCR‐

mediatedChemotaxis.XuehuaXu10.40‐11.10 coffeebreak11.10 41 A Cud‐type transcription factor regulates Dictyostelium spore

differentiation.YokoYamada11.30 42 Ras activationduring cellmovement in buffer and chemotaxis.Peter

vanHaastert11.50 43 The Protein Kinase C Orthologue PkcA Regulates the Actin

Cytoskeleton.DerrickBrazill12.10 44 Macropinosomes and pseudopods are associatedwith distinct spatial

patternsofPIP3andSCAR/WAVE.DouweVeltman12.30‐13.45 lunchDeparture

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Talks

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Talk1

DictyJails:anewtoolforlong‐termsingle‐cellimaging

AnaTeresaLópezJiménez1,MatthieuDélincé2,MonicaHagedorn3JohnD.McKinney2,ThierrySoldati1

1DepartmentofBiochemistry,SciencesII,UniversityofGeneva,30QuaiErnest‐Ansermet,CH‐1211Geneva‐4,Switzerland.

2SchoolofLifeSciences,ÉcolePolytechniqueFédéraldeLausanne(EPFL).1015Lausanne,Switzerland.

3SectionParasitology,BernhardNochtInstituteforTropicalMedicine,Bernhard‐Nocht‐Straße74

D‐20359Hamburg,Germany

Abstract

Recentprogress insingle‐cell techniqueshasevidenced thatcellpopulationsareintrinsicallyheterogeneous,andthattheaveragecellbehaviourcanbeirrelevantto the outcome of biological processes. This is especially relevant to themonitoring of intracellular infections, which result from the interaction ofpopulations of host and pathogens, and give rise to a variety of complexinterdependentresponses.

Weuse long‐termsingle‐cell imaging to study the infectionofDictyosteliumandthe intracellular pathogenMycobacteriummarinum, the causative agent of fishtuberculosis . Similarly to the human pathogenMycobacterium tuberculosis,M.marinum hampers the maturation and acidification of the phagosomalcompartment inwhich it resides. Afterwards,M.marinum breaks its vacuole togain access to the cytosol and eventually escapes by cell lysis or by a non‐lyticmechanismcalledejection.

Likemanyotherprofessionalphagocytes,Dictyosteliumishighlymotilemakingitunsuitable for single‐cell long‐term imaging. Therefore, a microfluidic device totrapinfectedcellswasdesigned.TheDictyosteliumcellcycleandmotilityarenotimpairedinthisdeviceandcanberecordedforseveraldays.

Wehavestarteddissectingthe infectionprocessusingdifferentcellularmarkerssuch as VacuolinA‐GFP, which accumulates at the mycobacterium‐containingcompartment. Exciting results showed that the induction of the differentiationcycle triggers the curing of the M. marinum infection. We thus engaged indeciphering how important pathways such as autophagy, exocytosis or ejectionspecificallyimpactonthefateoftheinfection,usingmutantssuchasatg1‐,wash‐orracH‐,respectively.

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Talk2

Src1aproteinoftheinnernuclearmembraneisinteractingwiththelamin‐likeproteinNE81in

Dictyosteliumdiscoideum

PetrosBatsios1,XiangRen2,IreneMeyer1,OttoBaumann1,DenisLarochelle2andRalphGräf1

1InstitutfürBiochemieundBiology,UniversitätPotsdam,Karl‐Liebknecht‐Str.24‐25,14476Potsdam,Deutschland

2DepartmentofBiochemistryandMolecularBiology,ClarkUniversity,15MaywoodSt.,Worcester,MA01610‐1477,USA

Abstract

The nuclear envelope (NE) consists of the outer and inner nuclear membrane(INM),wherebythelatterisboundtothenuclearlamina.WeidentifiedSrc1asaDictyostelium homologue of group II LEM‐domain proteins, such as the humanlamin and chromatin‐binding INMproteinMAN1.Yet, in contrast toMAN1 Src1lacksaLEM‐domain.BothendogenousSrc1andGFP‐Src1localizetotheNEduringthe entire cell cycle. During interphase Src1 is concentrated at sites associatedwithnucleoliasshownbyantibodystainingindicatingthatSrc1mayberequiredtobindnucleiattheNEinordertofacilitatetransportofribosomesubunitsintothe cytosol. As proven by immunogold‐EM stainings, GFP‐Src1 is exclusivelylocatedat the INM.CellswithhighGFP‐Src1overexpression formed filamentousnuclear protrusions similar in morphology and dynamics to those formed inmutants overexpressing a C‐terminal fragment of theDictyostelium laminNE81fused toGFPwhich led to the idea that Src1 andNE81 could interact. Thiswasconfirmed when soluble, truncated mRFP‐Src1 co‐localized at cytosolic clustersconsisting of an intentionally mis‐localized mutant of GFP‐NE81. Furthermore,Src1 interacted in a BioID biotinylation assay, independently proving aninteraction between both proteins. Our results show that lower eukaryotescontainconservedproteinsoftheinnerNEthatareinteractingwithalamin‐basednuclearlaminaasinhighercells.Thisisinterestingnotonlyfromanevolutionarypointofview,butalsomakestheseamoebaeaninterestingexperimentalplatformtostudytheroleoflamina‐chromatininteractionsinchromatindynamicsandcelldifferentiation.

 

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Talk3

Dictyosteliumasabiomedicalmodel:UnderstandingtheMCTketogenicdiet

PishanChang1,KatrinAugustin1,KimTan4,KarinBoddum2,SophieWilliams2,MinSun2,JohnTerschak3,JorgeD.Hardege3,KarinBorges4*,PhilipE.Chen1,Matthew

C.Walker2,RobinS.B.Williams1

1CentreforBiomedicalSciences,SchoolofBiologicalSciences,RoyalHollowayUniversityofLondon,Egham,TW200EX,UK.2DepartmentofClinicaland

ExperimentalEpilepsy,InstituteofNeurology,UniversityCollegeLondon,WC1N3BG,UK.3SchoolofBiological,BiomedicalandEnvironmentalSciences,UniversityofHull,

CottinghamRoad,HullHU67RX,UK.4DepartmentofPharmacology,SchoolofBiomedicalSciences,TheUniversityofQueensland,StLucia,QLD4072,Australia.

Abstract

OurresearchhasfocusedonemployingDictyosteliumasasimplemodelsystemforbiomedical research. Themodel provides a range of advantages including rapidgrowth, single and multiple cell stages, well characterized development, rapidgeneticablationorproteintaggingandtheuseofisogeniccultures.Thisresearchapproach is also consistent with changing funding priorities in the UK, with anincreasedfocusonbiomedicalorhealth‐relatedareas.Herewewilloutlinealong‐standing research area in our laboratory into epilepsy treatments that wasinitiatedinDictyosteliumandtranslatedtomammaliansystems.

We employed Dictyostelium in our initial studies to better understand themolecular mechanisms of the commonly used epilepsy treatment valproic acid.Using Dictyostelium, we showed that the drug blocked turnover ofphosphoinositides, and we were able to demonstrate a similar effect in themammalian brain during seizure progression. Using Dictyostelium, we thenidentifiedarangeofcompoundswithenhancedactivityinthismechanism.Oneofthesecompounds,decanoicacid,isamajorconstituentofadietusedtotreatdrugresistant epilepsy, themediumchain triglyceride (MCT)ketogenicdiet.We thenshowedthatdecanoicacid,whichiselevatedintheplasmaofpatientsonthediet,actstodirectlycontrolseizureactivityinmultipleanimalseizuremodels.Notably,thiseffectcouldnotbereproducedforketones,suggestingthat thedietmaynotactviaketogenesis.Wefurtheridentifiedaprimarytargetfordecanoicacidinthistherapeuticrole.

This project highlights the use ofDictyostelium as a simple and versatilemodelsystem for biomedical research, and successful translation to mammalian pre‐clinicalresearchmodels.

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Talk4

Roco4:AmodeltoinvestigateLRRK2inhibitorbinding

BerndK.Gilsbach1,GentaIto2,DarioR.Alessi2,PeterJ.MvanHaastert1,AlfredWittinghofer3ArjanKortholt1

1DepartmentofCellBiochemistry,UniversityofGroningen,9747AG,Groningen,TheNetherlands;

2MRCProteinPhosphorylationUnit,CollegeofLifeSciences,UniversityofDundee,DowStreet,DundeeDD15EH,Scotland,UnitedKingdom

3StructuralBiologyGroup,MaxPlanckInstitutfürMolekularePhysiologie,44227Dortmund,Germany

Abstract

LRRK2isamulti‐domainprotein,includingaGTPaseandkinasedomain,whichisfound to be mutated in Parkinson´s disease (PD) patients. So far there is noestablishedtherapyforPD.GivenitsprevalenceincasesofPD,andtheroleofthekinase and GTPase, LRRK2 represents a clear molecular target for therapeuticdevelopment.However,purificationofsuitableamountsandqualityofLRRK2toundertake structural and biochemical characterization is not feasible. Wepreviously have use Dictyostelium discoideum Roco4 as model to study thestructural and biochemical characteristics of the LRRK2 kinase domain (REF).Furthermore, the biochemical tractability of Roco4 allows in vitro screening ofinhibitor libraries,whereas the unique phenotype of roco4‐nullmutants and itsrescue by the Roco4‐LRRK2‐kinase chimera can be used for in vivo testing andscreening.

TheRoco4kinasedomaincanbepurifiedinhighamountsandcrystallizedundervariousconditions.Neverthelessthewild‐typeproteinbindsonlywithlowaffinitytoLRRK2specificinhibitorsthereforeweintroducedtwomutationsF1106LandF1161L which increased the inhibitor binding up to 175 fold. Furthermore thestructural analysis of the two co‐crystallized inhibitors LRRK2‐Inh1 andCompound19showedthepartsoftheinhibitorsdonotcontributetobindingandgivespaceforoptimizationofthesecompounds.

TogetherourdatashowthatRoco4isasuitablemodelsystemtoobtaininsightinthe binding mechanism, optimization of the current and identification of newLRRK2inhibitors.

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Talk5

ImpedancespectroscopyofDictyosteliumdiscoideumdevelopment:fromcollectiveoscillationstosinglecell

adhesiondynamicsLeonhardt,H.,Gerhardt,M.,Krüger,K.,Schäfer,E.,Aue,D.,Polo,E.,Westendorf,C.,

Oikawa,N.,Beta,C.,Geil,B.,Janshoff,A.,Bodenschatz,E.andTarantola,M.

Abstract D.d.synchronizesitselfduringearlydevelopmentwithwavesofcAMP.Wehavequantifiedthese collective phenomena on the intercellular level by applying electric impedancemeasurements. We showed that D. d. seeded on micrometer‐sized electrodes provokeimpedance oscillations, which can be attributed to cAMP dependent oscillations [1]. Werecorded timedependentcorrelationofElectricalCell‐substrate ImpedanceSensing (ECIS)signalsandchanges inheightdetectedbyTIRF‐microscopybycomparing light intensityofsubtractedBFimagesfrombothassays.AhighimpedancesignalIZIwasfoundtocorrelatewith a high fluorescence intensity and thus with a small cell‐surface‐distance. ThearrangementofD.d.analyzedonbrightfieldimagesrevealedsynchronousformationofcellaggregatescontributingtoimpedanceoscillations,whileadecreaseofcircularity<C>occursout‐of phase. Modeling calculations show that cells in united structures generate higherimpedance values than the same number of isolated cells [2]. In addition, we analyzestarving cells in microfluidic devices in combination with an impedimetric assay [3]. Theresultsprovideaquantitativeunderstandingof thecollectivecellmorphologyduringearlystarvation.Inarecentwork,wepresentelectricalimpedancemeasurementsofsingleamoeboidcellsonmicroelectrodes. Wild type cells and mutant strains are studied that differ in their cell‐substrateadhesionstrength.Werecordedtheprojectedcellareabytime‐lapsemicroscopyandobservedquasi‐periodicoscillationsofthecellshape.Thesewerecorrelatedwithlong‐termvariationsintheimpedancesignalanddidnotshowanysystematicdependenceontheadhesion strength. In contrast, short‐term impedance fluctuations were clearly correlatedwith the adhesion strength. Interference reflection microscopy recordings showed thatalteredcell‐substrateadhesioncorrespondstochangesintheareaofclosecontactbetweentheventralsurfaceofthecellandthesubstrate.Wethusconcludethatlong‐termtrendsinthe cellular impedance signal of single cells are caused by oscillatory changes in theprojected cell area,while short‐term fluctuations canbe attributed to local changes in thecell‐substrate distance that are related to the formation of adhesion sites. We propose amethodtoseparatebothcontributionstotheimpedancesignalbasedonband‐passfiltering.

[1] Schäfer, E., Westendorf, C., Bodenschatz, E., Beta, C., Geil, B. and Janshoff, A., "ShapeOscillations of Dictyostelium discoideum Cells on Ultramicroelectrodes Monitored byImpedanceAnalysis",Small,March2011,Vol.7(6),723pp.

[2]Schäfer,E.,Tarantola,M.,Polo,E.,Westendorf,C.,Oikawa,N.,Bodenschatz,E.,Geil,B.andJanshoff, A., "Chemotaxis of Dictyostelium discoideum: Collective Oscillation of CellularContacts",PLOSOne,January2013,Vol.8,e54172pp.

[3] Schäfer, E., Aue, D., Tarantola,M., Polo, E.,Westendorf, C., Oikawa,N., Bodenschatz, E.,Geil, B. and Janshoff, A., "Collective behavior of Dictyostelium discoideum monitored byimpedanceanalysis",Commun.Integr.Biol.,May2013,Vol.6(3),e23894pp.

[4] Leonhardt, H., Gerhardt, M., Krüger, K., Tarantola, M. and Beta, C., "Cell‐substrateimpedance fluctuations of single amoeboid cells encode cell‐shape and cell‐substrateadhesiondynamics",Phys.Rev.E,2015,submitted

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Talk6

CelldeathinDictyostelium:investigationsoncausality

PierreGolstein

Centred'ImmunologiedeMarseille‐Luminy,INSERM‐CNRS‐UniversitédelaMéditerranée,Case906,13288MarseilleCedex9,France

Abstract

Dictyostelium discoideum, a eukaryote, a protist, a slimemold, is unicellular infavorableconditions.Itshowsuponstarvationmulticellulardevelopmentleadingtofruitingbodies.Eachofthesefruitingbodiesismadeofamassofsporesontopofastalk.Thisstalkismadeofdyingordeadcells.

This cell death, developmental by definition, can be mimicked in vitro in cellmonolayers allowing morphological study and genetic manipulations.Dictyosteliumofferstwomainadvantagesforthestudyofthiscelldeath.First,itsgenome, now sequenced, is small, compact, and haploid, favoring insertionalmutagenesis.Second,fromacelldeathpointofviewthisgenomedoesnotencodecaspases or bcl‐2 family members, thus in this model organism there is noapoptosis machinery that could interfere with the non‐apoptotic cell deathmechanismsatplay.

The results to be reported include a description of successive steps of thisdevelopmental cell death asmimicked in vitro (including paddle cell formation,rounding,vacuolizationandcelluloseshellformation),theirgoverningexogenoustwo main signals, an insertional mutagenesis study partially identifying thecorresponding signaling pathways, and considerations ofwhere death proper islocatedinthesephenomenologicalandmolecularpathways.

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Talk7

HowLigandBreakdownShapesandPotentiatesChemotacticGradients

LukeTweedy1,DavidKnecht2,RobertInsall1

1BeatsonInstituteforCancerResearch,GlasgowG611BD,UK2UniversityofConnecticut,91N.EaglevilleRd.,Storrs,CT06269,USA

Abstract

Breakdown of chemoattractants such as cAMP is essential for their biologicalfunctions.DisruptionofcAMPphosphodiesterase,forexample,completelyblocksDictyosteliumdevelopmentascAMP levelsgrowtooconstitutivelyhigh toallowpulsatilesignalling.

Wehaverecentlyfoundinanothersystemthatuseschemotaxis‐melanomacellsmigratingtowardsLPA‐thatattractantbreakdowncancauseamorefundamentalbehaviour shift. Melanoma cells break down LPA that is globally present insaturatinglevels.Bydoingthistheycreatechemoattractantgradientsthatarelowwhere cell density is highest. These gradients have a number of unexpectedeffects ‐ they canoperateoverarbitrarily largedistances,unlike fixedgradients,whoserangeislimitedtoshortdistances.Althoughthepopulationrespondsasawhole,onlyavariableandcomplexproportionoftheindividualcellsmoveatanyone time; we provide computational models and direct measurements to showhowthisworks.

Consideration of the effects of attractant breakdown shows that even inestablishedassays,suchasDunnchamberandmicropipetteassays, theenzymesthatbreakdownattractantsfundamentallychangetheshapeofthegradientsandtheresponsesofthecells.

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Talk8

TowardstheApplicationofCRISPRforWholeGeneandNucleotide‐SpecificMutagenesis

WenliBai,AlanR.Kimmel

LaboratoryofCellularandDevelopmentalBiology

NationalInstituteofDiabetesandDigestiveandKidneyDiseases

NationalInstitutesofHealth,Bethesda,MD20892,US

Abstract

Althoughtheefficiencyoftargetedgenedisruptionbyhomologousrecombinationin Dictyostelium is high compared to more complex genomes, site‐specificmutagenesisandepitopetaggingarestillproblematic.Toovercometheseissues,we have been developing methods for Dictyostelium‐specific application of theCRISPR/Cas9genomeeditingsystem.Wewilldiscussvectordetails,experimentalguidelines,butalsocurrentlimitations.

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Talk9

AmoebaTastesBitter:ANovelnonAnimalModelforBitternessResearch

MarcoCocorocchio1,PaulL.R.Andrews2,RobinS.B.Williams1

1SchoolofBiologicalSciences,CentreofBiomedicalSciences,RoyalHollowayUniversityofLondon,Egham,UnitedKingdom,

2DivisionofBiomedicalScienceStGeorge’sUniversityofLondon,London,UnitedKingdom

Abstract

A large number of therapeutically active drugs have a bitter taste. This oftencauses an aversive reaction in both children and adults, leading to decreasedpatient compliance and in some cases to severe reactions such as nausea andvomiting.Researchintobittertasteperceptionoftenemploysanimalmodelsthatareexpensive,timeconsumingandcauses longlastingdistress.Thus, there isanurgentneedtodevelopnewstrategiesfortheidentificationofbittersubstancesinthe early phase of drug screening. Since we have shown that, like humans, thesocialamoebaDictyosteliumdiscoideumisabletorespondtobittermolecules,weset out to investigate if themodelwas able to predict the bitterness of specificmolecules. Here, twelve bitter substances (ibuprofen, azelastine hydrochloride,caffeine, chlorhexidine digluconate, potassium nitrate, paracetamol, quinine andfive blinded compounds) were employed to investigate the changes in cellbehaviour (inhibition of protrusion formation) as a read‐out for bitternessresponse. We show that Dictyostelium cell behaviour was inhibited by allcompounds in a dose dependent manner, with calculated IC50 values enablingcomparison to that obtained using the rat in vivo Brief Access Taste Aversion(BATA)testandhumansensorypanelmodelstest.Usingbothknownandblindedcompounds,Dictyostelium cell behaviour shows a significant correlation to bothrat and human bitterness response. Therefore,Dictyostelium shows promise asreplacementpre‐screeningplatformfortheinvestigationofbittertastants.

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Talk10

BacteriacaptureandkillingbyNOX‐dependentDNAextracellulartrapspredatestheevolutionofanimals

XuezhiZhang1,OlgaZhuchenko2,AdamKuspa2,andThierrySoldati1.

1DepartmentofBiochemistry,SciencesII,UniversityofGeneva,30QuaiErnest‐Ansermet,CH‐1211Geneva‐4,Switzerland.

2MolecularandHumanGenetics,BaylorCollegeofMedicine,HoustonTX770301,USA

Abstract

Thefirstlineofdefenceagainstbacteriaarephagocyticcellsoftheinnateimmunesystem. These cells kill bacteria via oxygen‐dependent (e.g. ROS) and oxygen‐independent (e.g. chemicals, enzymes and microbicidal peptides) mechanisms.While multicellular organisms use phagocytosis to kill microbes and initiate asustainedimmuneresponse,phagocyticamoebaeinternalisebacteriaasnutrients,via mechanisms of recognition, signalling and killing that are surprisinglyconserved.Inparticular,atthetransitiontomulticellularity,eukaryoticorganismsacquiredNOXenzymestogenerateROS.

Dictyosteliumisasocialamoebathatfeedsbyphagocytosisandhasarudimentarybut highly conserved cell‐intrinsic immune system. It is genetically andbiochemically tractable and has emerged as a powerful and experimentallyversatilemodelorganism.SolitaryDictyosteliumamoebaefeedonbacteriainthesoil until depletion of the food induces a developmental program in which~100,000 amoebae aggregate, form a migrating slug and eventually undergoterminaldifferentiation into thesporesandstalkcells thatcomprise the fruitingbody.Inaddition,theslugalsocontainsapopulationofSentinelcellsthataretheonlyslugcellsabletophagocytosebacteriatobeleftbehindduringslugmigration.We propose that Sentinel cells represent a form of amoebal innate immunityanalogoustomammalianneutrophils.

Extracellular traps (ETs) produced by neutrophils are reticulated nets of DNAdecoratedwithantimicrobialgranulesthatcontributetodefenseagainstbacteria.Sentinel cells are able to elaborate DNA‐based ETs morphologically andfunctionallysimilartothoseofneutrophils.TheproductionofETsisstimulatedbyLPS and by Gram‐negative bacteria.Most interestingly, ETs production requiresthegenerationofROSbySentinelcells,viaNOXenzymes.Theseresultsemphasisethat, like in neutrophils from CGD patients, NOX‐dependent ROS production isessential toET formation,andthat theoriginofDNA‐basedETsasamechanismforbacterialdefensepredatestheappearanceofanimals.

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Talk11

Identificationofmacrame–anovelregulatorofmicrotubuledynamicsandlysosomaltrafficking

JasonS.King

1DepartmentofBiomedicalSciences,UniversityofSheffield,FirthCourt,WesternBank,Sheffield.UK

Abstract

Autophagy is required by cells to capture and remove damaged cellularcomponentsaswellassurviveperiodsofstarvation.Ageneticscreento identifynew components required for autophagic degradation identified a new generequired for cells to degrade their cytosol, survive amino acid starvation andcompletedevelopment.Nullmutantshoweveralsohavepoorbacterialandaxenicgrowth, indicating a general lysosomaldefect.This gene (whichwehavenamedmacrame) encodes a highly conserved, but previously unstudied protein.Mechanistically,weshowthatmacrameisafundamentalregulatorofmicrotubuledynamics,andthusessentialforproperlysosomaltraffickinganddegradation.

22

Talk12

NutritionalimmunityandironhomeostasisinDictyostelium

BarbaraPeracino1,SimonaBuracco1,RaffaellaCinquetti2,AlessandraVollero2,ElenaSignoretto3,MichelaCastagna3,ElenaBossi2andSalvatoreBozzaro1

1Dept.ClinicalandBiologicalSciences,UniversityofTurin,AOUS.Luigi,10043Orbassano(TO),Italy,2Dept.BiotechnologyandLifeSciences,UniversityofInsubria,ViaDunant2,21100Varese(Italy),3Dept.PharmacologicalandBiomolecular

Sciences,UniversityofMilan,ViaTrentacoste,20133Milano,Italy

Abstract

Ironisanessentialelementforallcells,beinginvolvedinmanycellularprocessesas enzyme cofactor or component of Fe‐S or heme prosthetic groups. Bacteriaacquire iron from the environment by secreting siderophores, and manyintracellular pathogens, such as legionella or mycobacteria, scavenge iron fromthe phagosomal milieu. Professional phagocytes, such as macrophages orDictyostelium,havedevelopedmechanismstomanipulatemetalionconcentrationto control microbial growth, a process known as “nutritional immunity”. ThephagosomalirontransporterNramp1playsaparadigmaticroleinthisprocess.InDictyostelium,Nramp1displaysbothnutritiveandprotective function, favouringcytosolic accumulation of iron upon bacteria digestion and simultaneouslystarving ingested pathogens from iron. Such a dual role is similar to that of themacrophage ortholog, which recycles iron from ingested erythrocytes whilestarving engulfed pathogens.Dictyostelium Nramp1 shareswith themammalianortholog several features: their protein sequences are highly conserved, in bothcasestransport isregulatedbyaprotongradient, iselectrogenicandspecific forFe2+ and Mn2+, not Fe3+ or Cu2+. Dictyostelium also harbours Nramp2, which islocatedinthecontractilevacuoleandsynergisticallywithNramp1regulatesironhomeostasis. Nramp2 differs for a more divergent protein sequence and formediating non‐electrogenic transport of Fe2+ only. In vivo iron efflux fromphagosomesviaNramp1requiresreductionofFe3+toFe2+.Ofthreeputativeferricreductases,atleastoneisexpressedinplasmamembraneandphagosomes.Othermajorregulatorsofmammalianironhomeostasis,suchasmitoferrin,frataxinandAco‐1,areconservedinDictyostelium.

23

Talk13

Interactionsbetweenamoebaeandnon‐pathogenicbacteria:ageneticapproach

JadeLeiba,AymanSabra,RomainBodinier,PierreCosson

DepartmentofCellPhysiologyandMetabolism,UniversityofGenevaMedicalSchool,1211Geneva,Switzerland

Abstract

Dictyostelium amoebae can feed upon a variety of non‐pathogenic bacteria. Forthis,theymustingest,killanddigestthebacteriatowhichtheyareconfronted.Wehave previously isolated and characterized Dictyostelium mutants that lost theabilitytofeeduponbacteria.Forsomemutants, thiswasduetoadefect intheirability tokillbacteria (e.g. kil1, kil2mutantcells). Forothers (e.g. fspAmutant),thiswaslinkedtoaninabilitytosensethepresenceofbacteria.

Wehave recently isolatednewDictyosteliummutants unable to feeduponnon‐pathogenic bacteria, and are currently analyzing their phenotypes. Our currentresults reveal the existence of distinct mechanisms for sensing and killing ofdifferenttypesofbacteria.

24

Talk14

IstheTORpathwaycontrolledbyMycobacteriummarinum?

ElenaCardenal‐Muñoz1,CarolineBarisch,SoniaArafah,ThierrySoldati

1DepartmentofBiochemistry,UniversityofGeneva,SciencesII,30QuaiErnestAnsermet,1211,Genève‐4,Switzerland

Abstract

TheprofessionalphagocyteDictyosteliumhasbecomeagoodmodeltoinvestigatehoweukaryoticcellsrespondtobacterialinfection.WeuseDictyosteliumasahostmodel for tuberculosis studies. Like in macrophages, Mycobacterium marinum(bacterium causing a tuberculosis‐like disease in frogs and fish) modifies thephagosome ofDictyostelium to establish a replicative niche, whichmight shieldagainstintracellularimmuneresponsesbutlimitsthebacterialaccesstonutrients.Maybe as a consequence, early after the infectionM.marinum breaks its niche,leadingtotherecruitmentofhostautophagymarkerssuchasp62,Atg8/LC3andubiquitin.Autophagy ispartof the innate immuneresponseagainst intracellularpathogens,eventhoughsomebacteriabenefitfromautophagy‐derivednutrients.

Amoebae lacking Atg1/ULK1, the major kinase responsible for autophagyinduction, show increased numbers of lipid droplets (LDs, the main nutrientsource for mycobacteria), while proliferation ofM.marinum is enhanced. SinceTOR kinase regulates both autophagy and LDs homeostasis in eukaryotes, wewonderwhetherM.marinumactivelycontrolsTORinordertoobtainlipidsfromthehostandthussupportitsproliferation.Weobservechangesinthelocalizationof some TOR pathway components (e.g. Rheb, Lst8, Raptor) during infection,which mimic the changes observed under starvation. By applying severalbiochemicalandmicroscopyapproaches,weare trying todecipher thesignalingcascades responsible for the different localization of TOR pathway proteins, aswellasthecorrelationbetweenthesechangesinlocalizationandTORactivity.

25

Talk15

TheCropSowstheFarmer:Burkholderiabacteriainfectiouslyinducetheproto‐farmingsymbiosisof

Dictyosteliumamoebaeandfoodbacteria.

SusanneDiSalvo,TamaraS.Haselkorn,UsmanBashir,DanielaA.Jimenez,DebbieA.Brock,DavidC.Queller,JoanE.Strassmann

1DepartmentofBiology,WashingtonUniversityatSt.Louis,St.Louis,Missouri63130,USA

Abstract

Theinitiationofanewsymbioticassociationcanallowforanorganismtorapidlyacquire novel traits by gaining access to the genetic repertoire of their partner.However, symbionts may begin as pathogens that only subsequently becomebeneficial. In the Dictyostelium discoideum farming symbiosis, certain amoebas(farmers)persistentlyassociatewithbacterialpartners.Thesebacterialpartnerscan incur a reproductive cost but also provide their host with novel beneficialcapabilities,suchascarriageofbacterialfood(proto‐farming)anddefenseagainstcompetitors. Here, we explore the role of bacterial associates in the initiation,maintenance,andphenotypiceffects innewfarmingsymbioses.Wedemonstratethattwocladesof farmer‐associatedBurkholderia isolatescolonizeD.discoideumnon‐farmers and infectiously endow them with farmer‐like characteristics,suggesting that Burkholderia symbionts are a major driver of the farmingphenomenon. Under food rich conditions, Burkholderia colonized amoebasproducefewersporesthantheiruncolonizedcounterparts,buttheseverityofthiseffect differs betweenBurkholderia genotypes and in some cases, between newand old amoeba hosts, suggesting some role for co‐evolution within theassociation. However, Burkholderia colonization also leads to secondary foodcarriage by amoeba hosts, which may be conditionally adaptive because it canconferanadvantagetotheamoebahostwhengrowninfoodlimitingconditions.Burkholderiacanbefoundinsideamoebasporesdemonstratingthatitcaninvadeand survive within host cells. These results change our understanding of theDictyostelium farming symbiosis by establishing that the bacterial partner,Burkholderia,isanimportantcausativeagentofthefarmingphenomenon.

26

Talk16

Keynotespeaker

Systemsbiologyofcellularrhythms:frommicroorganismstomammaliancells

AlbertGoldbeter

UniversitéLibredeBruxelles(ULB),

CampusPlaine,CP231,Brussels,Belgium

Abstract

Rhythmic phenomena occur at all levels of biological organization,with periodsrangingfrommillisecondstoyears.Cellularrhythmsoriginatefromtheregulatoryfeedbackloopsthatcontrolthedynamicsofbiochemicalprocessesandrepresenta phenomenon of temporal self‐organization. They illustrate how an emergentproperty,autonomousoscillatorybehaviour,arisesfrommolecularinteractionsinregulatory networks. In my talk I will address the dynamical bases of cellularrhythmsinbothmicroorganismsandanimals.Inmicroorganisms,examplesrangefrom metabolic oscillations in yeast to cyclic AMP oscillations in Dictyosteliumcells,MinproteinoscillationsinE.coli,andcircadianoscillationsincyanobacteria.In plant and animal cells, circadian oscillations originate from transcription‐translation regulatory loops. The network of cyclin‐dependent kinases thatcontrolsprogressionthroughthemammaliancellcycleisregulatedinawaythatmakes itprone tooperate in aperiodicmanner.Focusingon the latter system Iwill address the balance between cell cycle arrest and cell proliferation. Thisbalance is controlled by the combined effect of growth factors, the levels ofactivators (oncogenes) and inhibitors (tumour suppressors) of cell cycleprogression, the extracellular matrix, and contact inhibition. Supra‐thresholdchanges in the levelofanyof these factorscantriggeraswitch inthedynamicalbehaviour of the Cdk network corresponding to a bifurcation between a stablesteady state, associated with cell cycle arrest, and sustained oscillations of thevariouscyclin‐dependentkinases,correspondingtocellproliferation.

References:

[1] A. Goldbeter, Biochemical Oscillations and Cellular Rhythms. The molecular bases ofperiodicandchaoticbehaviour.CambridgeUniv.Press,Cambridge,UK(1996).

[2]A.Goldbeter,Computationalapproachestocellularrhythms.Nature420,238(2002).

[3] A. Goldbeter, C. Gérard, D. Gonze, J.‐C. Leloup, G. Dupont, Systems biology of cellularrhythms.FEBSLett.586,2955(2012).

[4]C.Gérard,A.Goldbeter,Temporalself‐organizationofthecyclin/Cdknetworkdrivingthemammaliancellcycle.ProcNatlAcadSciUSA106,21643‐21648(2009).

[5] C. Gérard, A. Goldbeter, The balance between cell cycle arrest and cell proliferation:control by the extracellularmatrix and by contact inhibition. InterfaceFocus4: 20130075(2014).

27

Talk17

IdentificationofanovelchemoattractantGPCRthatregulatesbothchemotaxisandphagocytosis

MiaoPan1,XuehuaXu1,YongChen2andTianJin1

1ChemotaxisSignalSection,LaboratoryofImmunogenetics,NationalInstituteofAllergyandInfectiousdisease,NIH,Rockville,Maryland,20852

2ProteomicsCoreFacility,NationalHeart,Lung,andBloodInstitute,NationalInstitutesofHealth,Bethesda,MD20892

Abstract

Eukaryotic phagocytes search and destroy invading microorganisms viachemotaxisandphagocytosis. Differentreceptorsmediatesignalingpathwaystocontrol the remodeling of actin cytoskeleton that generates either chemotaxingleading frontsorphagocytic cups. Here,usingaquantitativephosphoproteomicapproach, we uncovered an orphan GPCR for folic acid, a chemoattractant forDictyosteliumdiscoideum. D. diccoideum cells are professional phagocytes thatchasebacteriaviachemotaxisandeatthemviaphagocytosis.WeshowedthatthisnewGPCRisrequired forbothchemotaxis toward folicacidandphagocytosisofbacteriaforfood.Thus,ourstudysuggeststhatachemoattractantGPCR‐mediatedsignaling pathways that control not only directional cell migration but alsophagocytosis.

28

Talk18

Apseudopod‐centredmodelforcellmigrationalsoreplicatesphagocytosis(plusabitofamplification)

MatthewNeilson1,RobertInsall1

1TheBeatsonInstituteforCancerResearch,GarscubeEstate,SwitchbackRoad,Glasgow,G66BD

Abstract

Phagocytosis(theprocessbywhichacellconsumesasolidparticle)isinvolvedina variety of processes, such as nutrient‐acquisition, immune‐response and theremovalofcelldebris.Uponencounteringaforeignparticle,theactincytoskeletonofaphagocyticcellundergoeslocalreorganisation,whichcausesthecelltostretchitselfaroundtheparticleuntil ithasbeenengulfed.Wedemonstratethat,withafew simple modifications, our existing computational model for pseudopod‐centredcellmigrationandchemotaxiscanbeusedtosimulatephagocytosis.Usingthismodifiedmodel,we investigate the effects of phagocytic signalling and cell‐particleadhesiononacell'sabilitytoperformphagocytosis.

Dictyostelium cells are capable of performing effective chemotaxis (that is,directedmigrationinresponsetoanexternalstimulus)overasurprisinglybroadrangeofchemicalconcentrations.Onewayinwhichacellcouldachievethislargedynamic range is by incorporating a variable amount of amplification into itssignalling system, such that the chemotactic sensitivity ismaximised relative totheambientconcentration.Weshowthat,withtheinclusionofadynamicsignal‐amplificationterm,ourexistingcellmigrationmodelcanproducechemotaxisoverawiderrangeofconcentrations.Thisproducesaformofadaptationthatisslower,andworksdifferently, fromtheshort‐termadaptationseeninLEGImodels. Ourmodel is thenused to investigate the effectsof variable amplificationona cell'sabilitytochemotaxinshallowgradients.

29

Talk19

Understandingcellmovementinthebackoftravelingwavesofchemoattractant

MonicaSkoge1,2,,Wouter‐JanRappel2,andWilliamF.Loomis1

1DepartmentsofBiologyUniversityofCalifornia,SanDiego,LaJolla,CA920932DepartmentsofPhysics,UniversityofCalifornia,SanDiego,LaJolla,CA92093

Abstract

During aggregation Dictyostelium cells migrate chemotactically towards thesourceoftravelingwavesofcAMP.Todoso,theymustfollowthespatialgradientin the front of the wave, which leads toward the wave source, and ignore theopposing spatial gradient in the back of thewave, which points away from thesource–the“back‐of‐the‐wave”problem.Ourrecentworkhasshownthat5‐hour‐developed, but not 3‐hour‐developed,cells continuemigrating toward thewavesourceinthebackofthewave‐thusrememberingthegradientdirectionseeninthe front of the wave ‐ for over 2 minutes in traveling waves with a “natural”period of 6minutes. Herewe explore themolecular basis for themovement ofcellsinthebackofthewaveusingmutants,inhibitors,andfluorescentreportersofthe chemotaxis signaling network. We found that caffeine strongly inhibitspersistenceandincreasesreversalinthebackofthewave,andtracedthiseffecttoimpairmentoftheTORC2‐PKBpathway.Incontrast,wefoundthatpersistenceisdecreased without increasing reversal in mutants lacking myosin II and cGMPproduction.

30

Talk20

InvestigatingtheeffectsofinositoldepletioninDictyostelium.

AnnaFrej1,JonathanClark2,CarolineLeRoy3,PeterThomason4,AndrewDavidson4,SergioLilla4,GrantChurchill5,SandrinePClaus3,RobertInsall4,Phillip

Hawkins2,LenStephens2andRobinSBWilliams1

1SchoolofBiologicalSciences,RoyalHollowayUniversityofLondon,Egham,UK

2TheBabrahamInstitute,Cambridge,UK

3DepartmentofFood&NutritionalSciences,TheUniversityofReading,Reading,UK

4TheBeatsonInstituteforCancerResearch,Glasgow,UK

5DepartmentofPharmacology,UniversityofOxford,Oxford,UK

Abstract

Regulating inositol signalling is a proposed mechanism of action for bipolardisorder treatments, includingvalproic acid (VPA) and lithium.VPAand lithiumreduceinositol(1,4,5)‐trisphosphate(IP3)levelsinDictyosteliumandmammalianneurons. VPAwas suggested to inhibit inositol‐3‐phosphate synthase (INO1) ininositolbiosynthesisalthoughthespecificmechanismremainsunknown.

We investigate consequences of inositol depletion and a role for INO1 in cellfunctionandasapotential target forVPA.Weshowthatacell linewithablatedino1 is unable to grow or develop unless supplemented with inositol. Inositoldepletionenhancesautophagy,lossofsubstrateadhesionandreducedcytokinesis.Overexpressionofino1rescuestheseeffects,confirminganessentialrequirementforINO1ingrowthanddevelopment.

WealsoexaminedthecellularandphysiologicaleffectsofINO1loss.TheabsenceofinositolandINO1proteincausedachangeinDictyosteliummetabolism.Inositoldepletionledtoadecreaseinthelevelsofaminoacidsandphospholipids,withtheexceptionofphosphatidylinositol4,5‐bisphosphate(PIP2).Tofurther investigatetheroleofINO1,wehaveidentifiedpotentialbindingpartnerstohelpdissectitsroleinbasiccellfunction.

OurdataprovidesanewinsightintothecellularandphysiologicalrolesofINO1,includingcellsurvival,metabolismandphospholipidsignalling.

31

Talk21

FunctionofRhoGTPaseinGradientSensing

HiroshiSenoo&MihoIijima

DepartmentofCellBiology,JohnsHopkinsUniversitySchoolofMedicine

Hunterian107,725N.WolfeStreet,Baltimore,MD21205

Abstract

Afundamentalquestioninchemotaxisishowcellstranslateunstableextracellularcuesintorobustintracellularsignaling?InDictyosteliumchemotaxis,gradientsofthechemoattractantcAMPareconvertedtothelocalactivationofRasGTPaseandthe production of phosphatidylinositol 3,4,5 triphosphate (PIP3) at the leadingedge of migrating cells. The conversion of extracellular chemical gradients tointracellular polarized signaling is called directional sensing and serves as aninternalcompasstodeterminethedirectionofcellmovements.

UsingbiosensorsforRasactivationandPIP3production,IfoundthatcellslackingaRhoGTPase,RacE,failtoorientRasandPIP3signalingeventsincAMPgradients.Asaresult,racE‐nullcellsfailedtomaintaintheproperpositionofRasandPIP3signalingandthereforeshowedimpairedchemotaxis.Importantly,anactiveformof RacE was located at the rear region facing away from cAMP gradients,suggestingthatRacEspatiallyrestrictsRasandPIP3signalingtothefrontofcellsfromtheback.

IdentificationandcharacterizationofRacEbindingpartnerswilllikelyhelprevealhowRacEspatiallyrestrictsRasactivationandPIP3production.IwasspecificallyinterestedinfindingproteinsthatbindtobothRacEandRas.Usingcombinationof immunoprecipitation and mass spectrometry, we identified a novel protein,GflB,thatbindstobothRacEandRas,asacandidatecomponentthatlinksRacEtoRas signaling. I generated gflB‐null cells and found that gflB‐null cells generateexcesslateralpseudopodsandareimpairedchemotaxis,similartoracE‐nullRacEcells.Theseresultssuggestdefects indirectionalsensingingflB‐nullcells.Atthemeeting, I will discuss how GflB controls directional sensing and chemotaxistowardcAMP.

32

Talk22

RegulationofPopulationDensityinDictyosteliumbyExtracellularPolyphosphate

PatrickSuessandRichardGomer

DepartmentofBiology,TexasA&MUniversity,CollegeStation,Texas,USA

Abstract

Muchremainstobeunderstoodabouthowapopulationofcellssensesitsdensityorthenumberofcells,andstopsproliferationwhenthedensityortotalnumberreachesapresetvalue.Inthe1970’s,YargerandSollfoundthatDictyosteliumcellssecrete a factor that induces stationaryphase in shaking culture.We found thatthefactorappearstobeinorganicpolyphosphate.Inshakingculture,extracellularpolyphosphate concentrations increase as the cell density increases, and if theconcentrationofpolyphosphatefoundatstationaryphaseisaddedtocellsatmid‐log, proliferation is inhibited. Adding an exopolyphosphatase to cell culturesdecreases extracellular polyphosphate and causes cells to proliferate toabnormally high cell densities. Biotinylated polyphosphate shows saturatablebinding at concentrations similar to those present at stationary phase, and thisbinding is competed by unlabeled polyphosphate as well as stationary phaseconditionedmedia. Despite the presence of polyphosphate in all eukaryotes, itssynthesisandregulationispoorlyunderstood.WefoundthatcellslackingInsP6K,whichaddsanextraphosphatetoIP6,orcellslackingthesecretedproteinsAprAandCfaD, reachabnormallyhigh celldensitites,donotentera stationaryphase,and showdecreasedextracellularpolyphosphate levels.Conversely, cells lackingthephospholipaseDPldBhave increasedextracellularpolyphosphate and reachstationary phase at abnormally low cell densities. These findings may provideinsight into polyphosphate synthesis and the nature of cell density and cellnumberregulationinhighereukaryotes.

33

Talk23

WhydoesDictyosteliumdiscoideumhavesomanyidenticalactingenes?

Tunnacliffe,E.1,Miermont,A.1,Santhanum,B.2,Rosengarten,R.2,Shaulsky,G.2andChubb,J.R.1

1MRCLaboratoryforMolecularCellBiology,UniversityCollegeLondon,GowerSt.,London,WC1E6BT,UnitedKingdom

2DepartmentofMolecularandHumanGenetics,BaylorCollegeofMedicine,OneBaylorPlaza,Houston,TX77030,USA

Abstract

Geneduplicationisimportantforthegenerationofnovelgenesandfunctionsandevolutionary theorypredictsentirely redundantgenesshoulddivergeover time.Dictyosteliumdiscoideumhasalargeactingenefamilywhichconsistsof32genes,17ofwhichencodeasingleprotein isoform.Othereukaryotessuchasyeasts (1gene),Drosophila (6) and humans (6) have far fewer genes, with each isoformencoded by a separate gene. Other Dictyostelids have a similar expanded actingene family, implying functional value. To investigate why this organism hasmaintainedsuchalargecomplementofgenesencodingidenticalproteins,weareusing1.RNAseqdatatoreevaluateearlyexpressiondatabasedonhybridisations,2.singlecelltranscriptomicstocharacterisethecellvariabilityinactinexpressionduring development, 3. live transcription imaging to monitor the acutetranscriptionaldynamicsofindividualfamilymembers,4.knockoutstoinvestigategenecrosstalkand5.localisationandstabilityassaystoaddresstheimportanceof3’UTRelements.

34

Talk24

Noisedampeningisrequiredforrobustcellfateoscillatordynamics

KokiNagayama1,NicoleGruenheit1,BalintStewart1,ThomasKeller1,WilliamSalvidge,WouterVanZon1andChrisThompson1

1FacultyofLifeSciences,UniversityofManchester,MichaelSmithBuilding,Manchester,M139PT

Abstract

Developmental cell fate choice and proportioning is characterised by itsrobustness and reproducibility. Consequently, heterogeneity (also termed noiseand stochasticity) in cell behaviours, cell signalling and responses are oftenthought to be a hindrance. However, recent observations of ‘salt‐and‐pepper’differentiation,which is seen in examples raging fromcompetence inB. subtilis,lineagespecificationinthemouseblastocyst,tostalkandsporedifferentiationinDictyosteliumhavechallengedthisview.Infact,inthesecasessuchheterogeneityhas been proposed to be required for normal cell fate choice and symmetrybreaking. Our research addresses the extent to which such heterogeneity isnecessary,howsomethingthatisinherentlyvariablecanbeharnessedtoresultinareproducibleoutcome,andwhatisthesourceofthisheterogeneity.

Ourmostrecentstudies(Chattwoodetal,eLife2014)revealedthattheinterplaybetween dynamic heterogeneity in Ras‐GTPase activity and heterogeneity innutritional status is required for normal lineage priming and salt and pepperdifferentiation. This is because such heterogeneity sets the intrinsic responsethreshold to lineage specific differentiation signals. Indeed new RNA seq datarevealsthatbothconditionsbiascellstowardsthesamecellfate(ietheoutcomeof the bias at the slug stage is identical). Despite this,we find that a surprisinginverse relationship between genes under Ras and nutritional control duringgrowth when lineage priming takes place (ie opposite gene expression profilesleadtothesamefateoutcome).Wehaveusedmodellingandexperimentationtoexplainthisobservation,whichrevealstheexistenceofanovelsystemrequiredtobuffer against extrinsic variation. We will describe evidence that this systemfacilitatestherobustrunningofanultradiancellfateoscillator.

35

Talk25

Generegulationbyc‐di‐GMPinDictyostelidsocialamoebas

Zhi‐huiChenandPaulineSchaap

DivisionofCellandDevelopmentalBiology,CollegeofLifeSciences,UniversityofDundee,DundeeDD15EH,UK

Abstract

Dictyostelidsocialamoebasaggregatetoformfruitingbodiesuponstarvation, inwhichthecellsdifferentiateintosporesandstalkcells.Wedetectedadiguanylatecyclase gene in this eukaryote,which proved to be highly similar to prokaryotediguanylatecyclases.TheDictyosteliumdiguanylatecyclase,DgcA,wasexpressedin prestalk cells and produced c‐di‐GMP that was secreted to induce stalk celldifferentiation(1,2).Wecomparedtranscriptionalprofilesofdgca‐andwild‐typecells to identify genes that are controlled by c‐di‐GMP signalling. We preparedfusion constructs of the promoters of the upregulated genes with LacZ andexaminedtheexpressionpatternsofthegenesindevelopingfruitingbodies.Apartfromgenesexpressedinthestalk,wedetectedseveralgenesthatwereexpressedvery late incells thatremainedamoeboidattheupper‐and lowerboundariesofthesporehead.Thissetdefinesanovelclassofdevelopmentallyregulatedgenesfor this organism. Invitro studies showed that theupper‐ and lower cupgeneswerestronglyupregulatedbystimulationwithc‐di‐GMP.Weexaminedexpressionofc‐di‐GMPregulatedgenesinseveralmutantsthatshowasimilardevelopmentaldefect as the dgca‐ mutant and identified a number of potential downstreamtargetsofc‐di‐GMP.

References

1.Chen,Z.‐H.andSchaap,P.(2012)Nature.488:680‐683

2.Schaap,P.(2013)IUBMBLife.65(11):897‐903.

36

Talk26

AnovelubiquitinlikedomaincontainingproteinisrequiredforearlydevelopmentandcAMPpropagationin

D.discoideum.

SatoshiKuwana1,RobertRKay2,MasashiFukuzawa1

1TheUnitedGraduateSchoolofAgriculturalSciences,IwateUniversity,Japan2MRCLaboratoryofMolecularBiology,UK

Abstract

PrestalkA(pstA)cellscomprisethetipregionoftheslug,andarelabeledwiththeecmAmarker(asubfragmentoftheecmAgene).Wehadpreviouslyshownthatatranscription factor MrfA is involved in pstA differentiation and suggested apossible function of pstA cells for tip dominance. However, further study usingomt12genemarkersuggeststhatthereisanotherpstApopulationintermingledinthe tip region. Unlike ecmA‐positive pstA cells, the novel pstA cells (pstA1)appeared in thevegetativephaseand thensorted into tipregion.Whengrowingcellswere separated into thepstA1 and the remaining cells, thepstA1‐enrichedpopulationdevelopsseveralhoursfasterthantheothercellpopulations,includingunsortedcells.

To investigate pstA1 differentiation, we used REMI mutagenesis and FACSenrichment to isolateamutantDG1037‐ thatexhibiteda significant reductionoftheomt12pmarkerexpression.SincetheDG1037encodesaubiquitin‐likedomaincontaining protein (UBL), we named the gene ubdA. UBL is also present inmammals,butitsbiologicalfunctionislargelyunknown.WhilestarvedubdA‐cellsshownormalchemotaxistoacAMPsource,theynevercreateaggregativestreambythemselves.cAMPpulsingdoesnotrescueubdA‐phenotypesincludingnotonlycsA and cAR1 expressions but also cAMP production. Interestingly, in chimerawith10%ofthepstA1cells,themutantcellscanaggregate.

These results suggest that ubdA is involved in early development via pstA1differentiationandweproposethatthepstA1actsasanearlyorganizertowardstipformationandcAMPwavepropagation.

37

Talk27

ThenewfaceofdictyBase

SiddharthaBasu,DavidJimenez‐Morales,PetraFey,RobertJ.Dodson,andRexL.Chisholm

dictyBase,NorthwesternUniversity.BiomedicalInformaticsCenterandCenterforGeneticMedicine,750N.LakeShoreDrive,Chicago,IL60611,USA

Abstract

dictyBase is the most comprehensive, current, and highly curated database forDictyostelid genomics available online. Our primary mission is to provide thebiomedical research community with high quality data and tools that enableoriginal research.However,outdated infrastructurehassignificantlyconstrainedour ability to implement the latest technological advances andnewdatasets. Toovercometheselimitations,amajoroverhauliscurrentlybeingcompleted[1].Inthisprocess,theuserinterfaceisbeingredesignedandmodernizedwiththegoalofimprovingnavigationandfacilitatingtheintegrationofnewtools.Weareusingmodern frameworks (AngularJS and ReactJS) for developing dynamic webapplicationsthatwillincreasetheadaptationtoscreensizesofdictyBaseonmostweb enabled devices. We will present the principal changes affecting the frontpageandmostpopularsectionsofdictyBase.AbetaversionofthenewdictyBasewillbereleasedtotheDictycommunityatthemeeting.Hands‐onsessionswillbeavailableduringthepostersession.

References

[1] Basu S, Fey P, Jimenez‐Morales D, Dodson RJ, ChisholmRL. dictyBase 2015:Expanding data and annotations in a new software environment. Genesis. 2015Jun19.doi:10.1002/dvg.22867

38

Talk28

CharacterizationofDictyosteliumgenesbyparallelphenotypingofbarcodedmutants.

ChristopherDinh1,ZhiyiLiu1,AnupParikh2,JoAnnOng1,JamieMartinez1,NhatMai1,TheresaLuong1,Vy‐ThaoDinh1,JuneHu1,JieSong1,ElizabethVillegas2,Bin

Liu1,RichardSucgang1,BlazZupan2,GadShaulsky2,andAdamKuspa1,2,3

1VernaandMarrsMcLeanDepartmentofBiochemistryandMolecularBiology,andthedepartmentsof2MolecularandHumanGenetics,and3Pharmacology,Baylor

CollegeofMedicine,OneBaylorPlaza,HoustonTX77030.

Abstract

The ability to phenotype mutants in parallel is useful for providing insights into genefunction at the whole‐genome level. We have produced a set of individually barcodedD.discoideum insertionmutants and have used them in parallel analyses of phenotypes thatprovide accurate inferences of gene function. In order tomake themwe constructed768barcodedRestrictionEnzyme‐Mediated Integration(REMI)plasmids,eachwithunique60‐meroligonucleotidetagsflankedby20‐merprimersites(barcodes)thatcanbeamplifiedbyPCR.The768plasmidswerelinearizedwithBamHI,EcoRI,orSphIrestrictionenzymesandused to mutagenize AX4 in >1,000 REMI transformations, using DpnII, ApoII, or NlaIII,respectively. Transformed cells were immediately cloned into 96‐well plates in order toobtain a broad sampling of genomic insertions sites, and 30 strains from each of the 768barcoded plasmids were stored individually, producing an indexed library of 23,040mutants.Toconductparallelphenotypingexperimentsweconsolidatedthemutantsinto30pools of 768 strains such that each strain in a pool carried a unique barcode. WedemonstratedthatwecouldidentifyeachofthebarcodesbycarryingoutPCRamplificationon DNA prepared from the pooled strains, followed either by hybridization to DNAmicroarrayscontainingthe76860‐mertagsfromeachbarcode(pluscontrol60‐mers),orbydirect sequencing of the population of amplicons. In various genetic enrichmentexperiments, we challenged triplicate pools of mutants through cycles of a particularcondition in order to revealmutants that showed a higher fitness under those conditionscomparedtoothermutants inthepool. Weinferredhigher fitnessbasedonthe increasedabundanceofamutant,asestimatedfromthechangeinabundanceofitsDNAbarcode.Wetestedgrowthinthepresenceofsub‐lethalconcentrationsof24differentdrugsthatinhibitthe growth of Dictyostelium, in order to find mutants that display relative resistance,identifyingpotentialdrug targets.Wealso tested for increases in sporulationefficiencybyputting the mutants through rounds of development in order to identify new cheatermutants,andweenrichedforresistancetoinfectionbyLegionellapneumophila.Weretested3‐10 strains from each pool that were enriched in each of the three replicates of theexperiment and we could confirm their phenotypes without exception. For example, weidentified dozens of mutants that were enriched after multiple rounds of infection by L.pneumophilaandidentifiedthemutatedgenes.Manyofthegenesencodeproteinsinvolvedinautophagy,signaling,andmembranetrafficking.SomeofthemhavehumanorthologsthathavenotbeenreportedtobeassociatedwithL.pneumophilapathogenesis.Wetestedoneofthese, the C2‐domain containing proteinKIAA0528, in anRNAi knockdown experiment inhuman macrophages derived from HL‐60 cells, using shRNAs targeting KIAA0528. Weobserved2.5‐to5‐foldreductioninKIAA0528mRNAinthesecells,dependingontheshRNAused,andobservedconcomitantresistancetokillingbyL.pneumophilainfection.

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Talk29

Workshop

IntroducinganewdictyExpress:fromrawreadstogeneexpressionbrowsing

MihaStajdohar1,2,3andBlazZupan2,3

1Genialisd.o.o.,Ljubljana2UniversityofLjubljana,Slovenia

3BaylorCollegeofMedicine,Houston

Abstract

Most likely, you have already been using dictyExpress [1], an interactive,exploratorygeneexpressiondataanalyticswebapp. ItsitsonthedictyBasewebpage,andisengagedeverytimeyouclickonagenexpressionlinkinanyofgenehome pages. dictyExpress has been serving gene expression profiles from over1000microarrayDictyexperiments fromBaylorCollegeofMedicine. In thepastmonth, we have replaced the old dictyExpress with a new app. Instead ofexpressionsfrommicroarrays,dictyExpressnowoffersonlydatafromNGSreads.The app has been entirely reimplemented, and yes, you can now view theexpressionofyourfavoritegeneontheiPhone.dictyExpressstillsupportsallthefamiliaroperations, includingexpressionprofileviewer,GObrowser,differentialexpression visualization, experiment comparison, hierarchical clustering. Underthehood,themostessentialchangeisthedatamanagement.OnecannowuploadNGSreads,processthemwithaclickofabuttonandpublishthemonline.Inthisworkshop,wewillintroducethenewdictyExpress,showhoweasyitistoaddnewNGSexperiments, discuss its computationalpipeline anddemo itsprogramminginterfacetodataminingtoolboxOrange[2].

References

[1]RotG,ParikhA,CurkT,KuspaA,ShaulskyG,ZupanB(2009)dictyExpress:aDictyostelium discoideum gene expression database with an explorative dataanalysisweb‐basedinterface,BMCBioinformatics,10:265.

[2] Demsar J, Curk T, Erjavec A, Gorup C, Hocevar T, Milutinovic M, MozinaM,PolajnarM,ToplakM,StaricA,StajdoharM,UmekL,ZagarL,ZbontarJ,ZitnikM,Zupan B (2013) Orange: data mining toolbox in Python, Journal of MachineLearningResearch,14:2349‐2353.

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Talk30

Keynotespeaker

Cellshapethroughthecellcycle

BuzzBaum

MRC‐LaboratoryofMolecularCellBiology,UniversityCollegeLondon,London,WC1E6BT,UK

Abstract

We are interested in the dynamics of cell shape control in animals. Morespecifically,weareinterestedinthemechanismsbywhichtheactincytoskeletoncontrols mitotic cell shape changes during normal and cancer divisions in cellculture and in a tissue context, where they are subject to external forces, andwhere division contributes to patterning andmorphogenesis. Asmodel systemswe use the fly, human cells in culture and patient samples. In addition, morerecently we have extended this work to study cell shape through the cycle inSulfolobus, since it turns out thatmuch of themachinery controlling eukaryoticcellarchitecturehashomologuesinArchaea.Ourstudiesuseacombinationoflivecellimaging,genetics,microfabricationandmechanicalperturbationstostudythemolecular, cellularandphysicalprocesses involved.My talkwill focuson recentworkinwhichwehavestudiedhoweukaryoticcellsactivelyremodeltheiractinandmicrotubule cytoskeletons as they enter and exitmitosis. I will discuss thewaysinwhichthesetwofilamentsystemsworktogether,viaeitherco‐regulationorcrosstalk,toensurethatgeneticmaterial,mitochondriaandcellmassarefairlyandaccuratelysegregatedatdivision.

41

Talk31

TheSTEgroupkinaseSepAcontrolscellmigrationandcleavagefurrowformation

AnnetteMüller‐Taubenberger

CellBiology(AnatomyIII),BiomedicalCenter,LudwigMaximilianUniversityofMunich,82152Planegg‐Martinsried,Germany

Abstract

Septase (SepA) is a STE20groupkinase that hadbeen identified in a screen forcytokinesis‐defective mutants in Dictyostelium discoideum. Functional studiesrevealed that SepA is crucial for proper cleavage furrow formation, and plays arole in development. SepA just as its upstream regulator, the GTPase Spg1, isassociated with centrosomes. Live‐cell imaging studies showed that SepA‐nullcells have unimpaired nuclear divisions and proceed normally throughmitosis.However, often cleavage furrowswere either not formed at all orwere atypicaland extremely asymmetric. Outside of the furrow, the cortical actin showed astrongrufflingactivity.TheseresultsledtotheconclusionthatSepAisinvolvedinthe spatial and temporal control system organizing cortical activities inmitoticandpost‐mitoticcells.

Morerecently,SepAhasbeenidentifiedasadownstreamtargetofErkB(NicholsandKay, inpreparation).ErkBregulateschemotaxis towards folateandcAMPinDictyostelium,andSepAbelongstothelistofproteinsthatarephosphorylatedinresponse to these chemoattractants. We therefore aimed at a more detailedanalysisofthemigratorybehaviourofSepA‐nullcells.Inunder‐agarfolateassays,thespeedoftheSepA‐nullcellsisstronglyreduced.Incontrasttowild‐typecells,the shape of SepA‐null cells is unusually elongated under these conditions.Expression of SepA in the null‐background rescues the defects of the mutantsalmost completely. In summary, our data suggest that SepA controls corticalactivitiesbothinmigrationandduringcelldivision.CurrentlyweaimatadeeperunderstandingofthesignallingnetworkregulatingSepA.

42

Talk32

ADP‐ribosylationofhistoneH2BinrepairofDNAdoublestrandbreaks

AlinaRakhimova,SeijiUra,Duen‐WeiHsu,HongYuWang,NicholasLakin,CatherinePears

DepartmentofBiochemistry,UniversityofOxford,SouthParksRoad,Oxford,OX13QU,UK

Abstract

DNA is susceptible tomany formsofdamage that, ifunrepaired, can lead to theaccumulation of mutations and diseases such as cancer. Double strand breaks(DSB)areoneofthemosttoxiclesionsandthecellhasevolvedmultiplewaystorepair this to prevent such damage. The two major repair pathways arehomologous recombination, in which the damage is repaired by copyinginformation from a sister chromatid or homologous chromosome, and non‐homologousend‐joininginwhichtheendsarerepairedandre‐ligated.Oneoftheearliest responses to DNA damage, including DSBs, is activation of ADP‐ribosyltransferases that ligate ADP moieties onto target proteins. Inhibitors of theseenzymeshavepotential for chemotherapyas they sensitize tumour cells toDNAdamaging agents such as ionizing radiation. ADP‐ribosyl transferases areconservedinDictyosteliumandwehavepreviouslyshownAdprt1atobethemajorform involved in DSB repair, influencing the choice between alternative repairpathways. Although histones are known to be ADP‐ribosylated following DNAdamage, the sites of modification and importance of this in repair of damage,includingDSBs,isnotknown.HerewereportthatDictyosteliumH2BisatargetformodificationbyAdprt1a invitroandmapsitesofmodification.H2BisalsoADP‐ribosylatedinresponsetoinductionofDSBsincellsandweusegenereplacementto investigate the importance of this post‐translationalmodification in repair ofDSBs.

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Talk33

Measurementandmodellingofstochastictranscriptiondynamics

AdamMCorrigan1,JonathanRChubb1

1Laboratory for Molecular Cell Biology & Department of Cell and Developmental Biology, University College London, Gower Street, London, WC1E 6BT, United

Kingdom

Abstract

What does it mean when we say that a gene is “on”? Standard approaches tomeasure transcription evaluate population averages of steady state RNA level.These methods, although highly useful, give a homogenous assessment oftranscriptionaloutputandmasktheunderlyingdynamicevents.Recentsinglecellstudieshave forceda re‐evaluationof thisview, revealing that transcriptioncanoccur in a series of irregular bursts, or pulses, with strong periods of activityinterspersedbylongperiodsofinactivity,andverylittletranscriptionoccurringinmost cells at any one time. Transcriptional bursts reflect the process oftranscriptionitself,andprovideaccesstodeeperunderstandingoftranscriptionalmechanism.Herewe investigate theprocesses underlying transcriptional burstsbyquantitative imagingof single gene transcriptional events in individual livingcells, in combinationwithmoleculargeneticsandmathematicalmodelling.Earlymodels based upon fixed cells suggest either a single permissive state oralternatinginactiveandpermissivestates.Incontrast,ourlivecelldataindicateacontinuum of transcriptional states, with a slowly fluctuating initiation rateconvertingthegenefromlowtohighlevelsofactivity,withthepropertiesofthefluctuationsdefinedbythecoretranscriptionmachinery.Thisbehaviourenablesa wide dynamic range of transcriptional responses, faithfully transmittingdevelopmental or environmental information in the highly stochastic cellenvironment.

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Talk34

SpacingRemodelersinDictyosteliumControlTranscriptionThroughDistinctEffectsonNucleosome

Size,PositioningandOccupancy.

MarkRobinson1,JamesPlatt1,2,NickKent1,AlanKimmel2andAdrianHarwood1

1CardiffSchoolofBiosciences,CardiffUniversity,Cardiff,UnitedKingdom2LaboratoryofCellularandDevelopmentalBiology,NIDDK,Bethesda,MD,USA

Abstract

Nucleosome positioning and occupancy are important determinants of thechromatin landscape, actively regulating transcription. Dynamic remodelling ofthislandscapeisessentialforcellulardifferentiation,replicationandDNArepair.TheCHDandISWIfamiliesofATP‐dependentchromatinremodelerspossesstheabilitytosampleDNAlinkerlengthandspacenucleosomesevenlyinvitro.Studiesin yeast have shown aberrant organisation of the chromatin landscape andtranscriptional disruption upon loss of these proteins, yet precisely how theformerresultsinthelaterremainsunclear.Knockoutmutantsweregeneratedforthemajor remodelers and their phenotypes examined.MNase‐seq and RNA‐seqwas used to investigate the relationship between chromatin structure and genetranscriptionofthefourspacingremodelers:ChdA,ChdB,ChdCandIsw.All fourremodeler mutants displayed distinct, non‐redundant phenotypes and variantRNA profiles. Nucleosome mapping further reveals distinct regions andmechanisms of activity for all four remodelers. Quantitative analysis ofnucleosome profiles allowed detection ofmutant‐specific relationships betweenthe size, positioning and occupancy of nucleosomes across genes and theirtranscriptionalregulation.

45

Talk35

ProteolyticregulationinDictoysteliummitochondria

MehakRafiqandElinorThompson

FacultyofEngineeringandScience,UniversityofGreenwich,ChathamMaritime,ME44TB,UK.Correspondingauthor:te30@gre.ac.uk

Abstract

Proteolysis is increasingly recognised as a key regulatory mechanism in cellbiology,proteasescomprising2‐5%oforganismgenomesacrossevolution.Theygovern protein activation, localisation, exposure of cryptic binding sites andrelease of neoproteins: indeed, altered protease expression and substrate‐proteolysis are important in pathogenesis, including Parkinson’s and otherneurodegenerative diseases. Proteolysis takes place within cell membranes viafamilies of evolutionarily well‐conserved intermembrane proteases. Amongstthese are the ‘rhomboid’ serine proteases, enzymes known to influencedevelopment,signallingandinfectioninarangeofeukaryotesandprokaryotes.Offour predicted enzymatically‐active rhomboids inD. discoideum, we have foundthree to be important in the mitochondrion. The conserved eukaryotic,mitochondrial ‘PARL’‐typeprotease(RhmD)cannotbe inactivatedbut therhmA‐rhomboid knockout responds poorly to chemoattractant, shows decreasedmotility and displays aberrant mitochondrial ultrastructure and alteredrespiration. A further rhomboid knockout, rhmB‐, has reduced growth rates andfolate chemotaxis and, interestingly, a double A/B‐mutant is unable tophagocytose prey bacteria. Whereas RhmA‐GFP is visualised at the contractilevacuole, RhmB‐GFP colocalises with MitoTracker to the mitochondrion, buttranscription of A and B both peak duringmulticellular growth and, in qPCR,rhmB transcription is differentially regulated in rhmA‐ cells. Since transcriptionlevels are also altered of our predicted Dictyostelium orthologues ofSaccharomyces cerevisiaemitochondrial rhomboid‐substrates, we are examining(co‐)localisation of orthologue‐RFP in GFP‐expressing and rhmA‐ and B‐ cells,whileconductingreporterstudieswiththeremainingrhomboidsCandD.

46

Talk36

C‐module‐bindingfactorsupportsretrotranspositionofTRE5‐AbysuppressinganRNAipathway

AnikaSchmith1,ThomasSpaller1,ÅsaFransson2,JonasKjellin3,BenjaminBoesler4,SandeepOjha5,WolfgangNellen4,ChristianHammann5,FredrikSöderbom3,

ThomasWinckler1

1DepartmentofPharmaceuticalBiology,InstituteofPharmacy,UniversityofJena,Germany

2DepartmentofMolecularBiology,BiomedicalCenter,SwedishUniversityofAgriculturalSciences,Uppsala,Sweden

3DepartmentofCellandMolecularBiology,BiomedicalCenter,UppsalaUniversity,Sweden

4InstituteofBiology–Genetics,UniversityofKassel,Germany5Ribogenetics@BiochemistryLab,DepartmentofLifeSciencesandChemistry,MolecularLifeSciencesResearchCenter,JacobsUniversityBremen,Germany

Abstract

Transposableelementsarefoundinalmostallorganismsandplayacentralroleinshapingtheirhost'sgenomes.BecauseintegrationintogenomicDNAisimmanentto transposition,mobile elements represent a constant threat to the viability oftheirhosts.ThegenomeofDictyosteliumdiscoideumishaploidanddenselypackedwith genes, and, therefore, control of transposon activity is of particularimportance for maintaining genome integrity in this organism. Previous RNAsequencing experiments revealed that the expression of several transposableelements in theD.discoideum genome is deregulated in amutant lacking theC‐module‐bindingfactor(CbfA).Atthesametime,theCbfAmutantshowsa~200‐fold overexpression of the gene coding for the Argonaute‐like protein AgnC,suggesting that CbfA may indirectly support the amplification of transposableelements by limiting the availability of AgnC in a distinct RNA interferencepathway. We used the retrotransposon TRE5‐A, which maintains an activepopulation in D. discoideum cells and for which we have an in vivoretrotransposition assay, as amodel to evaluate this assumption.We show thatTRE5‐AtranscriptsaccumulateinanagnCknockoutstraintohigherlevelthaninwild‐typecells.Incontrast,TRE5‐AexpressionvanishesincellsthatoverexpressagnC in a CbfAwild‐type background, and this is followed by amore than90%reduction of the retrotransposition activity of the TRE5‐A population. It thusappearsthattheendogenecbfAwas“hijacked”bytheretrotransposonmachineryto eliminate a defense mechanism against transposition, which certainly had aprofoundeffectongenomeevolution.

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Talk37

REMI‐seq–generationofagenome‐widemutantresourceforDictyosteliumfunctionalgenomics

AmyBaldwin1,NicoleGruenheit2,SarahJaques1,ThomasKeller2,RexChisholm3,ChristopherThompson2andAdrianHarwood1

1CardiffSchoolofBiosciences,HadynEllisBuilding,MaindyRoad,CardiffUniversity,Cardiff,UK,CF244HQ

2FacultyofLifeSciences,MichaelSmithBuilding,OxfordRoad,TheUniversityofManchester,Manchester,UK,M139PT

3FeinbergSchoolofMedicine,NorthwesternUniversity,Chicago,Illinois,USA,60611

Abstract

WearecombiningRestrictionEnzymeMediatedIntegration(REMI)mutagenesiswithNGStechnologytocreateagenome‐widesetofgeneknockoutmutants.Usingthisprocess,weaimtocreateaneartotalcollectionofDictyosteliummutantsasanew resource for the Dictyostelium community. The resource will compriseindividual, setsand largepoolsofmutants,with thepositionofREMImutationssearchableviadictyBase.

To date, we have generated more than 11,000 insertion mutants by REMI andstored them in a gridded format for later access. The REMI insertion sites for~1,000of thesemutantshavebeen identifiedand include280mutantswith ‘in‐gene’insertionsthatarenotcurrentlycataloguedintheDictystockcentre.Ofthe~1,000mutantsprocessedtodate,63%havein‐geneinsertions(37%haveinter‐genic insertions) and 4% contain insertions attributed to hotspots. 84% of thestockscontainasinglemutantwithasingleinsertion.

This new resource will produce a step change in Dictyostelium genetics. Theprinciplebenefitswillbe theon‐lineavailabilityof independentandmulti‐allelicmutants for nearly allDictyostelium genes and the capacity to conduct complexphenotyping of protein families. Another key benefit will be the ease at whichwhole genome phenotypic screens can be conducted; parallel sequencing andpopulationfitnessstudiesonbar‐codedpoolsofrandommutantswillprobebothlethal and sub‐lethal sensitivity changes, for example to developmental signals,toxins or drugs. Proof of principle experiments have demonstrated a linearresponseanddeterminedabroaddynamicrangeofthesystem.

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Talk38

ForminAfunctionduringmigrationinconfinedenvironments

NagendranRamalingam1,2,ChristofFranke3,EvelinJaschinski4,,MoritzWinterhoff3,YaoLu5,StefanBrühmann3,AlexanderJunemann3,HelenaMeier3,AngelikaA.Noegel6,IgorWeber7,HongxiaZhao5,RudolfMerkel4,Michael

Schleicher1,andJanFaix3

1AnatomyIII/CellBiology,Ludwig‐Maximilians‐University,Schillerstr.42,80336Munich,Germany;

2presentaddress:DepartmentofNeurology,ColumbiaUniversity,650West168thStreet,NewYork,NY10032,USA;

3InstituteforBiophysicalChemistry,HannoverMedicalSchool,Carl‐Neuberg‐Str.1,30625Hannover,Germany;

4InstituteofComplexSystems7:BiomechanicsForschungszentrumJülichGmbH,52425Jülich,Germany;

5InstituteofBiotechnology,POBox56,UniversityofHelsinki,Helsinki00014,Finland;6CenterforBiochemistry,MedicalFaculty,UniversityofCologne,50931Köln,Germany;

7DivisionofMolecularBiology,RuderBoškovićInstitute,Bijenička54,10000Zagreb,Croatia.

Abstract

Eukaryotic cells can move by distinct modes of action. Fast amoeboid cellmigration,asutilizedbyleukocytesorDictyosteliumamoebae,ischaracterizedbyweakadhesion,formationofactin‐richpseudopodsorhydrostaticpressure‐drivenblebs in their fronts and myosin II‐driven contractility in the rear. However, itremainedelusivehowthecontractilemachinery in the trailingedge is regulatedandhow this system is localized.Herewe identifyDiaphanous‐related forminA(ForA) fromDictyosteliumdiscoideumasacritical regulatorycomponentof thismachinery also comprising the actin crosslinker cortexillin and IQGAP‐relatedproteins.ForAexertscanonicalforminactivityinvitro,rapidlyrelocalisestonewprospective ends in repolarizing cells and is required for cortical integrity.Intriguingly,thespeedofrandomlymigratingforA‐nullcellsismarkedlyincreasedin unconfined environments,whereas compression under agar strongly impairsmotility due to extensive, unproductive blebbing in their rear. Our findingsthereforestronglysuggestForAtogenerateaspecializedsubsetoffilamentsasthebasis of a protective cortical actin sheath in the cell rear to withstand theincreasedcontractileforcesimposedbymyosinIIinresponsetomechanicalstressthatarerequiredforefficientcellmigrationin2D‐confinedenvironments.Finally,weshowthatthelocalizationofForAtotherearofpolarizedcellsismediatedbyitsPI(4,5)P2‐specificC2domain.

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Talk39

Polychromatic‘greenbeard’genesdeterminepatternsofaggregationinasocialamoeba

NicoleGruenheit1,KatieParkinson1,JenniferA.Howie1,JasonB.Wolf2andChristopherR.L.Thompson1

1FacultyofLifeSciences,MichaelSmithBuilding,UniversityofManchester,OxfordRd,Manchester,M139PT,UK

2DepartmentofBiologyandBiochemistry,UniversityofBath,ClavertonDown,Bath,BA27AY,UK

Abstract

Multicellularity requires agreatdegreeof cooperationamongcells toultimatelybenefit the multicellular individual. This can be achieved using kin recognitionmechanisms, which follow genome wide relatedness, or a phenotypic marker,whereageneencodesbotha recognizable characteristic, suchasagreenbeard,and altruistic behaviour towards others bearing the same mark. In this caseindividuals need only exhibit a genetic relationship at this ‘greenbeard’ locus.However,amarklikethiswillinevitablysweeptofixation,simultaneouslyloosingitsdiscriminatorynatureunlessmultiplevariationsofthesamemark,e.g.multiplebeardcolours,areused.

In D. discoideum aggregations can consist of multiple different genotypes. Thisraises the question of what prevents the emergence of disruptive cheaters thatcontributeasmallerproportionofcellstothestalk.Also,natural isolatesvaryintheirabilitytoco‐aggregate,withsomestrainspartiallyexcludingoneanother.Itiscurrentlyunknownwhetherthisisdrivenbyakinrecognitionoragreenbeardmechanism.

Here we tested whether tgrB1 and tgrC1 encode a muliticoloured greenbeardusing co‐occurring strains known to contain cheaters and losers. These strainsexhibit complex partner specific non‐transitive patterns of segregation, withsegregation most prevalent in cases where facultative occur in chimericdevelopment.Overallgeneticdistance is insufficient toexplainthesesegregationpatterns, which is inconsistent with a kin recognition mechanism. Instead,extensive patterns of sequence divergence, which affect the strength of in vitroTgrB1 and TgrC1 binding, can explain the observed segregation patternssuggesting amechanism for cheater avoidance by providing a phenotypicmarkakintoamulticolouredgreenbeard.

50

Talk40

LocallyRegulatedRasSignalingRevealsInhibitoryProcessinGPCR‐mediatedChemotaxis

XuehuaXuandTianJin

ChemotaxisSignalingSection,LaboratoryofImmunogenetics,NationalInstituteofAllergyandInfectiousDiseases,NationalInstitutesofHealth,12441ParklawnDr.

Rockville,MD20852,USA.

Abstract

Optimal polarization and chemotaxis in a wide range of concentration gradientrequireinhibitionprocesses.Nomolecularmechanismofinhibitionhaseverbeenidentified.Here,werevealalocallycontrolledinhibitoryprocessatthestepofRasactivationbyitsnegativeregulator,aC2domaincontainingRasGAP,C2RasGAP1.C2RasGAP1localizesatleadingedgeofchemotaxingcellsandplaysessentialrolesinpersistentadaptationofRasactivation.ThealterationofRasactivationresultsin impaireddirectional sensingandanultimatelyexcessivepolymerizationofF‐actininthecells,leadingtoimpairedchemotaxis.Remarkably,theimpairedabilityof directional sensing and chemotaxis in c2rasgap knockout cells waschemoattractantconcentrationdependent.Takentogether,ourresultsuncoveramechanism by which cells achieve optimal polarization and chemotaxis in aconcentration‐independentmanner,theessenceofaninhibitoryprocessrequiredfordirectionalsensingandchemotaxis.

51

Talk41

ACud‐typetranscriptionfactorregulatesDictyosteliumsporedifferentiation

YokoYamadaandPaulineSchaap

CollegeofLifeSciences,UniversityofDundee,Dundee,UK

Abstract

Spore differentiation of social Dictyostelid amoebas likely evolved fromencystationofsolitaryamoebasbyadoptingtheencapsulationmechanismintoamulticellular developmental process. At the time of spore maturation, signalsexchanged between prestalk and prespore cells ultimately converge on theactivation of PKA in prespore cells, which subsequently induces expression ofspore‐specific genes, exocytosis of prespore specific vescicles that containpreassembledsporecoatproteinsandcellulosesynthesisatplasmamembranetoform fully encapsulated spores. Many components of the pathways controllingspore formation are still unknown andmost notably the downstream targets ofPKA.To identifygenes in thesporepathway,we isolatedamutantwitha sporedefect after REMI mutagenesis of D. discoideum, and identified a Cud‐typetranscription factor, spdA (sporulation defective A) as the defective gene. Cudfamily genes are found in Amoebozoa and plants and are conserved betweenDictyostelid taxon groups. spdA‐ cells differentiate into prespore cells but formvirtually no viable spores. We show that spdA acts cell‐autonomously in sporeformationandisessentialforinductionofsomespore‐specificgenesbyPKA.

52

Talk42

Rasactivationduringcellmovementinbufferandchemotaxis

PetervanHaastert,InekeKeizer‐GunninkandArjanKortholt

DepartmentofCellBiochemistry,UniversityofGroningen,theNetherlands

Abstract

ActivationofRasplaysanimportantroleduringchemotaxis.Inashallowgradientof cAMPactivationof receptor andheterotrimericG‐proteins are approximatelyproportionaltothelocalconcentrationofcAMP,whereastheactivationofRasismuchstrongerattheleadingthanatthebackofthecell.Previouslywehaveusedaverysensitiveassay todetectRasactivationduringchemotaxis (Kortholtetal,2013, JCellSci,126,4502‐13).Thisassay isbasedon theco‐expressionofRBD‐Raf‐GFP and cytosolic RFP. RBD‐Raf‐GFP only binds to active Ras‐GTP and thustranslocates to the membrane upon Ras activation. Cytosolic RFP is used todeterminethecytosolicvolumeoftheseboundarypixels;theGFP‐RFPdifferenceyieldsquantitativeandverysensitiveinformationonRasactivation.WehavenowusedthisassaytoinvestigatedindetailhowdynamicRaspatchesofcellsinbufferare regulated by the cytoskeleton, and how gradients of chemoattractantinfluence Ras patches. A picture emerges of excitability, symmetry braking andphase separation of Ras/cytoskeleton interactions that is coupled to basal cellmovement.Inthispicture,chemotaxisisarelativelysimplepositionalbiasofRasactivationbythegradientofchemoattractanttomediatedirectionalmovement.

53

Talk43

TheProteinKinaseCOrthologuePkcARegulatestheActinCytoskeleton

Singh,S.,Garcia,R.,Khan,M.,Shah,S.,Brazill,D.

HunterCollegeandTheGraduateCenter,CityUniversityofNewYork,DepartmentofBiologicalSciences,927N,695ParkAve,NewYork,NY10065,USA

Abstract

Quorum sensing, the ability of to monitor the local density of cells andmodifybehavioraccordingly,isimperativeforregulatedtissuedevelopmentandupkeep.While cell density‐sensing (quorum‐sensing) pathways have been well‐characterizedinbacteria,thesepathwaysarepoorlyunderstoodineukaryotes.InDictyostelium discoideum, the CMF quorum‐sensing pathway ensures properdevelopment by preventing the initiation of development until enough starvingcells are present. This is accomplished, in part, through regulating the actincytoskeleton. Here we describe a putative protein kinase C orthologue, PkcA,whichregulatesquorumsensingandactin inD.discoideum. We foundthatcellsoverproducingPkcAareunabletoaggregateevenathighcelldensity,asiftheyareblindtoCMF.Conversely,cellslackingPkcAaggregateatlowcelldensity,asifinthepresenceofCMF.However,thisaggregationoccursintheabsenceofcoherentstreaming. The aberrant aggregation in bothmutants appears to be caused bydefects in cAMP chemotaxis and adhesion. Cells overexpressing PkcA havedefective cAMP chemotaxis and enhanced cell‐substrate adhesion. Cells lackingPkcAhavedecreasedcell‐cellandcell‐substrateadhesion.TheadhesiondefectsinthePkcAmutantcellsappeartobecausedbyF‐actinmislocalization. Inkeepingwith a role in actin regulation, cells lackingPkcA alsohave a cytokinesis defect.Takentogether,theresultssuggestthatPkcAmayregulateactinduringvegetativegrowth,aswellasduringstarvationforCMFquorumsensing.

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Talk44

MacropinosomesandpseudopodsareassociatedwithdistinctspatialpatternsofPIP3andSCAR/WAVE.

1DouweM.Veltman,2Bi‐ChangChen,2EricBetzig,3RobertH.Insalland1RobertR.Kay.

1MRCLaboratoryofMolecularBiology,Cambridge,CB20QH,UK2JaneliaFarm,Ashburn,Virginia20147,USA

3BeatsonInstituteforCancerResearch,GlasgowG611BD,UK

Abstract

Actin pseudopods and macropinosomes contain similar components, but arearchitecturallyandfunctionallydifferent.WeshowtheyaredifferentiatedbyPIP3andthekeyactinregulator,SCAR/WAVE.PIP3patches,historicallyassociatedwithchemotacticsignalprocessing,invariablyexcludeSCARfromtheircentres,leadingtoaringofSCARandactinaroundtheedgeofthepatch.Thiscausesprotrusionofcircularrufflesandmacropinosomes,butnotpseudopods.PIP3patchesthereforecause macropinocytosis and oppose migration by blocking pseudopodprogression.PIP3patchesalwaysleadtoringsofSCAR,whethertheyarebasal,atcell‐cell contacts, or caused by uniform chemottractant stimulation. In contrast,motilepseudopodsshowcontinuousrecruitmentofSCAR,accompaniedbymuchlower levels of PIP3, in shallow gradients and only modestly increased bychemoattractants.PIP3patchesarecommoninchemotacticcellsofaxenicstrains,butareabsentfromwildtypecells.WeproposethatcircularSCARrecruitmentatthe edges of PIP3 patches provides a conserved template for cupped actinstructures.

55

Posters

56

Poster1

Antibodiesforeverybody

MadeleineZufferey,PhilippeHammel,PierreCosson

DepartmentofCellPhysiologyandMetabolism,UniversityofGenevaMedicalSchool,1211Geneva,Switzerland

Abstract

Antibodiesareessentialreagentsforallaspectsofbiologicalresearch.Monoclonalantibodies have proven valuable to study a wide variety of processes inDictyostelium discoideum. However the number of antibodies available hasremained relatively limited, and previously characterized hybridomas aresometimeslost.

WearedevelopinganewserviceattheUniversityofGeneva,opentoallacademiclaboratories: our service generates recombinant antibodies againstnew targets,storesindefinitelytheplasmidsencodingtheantibodies,producestheantibodies,and distributes them to the scientific community. If adopted widely by ourscientific community, this could represent a valuable new tool for theDictyosteliumcommunity.

57

Poster2

Understandingtheroleofvacuolins/flotillinsinthebiogenesisoftheMycobacteriummarinumniche

CristinaBosmani1,MonicaHagedorn2,ThierrySoldati1

1DepartmentofBiochemistry,UniversityofGeneva,Geneva,Switzerland2BernhardNochtInstituteforTropicalMedicine,Hamburg,Germany

Abstract

We useDictyostelium as a host to study mycobacterial infections.Dictyosteliumencodes three vacuolins (A, B and C), found on the compartment containingMycobacteriummarinum,aclosecousinofMycobacteriumtuberculosis.Inaddition,vacuolin B knockout (KO) cells appeared more resistant to mycobacterialinfections.Therefore,westudywhethervacuolinsplayaroleintheestablishmentofapermissiveniche.

Vacuolins are homologous to the metazoan flotillins. Recently, flotillins wereshown to interact with the recycling machinery and be involved in receptorrecycling. Consequently, we also want to understand whether vacuolins areinvolvedinrecyclinginDictyostelium.

We demonstrate that vacuolin C is mainly localized to lysosomes, whereasvacuolin A and B are found in postlysosomes. In addition, biochemicalfractionation experiments show that vacuolins, like flotillins, are stronglyassociatedwithmembranes,arepalmitoylatedandcanbefoundinaTritonX‐100insolublefraction.

Furthermore, our GTP‐Trap pulldown experiments suggest that vacuolins caninteractwithseveralsubunitsofthev‐ATPaseanddifferentRabproteins.Wearefurthercharacterizingtheseinteractionsandinvestigatingifvacuolinscaninteractwiththerecyclingmachinery.

After showing that the vacuolin B KO cells previously described were in fact adoublevacuolinBandCmutant,wedecidedtogeneratenewsingleandmultipleKO mutants. The new single vacuolin B KOs only show a mild resistance toinfection, conforming that we need to further explore the role of vacuolins ininfectionusingmultipleKOstrains.

58

Poster3

DelineatingtheImmunityFunctionsofReactiveOxygenSpeciesUsingDictyosteliumdiscoideumasaModel

Phagocyte

JoeDanDunn,XuezhiZhang,andThierrySoldati

DepartmentofBiochemistry,UniversityofGeneva,30quaiErnestAnsermat,SciencesII

1211Geneva,Switzerland

Abstract

Reactive oxygen species (ROS) are key components of the immune response tointracellular pathogens. Deleteriousmutations in the ROS‐generating phagocyteNADPHoxidase(NOX)underliechronicgranulomatousdisease,markedbysevere,recurring bacterial and fungal infections. NOX comprises a heterodimer oftransmembraneproteins:Nox2,thecatalyticsubunit,andp22phox,whichrecruitscytosolic proteins required for activation. Vegetative Dictyostelium expressesNoxA, CybA, andNcfA, homologs of Nox2, of p22 phox, and of theNOX regulatorp67phox, respectively. Expression of two additional Nox homologs, NoxB and C,appearstobelimitedtodevelopmentalstages.

Using fluorescence‐based assays,we have observed thatDictyostelium producesROSwhenexposedtobacterialproductssuchaslipopolysaccharide(LPS)andthatthe rate of ROS production varies based on the type of LPS. ROS production isdecreased in noxabc triple null or cyba null mutants.We expressed fluorescentprotein fusions of CybA and of NcfA in wild‐type and mutant amoebae. CybAlocalizes to the plasma membrane, macropinosomes, and bead‐containingphagosomes independently of NoxA. NcfA is cytosolic and is enriched at theleading edge, forming macropinosomes, and phagocytic cups but disappearsrapidly after closure. Enrichment ofNcfA is independent of CybA andNoxA andrequiresapredictedRac‐bindingdomaininitsN‐terminus.Theseresultssuggestthat Rac activity recruits NcfA to phagocytic/macropinocytic sites to facilitateinteractionwithmembrane‐localizedCybAandNoxAand that, in theabsenceofactivating signals, NcfA is not retained. We are currently imaging infections todeterminewhetherNcfAisrecruitedtomicrobe‐containingphagosomes.

59

Poster4

PackagingofnonpathogenicbacteriainsecretedmultilamellarbodiesbyDictyosteliumdiscoideum

ValérieE.Paquet1,2,MariusDybwad3,JanetM.Blatny3andSteveJ.Charette1,2

1InstitutdeBiologieIntégrativeetdesSystèmes(IBIS),UniversitéLaval,1030avenuedelamedicine,Québec,Canada,G1V0A6

2CentredeRecherchedel’InstitutuniveritairedecardiologieetdepneumologiedeQuébec(CRIUCPQ),2725ch.St‐Foy,Québec,Canada,G1V4G5

3 ForsvaretsForskningsinstitutt(FFI),NorwegianDefenceResearchEstablishment,box25,2027Kjeller,Norway

Abstract

In the environment, amoebae chase bacteria as food source. Some bacteria areable to avoid the enzymatic degradation by the phagocytic pathway throughvarious mechanisms. Bacteria that can survive amoeba predation are calledamoeba resistant bacteria (ARB). It is already known that some ARB findthemselves packaged in multilamellar bodies (MLB), which are composed ofmultiple concentric membrane layers produced and secreted by amoebae.Packagedbacteriaareprotectedfromharshconditionsandthepackagingprocessissuspectedtopromotethepersistenceofbacteriaintheenvironment.Untilnow,only pathogenic bacteria were studied regarding their ARB capacity and thepackagingprocess.Weproposethatalinkexistsbetweenthecapacityofbacteriato resist to amoebagrazing and their capacity tobe included inMLB regardlesstheirpathogenicstatus.UsingDictyosteliumdiscoideumamoebamodel,thegrazingresistance have been assessed for about one hundred environmental bacterialisolates. Bacteria displaying an ARB phenotype have been then tested for theircapacitytobepackagedandconfirmedbytwomicroscopicmethods.TenbacteriafromCupriavidus,Micrococcus andStaphylococcus generawerepackaged inMLBsecreted by D. discoideum. These results confirm that bacteria packaging byamoebaisnotrestrictedtopathogenicbacteriaandsuggestamorecomplexroleforthisprocessinmicrobialecology.

60

Poster5

IdentificationofproteinsassociatedwithmultilamellarbodiesproducedbyDictyosteliumdiscoideum

AlixM.Denoncourt1,2,3,ValérieE.Paquet1,2,3,AhmadrezaSedighi1,2,3,andSteveJ.Charette1,2,3

1.InstitutdeBiologieIntégrativeetdesSystèmes,PavillonCharles‐Eugène‐Marchand,UniversitéLaval,QuebecCity,QC,Canada

2.Centrederecherchedel’InstitutuniversitairedecardiologieetdepneumologiedeQuébec(HôpitalLaval),QuebecCity,QC,Canada

3.Départementdebiochimie,demicrobiologieetdebio‐informatique,Facultédessciencesetdegénie,UniversitéLaval,QuebecCity,QC,Canada

Abstract

Dictyosteliumdiscoideumamoebaeproducemultilamellarbodies(MLBs)whenfeddigestiblebacteriaandsecretetheminthemilieu.ThelipidcompositionofMLBsismainlyamoebal inorigin, suggesting thatMLB formation is aprotozoa‐drivenprocess,whichcouldplaysignificantrolesinamoeba’sphysiology.Therefore,themain objective of this studywas to identifymajor proteins present onMLBs inordertogetabetterunderstandingofthemolecularmechanismsgoverningMLBformation. MLBs were purified from a co‐culture of amoebae and digestiblebacteria using a sodium bromide density gradient. Using SDS‐PAGE and massspectrometry, fourmajorproteinswere identified, including theGP17protein, aproteinwithanunknownfunction.ThepresenceofGP17onMLBswasconfirmedby immunofluorescence using a specific anti‐GP17 antibody. The localization ofGP17wasassessedinaxenicconditionandinco‐culturewithdigestiblebacteria.Another major protein found on MLB is one with an unknown function and isrecognized by theH36 antibody. This antibodywas used as aMLBmarker in apreviousstudy.Finally, identificationofproteinsassociatedwithMLBsandtheirfunctionsmayhelptoattributeapotentialphysiologicalrole to thesestructures.The elucidation of the biochemical composition of MLBs and the subsequentdevelopmentofmoleculartoolssuchasMLBmarkersmayallowfurtheranalysestobetterunderstandthemolecularmechanismsofMLBformation.

61

Poster6

AnalysesofDrkAkinaseinSTATaactivation

YukikaSaga1,YumiIwade1,MegumiIshikawa1,TsuyoshiAraki2,JeffreyG.Williams2andTakefumiKawata1

1DepartmentofBiology,FacultyofScience,TohoUniversity,2‐2‐1Funabashi,Chiba274‐8510,Japan

2DivisionofCellandDevelopmentalBiology,CollegeofLifeSciences,UniversityofDundee,DowStreet,DundeeDD15EH,UK

Abstract

DictyosteliumSTATaisastructuralandfunctionalhomologueofmetazoansignaltransducers and activators of transcription (STATs) and controls stalk celldifferentiation.STATa is activatedbyphosphorylationonTyr702whencells areexposedtoextracellularcAMP.Althoughtwotyrosinekinase‐like(TKL)proteins,Pyk2 and Pyk3, have been definitively identified as STATc kinases no kinase isknown for STATa. From the screening of a kinase overexpressor library, weidentifiedDrkA, amemberof theTKL familyand theDictyostelium receptor‐likekinase(DRK)subfamily,asacandidateSTATakinase.ThedrkAgeneisselectivelyexpressedinprestalkA(pstA)cells,whereSTATaisactivated.InanullmutantfordrkA, cAMP induced STATa activation is almost completely eliminated.Recombinant DrkA protein is auto‐phosphorylated on tyrosine, and an in vitrokinase assay shows thatDrkA can phosphorylate STATa onTyr702 in a STATa‐SH2 (phophotyrosine binding) domain dependent manner. These observationsstrongly suggest that DrkA is one of the key regulators of STATa tyrosinephosphorylation and are consistent with it being the direct activating STATakinase.

62

Poster7

MetabolismofthesignalingmoleculeglorinduringmulticellulardevelopmentofPolysphondyliumspecies

DanielRaszkowski1,DirkHoffmeister2,3,ThomasWinckler1

1DepartmentofPharmaceuticalBiology,InstituteofPharmacy,UniversityofJena,Germany

2DepartmentofPharmaceuticalMicrobiology,InstituteofPharmacy,UniversityofJena,Germany

3LeibnizInstituteforNaturalProductResearchandInfectionBiology–Hans‐Knöll‐Institute,Jena,Germany

Abstract

Signaling with the unconventional dipeptide glorin (N‐Propionyl‐gamma‐L‐glutamyl‐L‐ornithin‐delta‐lactam‐ethylester) may be the most ancient form ofintercellularcommunicationintheevolutionofmulticellularityintheDictyostelidsocialamoebae.Wepreviouslyobservedthatseveraltaxaareresponsivetoglorininadropletchemotaxisassayunderconditionswhichrequiredthegenerationofaglorin gradient by secreted glorin‐degrading enzymes known as glorinases. WeusedPolysphondyliumviolaceum andP.pallidum asmodel species andHPLC‐MStechnologytodetermineglorinaseactivitiesproducedbythecellsduringthefirsthours of starvation. Bioactivity‐guided fractionation of buffer supernatants andproteomicsmethodsarebeingappliedtoidentifyglorinasesfromP.pallidumcells.

63

Poster8

TheFutureofCurationatdictyBase

PetraFey,SiddharthaBasu,RobertJ.Dodson,DavidJimenez‐Morales,andRexL.Chisholm

dictyBase,NorthwesternUniversity.BiomedicalInformaticsCenterandCenterforGeneticMedicine,750N.LakeShoreDrive,Chicago,IL60611,USA

Abstract

The complete dictyBase overhaul and introduction of state of the art softwareinfrastructurewillallowcuratorstobeginannotatingnewbiologicalfeaturesanduse existing annotations to represent and connect data in novel ways. CuratedproteininteractionsviatheGeneOntology(GO)willbeusedtorepresentprotein‐protein interactions. Curatorsalreadyprivately annotate spatial expressionwiththe Dictyostelium anatomy ontology and we recently started annotatingDictyostelium disease orthologues with their respective disease ontology (DO)terms.TheupdateddatabasewillalsoallowrepresentingGOannotationswith‘GOextensions’, which add deeper context to those annotations. In the near futureHTML5 technology will revolutionize the way curators add annotations to thedatabase,allowingthedirecteditingofgenepages.Furthermore, itwillopenthedoorfordirectcommunityannotationsonthegenepageforinterestedusers.

Basu S, Fey P, Jimenez‐Morales D, Dodson RJ, Chisholm RL. dictyBase 2015:Expanding data and annotations in a new software environment. Genesis. 2015Jun19.doi:10.1002/dvg.22867

64

Poster9

TheregulationofmacropinosomematurationbyPIKfyve

CatherineBuckleyandJasonKing

1DepartmentofBiomedicalSciences,UniversityofSheffield,FirthCourt,WesternBank,Sheffield.UK

Abstract

The macropinocytic pathway is poorly understood, yet plays pivotal roles innutrientacquisition, immunedefenceand is subverted inmanydiseasessuchascancer, atherosclerosis and neurodegenerative diseases. Macropinocytosis is acriticalcomponentoftheinnateimmunesystem;dendriticcellsandmacrophagesactassentinels,surveyingtheirenvironmentforthreatsthroughmacropinocyticsampling.Althoughimportantindefenceagainstinfection,manypathogenshijackthe macropinocytic pathway, using it as an entry mechanism and preventingmaturationtoallowestablishmentofintracellularniches.

Despite its importance, the molecular mechansisms of maturation and proteinrecyclingremainelusive.Wehavesofaridentifiedtwoproteinsthatareimportantin the maturation pathway. PIKfyve is a phosphatidylinositol 5 kinase that isessential for the production of PI(3,5)P2. Inhibition of PIKfyve manifests in aswollen vesicle phenotype and severe defects in vesicle trafficking. A possiblePIKfyve effector protein, SNXA, localises to macropinosomes in a PIKfyvedependentmanner. Furthermore SNXAknockout cells appear to havedefects inmacropinosomematuration.IaimtoexploretherolebetweenPIKfyveandSNXAandtodeciphertheirroleincontrollingmacropinosomematuration.

65

Poster10

ThemitochondrialTOB/SAMcomplexindifferentstagesofDictyosteliumdiscoideumlifecycle.

MonikaAntoniewicz1,MarcinSkalski1,MalgorzataSłocińska2,HannaKmita1,MalgorzataWojtkowska1

1InstituteofMolecularBiologyandBiotechnology,DepartmentofBioenergetics,Adam

MickiewiczUniversity,Poznan,Poland;2InstituteofExperimentalBiology,Departmentof

AnimalPhysiologyandDevelopment,AdamMickiewiczUniversity,Poznan,Poland;

Abstract

TheslimemoldDictyosteliumdiscoideumlifecyclegivesaninterestinginsightintocomplexityofchangeswhenunicellularorganismis transformedtomulticellularone.

InourresearchwefocusedontheTOB/SAM(TopogenesisoftheMitochondrialOuterMembraneβ‐barrelProteins/SortingandAssemblyMachinery)complex.Itislocatedintheoutermitochondrialmembraneandisinvolvedininsertionofmitochondrialβ‐barrelproteinsaswellasassemblyoftheTOMcomplex

(TranslocaseoftheOuterMembrane)regardedasageneralgateforproteinsimportedintomitochondria.Interestingly,availabledataindicatethatthe

TOB/SAMcomplexexistsinvariousformsthatmaydifferinsubunitidentity,sizeandnumberandthenumberoftheTOB/SAMcomplexformsincreasesin

mitochondriaofmulticellularorganisms.ConsequentlyonecouldspeculateaboutthefunctionalrelationshipbetweentheTOB/SAMcomplexandmulticellularity.

Therefore we studied the TOB/SAM complex in the following four stages ofD.discoideum life cycle: unicellular, early starvation, aggregation and culmination.The obtained results indicate that the complex consists of two subunits, namelyTob55/Sam50(~50kDa)andMetaxin(~50kDa).Thelatter(DDB_G0276899)isahomologue of human Metaxin. It was detected by antibody against humanMetaxin1(Mtx1)anddisplaysdistinctsimilaritytotheprotein.MolecularweightoftheTOB/SAMcomplexisabout250‐300kDainmitochondriaisolatedfromcellsfromallstudiedstagesexceptfromtheaggregationstagewherethecomplexseemtohaveanadditionalformwithmolecularweightofabout450kDa.

Thestudiesweresupportedby:NCN2012/05/N/NZ3/00293.

66

Poster11

Pks26nullmutantshowedloserphenotype

AyakaMatsumoto1andTamaoSaito2

1 GraduateSchoolofScienceandTechnology,SophiaUniversity,Tokyo,Japan2 FacultyofScienceandTechnology,SophiaUniversity,Tokyo,Japan

Abstract

Altruismisacooperativebehaviorthatincreasesthefitnessofotherindividuals.Itis important andubiquitous system innature, but its geneticbasis isdifficult toexamine.SocialamoebaeDictyosteliumdiscoideumshowsaltruisticbehavior,andisamenabletogeneticmanipulation.

In 2008, more than 100 genes have been reported to be involved in altruisticbehavior inDictyosteliumby theuseofREMImutants.Oneof them,pks26REMImutant was suggested to be a loser, which differentiate less spores than itsproportionalsharewhenmixedwithitsparentalstrain.

We focused on this gene to examine the involvement of polyketide in themechanisms of Altruism.We created knockoutmutant ofpke26 by homologousrecombinationandexamineditsphenotype.Thegrowthofpks26nullmutantwasnormally,but itsdevelopmentwasslightlydifferentfromitsparentalstrainAx2.Thepks26nullmutantshowedrapiddevelopmentanditsfruitingbodyhadlongerstalkthanthatofAx2.Wemixedpks26nullcellswithAx2cellsandfoundthenullcells decreased in its frequency through successive generations. This resultsuggests that pks26 null mutant is a loser mutant. The detailed developmentalfeaturesofpks26nullmutantwillbediscussed.

67

Poster12

AutophagyandESCRTmachinery:aplasmamembranerepairmechanismduringmycobacterialegress?

LilliGerstenmaier1,RachelPilla1,MonicaPrado1,HendrikHerrmann1,RudiReimer2,ThierrySoldati3,JasonKing4andMonicaHagedorn1

1Bernhard‐Nocht‐InstituteforTropicalMedicine,Bernhard‐Nocht‐Str.74,Hamburg,Germany

2HeinrichPetteInstitute,LeibnizInstituteforExperimentalVirology,Hamburg,Germany

3UniversityofGeneva,DepartmentofBiochemistry,Geneva,Switzerland43UniversityofSheffield,DepartmentofBiomedicalSciences,Sheffield,UK

Abstract

Nearlytwomillionpeoplearedyingfromtuberculosiseveryyear(WHO)andtheglobalburdenof the infectionwithMycobacteriatuberculosis remainsenormous.Despite intense research no effective vaccines or treatment targets have beendevelopedinthelastdecades.Incontrasttothedifficultandtime‐consumingworkwiththestrictlyhumanpathogenM.tuberculosis,itscloserelativeM.marinumincombination with the amoeba Dictyostelium discoideum was established as atractable model to study the mechanisms of mycobacterial pathogenicity andcellularhostdefense.

Whilehostcellentryandtheestablishmentofareplicationcompartmentarewellunderstood,littleisknownabouttheegressofmycobacteriafromtheirhostcellsand how the infection spreads between cells. In Dictyostelium, a F‐actin basedstructure, the ejectosome, has been shown to allow non‐lytic egress ofmycobacteria (Hagedorn et al., 2009). It remains unclear how the host cellmaintains its integrity during the process of bacterial egress in which the hostplasmamembraneruptureslocally.Applyingmicroscopytechniqueswewereabletoshowthattheautophagicmachineryisspecificallyrecruitedtothedistalpoleofejectingbacteria.Ifautophagyisimpaired,cell‐to‐celltransmissionisreduced,theplasma membrane becomes compromised and the host cells eventually die(Gerstenmaieretal.,2015).Furthermore,wecouldshowthatfactorsoftheESCRTmachinery localize to ejecting bacteria in a similar pattern as the autophagymachinery does. We propose that an interplay of the autophagic and ESCRTmachinerysealsthe"large"woundthatisgeneratedbyegressingmycobacteria.

68

Poster13

Gip1regulatesthedynamicrangesofchemotaxisthroughshuttlingofheterotrimeircGproteins.

YoichiroKamimura1,YukihiroMiyanaga2,MasahiroUeda1,2

1LaboratoryforCellSignalingDynamics,QbiC,RIKEN

6‐2‐3,Furuedai,Suita,Osaka,Japan,565‐08742LaboratoryofSingleMoleculeBiology,OsakaUniversity

1‐1,Machikane‐cho,Toyonaka,Osaka,Japan,560‐0043

Abstract

Chemotaxisisthecellularbehaviorthatcombinesmotilitywiththeperceptionofchemical gradient. Dictyostelium discoideum display a typical chemotacticresponse to cAMP at the early stage of multicellular development. In this case,cAMP is recognized by a sensor unit which sends signals via several signalingmodules to cellular motility unit. Trimeric G protein, Gα2 and Gβγ, is essentialcomponentofthesensorunit.Weareinterestedinhowthisunitprocesschemicalinformation and found a novel regulator of heterotrimeric G proteins, Gip1(trimericGprtoteininteractingprotein1).Althoughacelllackingthisnovelfactorcancompletethedevelopmentprocesstomakefruitingbodies,earlyonabnormalaggregationpatternwasobserved.CloserexaminationhasshownthatchemotaxiswasabrogatedespeciallyathigherconcentrationsofcAMP.ThisdatasuggeststhatGip1 extends the dynamic ranges of chemotaxis to higher cAMP concentrations.We also noticed that Gip1 controls trimeric G proteins shuttling between theplasmamembraneandthecytosolandmediatesthecAMPtriggeredtranslocationof trimeric G proteins. These behaviors appear to explain the defects of Gip1perturbationingradientsensing.

69

Poster14

Structureactivitycorrelationof4‐methyl‐5‐pentylbenzene‐1,3‐diol(MPBD),adifferentiation‐

inducingfactorofDictyosteliumdiscoideum.

AnnaKondo1,NatsumiIwasaki1,TakaakiNarita2,ChihiroMurata1,ToruOgura1,ToyonobuUsuki2,TamaoSaito2.

1Sophiagraduateschool,7‐1Kioi‐cho,Chiyoda‐ku,Tokyo2Sophiauniversity,departmentofmaterialsandlifescience,7‐1Kioi‐cho,Chiyoda‐

ku,Tokyo

Abstract

4‐methyl‐5‐pentylbenzene‐1,3‐diol(MPBD) is a polyketide produced by SteelyApolyketide synthase. The functional analysisofMPBDrevealed that it regulatedchemotaxis in aggregation stage and spore maturation in the last step of thedevelopment. To understand the structure‐activity correlation of MPBD, wecreated 4 derivatives of MPBD and investigated their chemotaxis regulation inDictyosteliumcells.

One of them, 4‐ethyl‐5‐pentylbenzene‐1, 3‐diol (EtPBD), recovered chemotaxisactivity in stlA null mutant to the same level with MPBD in vitro. Two of thederivatives,1,5‐dimethoxy‐2‐methyl‐3‐pentyl‐benzene(MeprotectedMPBD),and4‐methyl‐3‐pentyl‐phenol (Dehydroxy‐MPBD) recovered moderate level ofchemotaxis in stlA null mutant. These results suggest that chain length at 4position of these molecules is important for chemotaxis. The spore maturationactivityofthesederivativeswillalsobediscussed.

70

Poster15

GradientresponseofgiantDictyosteliumdiscoideumcells

KirstenKrüger,MatthiasGerhardt,CarstenBeta

InstituteofPhysicsandAstronomy,UniversityofPotsdam,Potsdam,Germany

Abstract

Many current models of eukaryotic gradient sensing rely on the interplay of alocalizedactivatorwithaglobal,fastdiffusinginhibitor.Basedonthismechanism,weexpectthatgradientsensingiscellsizedependent.Toprobethisdependence,giant cells of the social amoeba Dictyostelium discoideum offer a promisingperspective. They can be generated in different sizes by electric pulse‐inducedfusion from a suspension of native, normal‐sized amoeba. We have previouslyusedthissystemtostudythedynamicsofcombinedactinandPIP3wavesinlargeunboundeddomains.Here,weshowfirstresultsofgiantcellsthatareexposedtogradientsofcAMP.Ourexperimentsindicatethat,incontrasttonormal‐sizedcells,thegradientresponseofgiantcellsinitiallydoesnotinvolvetheentirecellbody.Instead, individual protrusions are formed that are comparable in size toindividualnativecells.Theymigratetowardsthechemicalsource,whilethemainbodyofthecellsremainsstaticorevenmovesinanotherdirection.Onlyafter1to2 minutes also the movement of the main body is affected and follows theprotrusion in gradient direction, suggesting that initial gradient sensing isconfinedtosmallsubsetsofthegiantcellthateventuallybiasthemovementofthemaincellbody.

71

Poster16

Axeniccultivationof'non‐axenic'Dictyosteliumcells

GarethBloomfield1

1MRCLaboratoryofMolecularBiology,Cambridge,UK

Abstract

Methods and media for the short term cultivation of wildtype ('non‐axenic')Dictyosteliumdiscoideumcellsarereported.

72

Poster17

PresporeCudArequiresCudDfornuclearaccumulation

MasashiFukuzawa,AyakaIshizaka

DepartmentofBiology,FacultyofAgricultureandLifeScience,

HirosakiUniversity,JAPAN

Abstract

CudA (culmination‐defective A) is a transcription factor involved in stalk tubeinitiationandsporematurationatthefinalstepofD.discoideummorphogenesis.The expression of cudA and its protein coincides in the tip and in the presporeregionofaslug.WhencudA iscomplementedintheprestalkregionofcudAnull,culmination is restored but no spores are formed (glassy spore head). Duringanalysis of other cud familymembers,wehave isolated a cudDmutant showingglassysporeheads.ThisledusexaminethecorrelationbetweenCudAandCudD.

CudAisabolishedfromnuclei inpresporecellsof thecudDmutantwhile thetipCudAmaintains nuclear localization. CudA in the cudDmutant becomes nuclearlocalized when cells are treated with leptomycin B, an inhibitor for exportin,suggestingthatthenuclearaccumulationofCudAisregulatedvianuclearexport.AmoderatelevelofinteractionbetweenCudAandCudDisdetectedinyeasttwo‐hybridanalysis,suggestingthatCudDmayactasanexportinhibitorforCudA.

InthecudDmutant,theexpressionofcudA‐dependentcotC isreducedto20%ofthat in wild‐type, while in a cudD overexpressor that increases to 260%. TheexpressionofcudA‐independentpspA isalmostunaffected. Interestingly, similarresultsareobtainedincudAexpression,suggestingthatCudAitselfregulatescudAexpression.

CudD localizes in nuclei of disaggregated cudA‐ slug cells. Taken together, ourresultsindicatethatCudDdoesnotrequireCudAfornuclearaccumulation,butisnecessaryforinhibitingexportofCudAfromnucleus.

73

Poster18

ExploringaPotentialRoleforFbxAinDIFSignaling

KarinaD.Sarver,AllysonH.Bartlett,andMargaretK.Nelson

DepartmentofBiology;AlleghenyCollege;MeadvillePA16335USA

Abstract

The original characterization ofmutants lacking FbxA, an F‐boxWD‐40 proteinthat ispart of an SCFE3ubiquitin ligase complex, focusedon the resulting cell‐autonomous sorting defect (“cheater” phenotype), decreased prestalk:presporeratio, and tendency to arrest as slugs. However, several aspects of the fbxA‐phenotype(fragileslugs;reducedpstOpopulation;culminantswithslippedsporeheads)aresimilartothosesubsequentlyreportedformutantswithdefectsinDIFsynthesisorDIF‐responsivetranscriptionfactors.DevelopmentonDIF‐containingagardidnot rescue the fragile slugsoraberrantculminantscharacteristicof thefbxA‐nullmutant,aswouldhavebeenpredictedforamutantwithadefectinDIFsynthesis. Hence,wehypothesizedthatFbxA insteadplaysarole inresponsetoDIF.We used the ecmB‐lacZ reporter to confirm that fbxA‐ mutants display thealteredlowercupexpressionandreducedbasaldisccharacteristicofDIFresponsemutants. However, a global effect on DIF responses seems unlikely as neitherfbxA‐mutantsnorcellsoverexpressingFbxAshowedanystrikingalterationinDIF‐induced nuclear localization of DimB or StatC in cells starved in shakingsuspensionforeither4or8hours.Whetheranydifferenceinrelocalizationoccurslater in development (when fbxA ismorehighly expressed), or if the kinetics ofDIF‐inducedphosphorylationvary, iscurrentlyunknown. Similarly,wehaveyettodirectlymeasure theeffect of alteredFbxA levelsonDIF’s regulationofpspA,ecmA, and ecmB transcription. Our analysesmay eventually reveal a previouslyunappreciatedroleforFbxAinthiscomplexsignalingnetwork.

74

Poster19

FunctionalconservationofadenylatecyclaseA(ACA)throughoutDictyostelia

YoshinoriKawabe1,Zhi‐huiChen1,PaulineSchaap1

1CollegeofLifeSciences,UniversityofDundee,MSI/WTB/JBCcomplex,DowStreet,DundeeDD15EH,UK.

Abstract

In Dictyostelium discoideum, ACA produces extracellular cAMP which acts aschemoattractant in the coordination of aggregation and culmination. AllDictyostelids species are subdivided into fourmajor groups. LikeD. discoideum,group 4 species use cAMP as chemoattractant. Other group species do not usecAMPaschemoattractant.However,cAMPreceptornullmutantingroup2speciesPolysphondylium pallidum revealed that extracellular cAMP is also required forfruitingbodyformation.

To understand evolution of cAMP signaling in Dictyostelia, we analyzed ACAfunctioninothergroupspecies.RecentgenomeprojectsofsixDictyosteliaspeciesrevealed that ACA orthologous genes are conserved throughout theDictyostelidphylogeny. Most Dictyostelia have a single ACA gene, except for the group 2species, which have three ACA genes. Expression of D. lacteum ACA in D. discoideum ACA null mutant partially restored aggregation and fruiting bodyformation, showing that these orthologuesproduce cAMP. Single or doubleACAknockoutmutantinP. pallidumstillformedfruitingbodiesandnormalsporesandstalk cells, although one of the double ACA mutants formed abnormal fruitingbodies. This indicates that theP.pallidum ACAs have overlapping functions. ThespecificACAinhibitorSQ22536completelyblockedaggregationandalsoinhibitedpost‐aggregative development in P.pallidum, indicating that ACA has functionsboth in aggregation and post‐aggregative development in P. pallidum. SQ22536alsoinhibitedaggregationofgroup1and3species,suggestingthattheroleofACAintheaggregationstageisconservedthroughouttheDictyostelidphylogeny,eventhoughgroup1‐3speciesdonotusecAMPaschemoattractant.

75

Poster20

Effectsoftemperatureongrowthanddevelopmentofdictyostelids

‐Theirdiversityandecologicalimplications

HidenoriHashimura1,2,KeiInouye1

1DepartmentofBotany,GraduateSchoolofScience,KyotoUniversity,Japan2Presentaddress:DepartmentofBiologicalSciences,GraduateSchoolofScience,

OsakaUniversity,Japan

Abstract

The distribution of dictyostelids isworldwide, inhabiting various climatic zonesfrom the tundra to the tropics. To see whether the global distributions ofdictyostelids are correlated with their temperature tolerance, we examined theeffectsoftemperatureovertherangeof0~37˚Congrowthanddevelopmentof30species isolated fromthetemperate, tropicorsubarcticzones.Wefoundthatsome“thermophilic”speciesgrewoptimallyandformedfruitingbodiesat28˚Corabove,while some “cold resistant” species grew and developed normally at 4˚C.Surprisingly, someof the lattergrewand formednormal fruitingbodiesevenat0˚C. Allspecies isolated fromthesubarcticzoneshowedcoldresistanceanddidnot survive at temperatures above 28˚C. Species in group 3, clades 2A and 2Btended tobe thermophilic andmanycold resistant speciesbelonged togroup4.Thisisconsistentwiththefactthatmanyspeciesisolatedfromthetropicregionsaregroup3speciesandthatthemanyofthoseisolatedinthecoldregionsbelongtogroup4.Additionally,comparisonbetweenthehighlycold‐resistantspeciesD.rosarium and a modestly cold‐resistant D. discoideum revealed that there aredifferencesincellshapeandF‐actinlocalizationat0˚C.

76

Poster21

Mechanicalbasisofcellsortingphenomena

KeiInouye,IkumiShibano‐HAYAKAWA

DepartmentofBotany,GraduateSchoolofScience,KyotoUniversity,Japan

Abstract

"Cell sorting", changes in the relative position of cells within a tissue, is animportant component of development, and its role in the establishment of theprestalk‐presporepatterninDictyosteliumdiscoideumhasbeenwelldocumented.Itwillalsobeinvolvedinsegregationbetweengeneticallydistinctcellsofthesamespecies and between cells of closely related species that share a commonchemoattractantforaggregation.

In past studies of cell sorting, cell adhesion has been the main subject to befocused. However, cell sorting is an active process, and in Dictyosteliumdevelopment in particular, it takes place while the cells are exhibiting massivemovement. Then, two questions arise concerning itsmechanism; (1) how a cellchanges position when embedded in a tissue of motile cells, and (2) how thedifference between cell types affect their movement. To answer them, weperformedvarioustypesofmixingexperiments,includingtheonedemonstratingsuppressionofinterspecificsegregationbyheterologousexpressionofatgrgene,andalsosome theoreticalanalysis.On thebasisof theseandold results,weputforwardahypotheticalmechanismforcellsorting.

77

Poster22

Thebotanicalcurcuminnegativelyregulatestranscriptionofantioxidantgenesandincreasesreactive

oxygenspeciesinDictyosteliumdiscoideum

WilliamSwatson1,StephenAlexander1

1DivisionofBiologicalSciences,UniversityofMissouri,Columbia,MO65211,USA

Abstract

Herbaldietarysupplementsrepresenta$20billionperyear industry. However,little is known about their safety and efficacy. An example of this is thephytochemicalcurcumin,whichisextensivelyusedworldwidewithclaimsthatithas curative effects against microbial, viral, and inflammatory diseases andpreventative effects against cancer and Alzheimer’s disease. The therapeuticeffectsofcurcuminareoftenattributedtoantioxidantproperties,althoughothermechanismshavealsobeensuggested.Dictyosteliumdiscoideumhasbeenproventobeanexcellentleadgeneticsystemforinterrogatingthemechanismofactionofanumberofdrugsusedinhumanmedicine.Therefore,wehaveusedthismodelsystem to investigate the mechanism of action of the botanical curcumin. Ourstudies show that: 1) Curcumin has a dose‐dependent inhibitory effect on cellproliferation;2)Thetranscriptionoftheantioxidantcatalase(cat)Agene,andthesuperoxide dismutase (sod) A, B and 2 genes are reduced in the presence ofcurcumin; 3) Catalase A specific enzyme activity is lowered in response tocurcumin;4)Thelevelofsuperoxideincreasesinthepresenceofcurcumin;5)Theanti‐oxidant N‐acetylcysteine does not have an effect on cell proliferation orcatalaseAspecificactivity;and6)CurcuminhasnoeffectoncellproliferationinDictyosteliumpurpureum.TheseresultsindicatethatDictyosteliumdiscoideumcanbe used as a non‐mammalian model to assess the molecular mechanismsunderlyingthepharmacologicaleffectsofbotanicalsandalsotobetterunderstandtherolesofoxidantsonamoebalphysiology.

78

Poster23

Amoeboidcellsasatransportsystemformicroobjects

OliverNagel,MatthiasGerhardt,andCarstenBeta

InstituteofPhysicsandAstronomy,UniversityofPotsdam,Potsdam,Germany

E‐mail:olnagel@uni‐potsdam.de

Abstract

The transport and positioning ofmicron‐sized objects in complex geometries isoftenaccomplishedbyfluidflow.However,undergeometricconstrains,likedeadend structures, this isdifficult to achieve.Analternative approachwouldbe theuseofmagneticoropticaltweezers.Yetthesetechniquesrequirealotoftimetorearrangemanyobjects, since ithas tobedoneonebyone.Here,wepropose anovelapproachexploitingthechemotacticbehaviorofsinglecells.Weusecellsofthe social amoeba Dictyostelium discoideum to transport particles of differentsizes.ThecellsareguidedbyagradientofthechemoattractantcAMP,whichcanbeestablishedwiththehelpofagradientchamberorthroughphoto‐uncagingtodirectthecellsinamorespecificfashion.Toanalyzethistransportinmoredetail,theforceappliedtoamicron‐sizedbeadbyaDictyosteliumcellismeasuredwiththehelpofanopticaltrap.

79

Poster24

Developinganon‐animalmodelsystemtoinvestigatebittertastantsasnewtreatmentsforasthma

Otto,GP1,CocorocchioM1,Manson,M2,James,A2,Adner,M2,Dahlen,S‐E2,andWilliams,RSB1.

1SchoolofBiologicalSciences,RoyalHollowayUniversityofLondon,Egham,UK,TW200EX.

2InstituteofEnvironmentalMedicine(IMM),C6,Experimentalasthmaandallergyresearch,Box28717177Stockholm,Sweden.

Abstract

Bitter tasting compounds have recently been heralded as potential newtreatments for asthma, as they relax airways and inhibit inflammatory cellfunction. A major obstacle in the development of bitter tastants as newmedicationsforasthmaisalackofknowledgeaboutthemolecularmechanismoftheiractioninairwaymusclecells.AlthoughbittercompoundsareagonistsfortheG protein‐coupled bitter taste receptors (TAS2Rs), our preliminary findings inhuman bronchi suggest that the bitter tastants may act via distinct signallingpathways.WeareemployingDictyosteliumdiscoideum asanon‐animalmodel toidentifythesedistinctmechanisms,sinceDictyosteliumdoesnotexpressTAS2Rs.WewillscreenaDictyosteliumREMIlibraryformutantsresistanttothegrowth‐inhibitoryeffectsof candidatebitter tastants,whichwillprovide insight into themolecular identityof the signallingpathways thatmediate theseeffects.Wewillalso investigate the conservation of signalling pathways by expressing humanTAS2Rs inDictyostelium and observing whether they confer sensitivity to theircognate bitter tastant agonists. Ultimately,wewish to identify novel treatmentsand theirmolecular targets to aid in the treatmentof asthmaandother airway‐obstructivediseases,withouttheuseofanimals.

80

Poster25

Findingthetargetofvalproicacid

ElizabethF.Kelly.1,MatthewC.Walker.2,Robin,S.B.Williams1

1CentreforBiomedicalSciences,SchoolofBiologicalSciences,RoyalHollowayUniversityofLondon,Egham,Surrey,TW200EX,UK.2DepartmentofClinicaland

ExperimentalEpilepsy,InstituteofNeurology,UniversityCollegeLondon,WC1N3BG,UK.

Abstract

Valproic acid (VPA) is a broad spectrum anti‐epileptic drug that is also used totreat bipolar disorder and migraine. Although VPA is very effective in seizurecontrol, it has a number of adverse side effects highlighting a need to developimprovedtherapeuticagents.Researchinthisareahasbeenprimarilyfocusedonusing animalmodels, however,Dictyosteliumhas recently shown promise as analternativemodeltostudythemolecularmechanismofVPA.UsingD.discoideum,VPAhasbeenshowntoblockphosphoinositidesignalling,andthiseffecthasbeenverified in animal seizure models, where VPA blocks the rapid reduction inphosphoinositideturnoverduringepilepticseizures.InD.discoideum,thiseffectofVPA is independent of phospholipid kinase activity and inositol synthesis andrecycling, suggesting the molecular target may be a component of thephosphoinositidesalvagepathway.TheaimofthisprojectistouseD.discoideumtoinvestigatethreeproteinsinvolvedinthephosphoinositidesalvagepathwayaspotential VPA targets. These proteins are cytidine‐diphosphate diacylglycerolsynthase(CDS),diacylglycerolkinase‐α(DGKA)andphosphatidylinositolsynthase(CDIPT). Knockoutmutants for each encoding genewill therefore be tested forresistance to the effect ofVPA indevelopment andoverexpressionof each genewillrescuethewildtypephenotype.Preliminarydata,usingasingleknockoutforDGKA, shows gene ablation gives rise to an aberrant developmentmorphology,but themutant does not show altered resistance to VPA during developmental.ContinuedanalysisoftheinositolsalvagepathwayasatargetofVPAwillexaminetheremainingproteinstoidentifythetargetofVPAinthetreatmentofepilepsy.

81

Poster26

Analysingthe‐secretaseComplexUsingtheSimpleModelOrganismDictyostelium

DevduttSharma,GrantOtto,PhilipBeesley,RobinS.B.Williams

CentreforBiomedicalSciences,SchoolofBiologicalSciences,RoyalHollowayUniversityofLondon,Egham,Surrey,TW200EX,UK

Abstract

Alzheimer’sdisease (AD) is aneurodegenerative condition resulting in cognitivedecline,dementia,anddecreasedqualityoflife.ADaffects35millionpeopleintheover‐60 populationworldwide, with a predicted 115million people affected by2050. The causes of AD are multifactorial and mutations in the ‐secretasecomplex contribute to thepathogenesisandprogressionof the condition.The‐secretase complex is composed of four subunits: one of two catalytic Presenilinproteins (Psen1/2);Nicastrin (Ncstn);Anteriorpharynxdefective1 (Aph‐1)andPresenilin enhancer 2 (Pen‐2). The complex acts via a proteolytic mechanismleading to generation of amyloid‐, and via a non‐proteolytic (scaffolding)mechanism.Whilst Presenilin proteins have been extensively researched, fewerstudieshaveinvestigatedtherolesoftheremainingthreeproteins.Inmammalianmodels it is difficult to study this complex as deletion of subunits results inembryonic lethality. Dictyostelium shares all ‐secretase components withmammalsand is thesimplestmodelorganismwithtwopresenilinproteins.Thisproject aims to determine the function of Ncstn, Aph‐1 and Pen‐2 in theDictyostelium ‐secretase complex by genetic ablation of individual components.The resultant mutants will be characterised, and rescued by overexpression ofDictyostelium and human proteins. The non‐proteolytic role of the complexwillalso be analysed bymutating keymotifs and domains to better understand thestructural role of the complex and its components. Finally, formation of thecomplexinDictyosteliumwillbeanalysedthroughtheuseoffluorescentlytaggedcomplex components. This work will enhance understanding of proteolytic andnon‐proteolyticfunctionsofthe‐secretasecomplex,anditsroleinAD.

82

Poster27

Roco4diffusionkineticsinvivo:dimerization,complexformationand/ororganellebinding?

LauraNederveen‐SchippersandArjanKortholt

UniversityofGroningen,TheNetherlands

Abstract

LRRK2 is a large multidomain protein, consisting of several protein bindingdomains,adimerizationdomainandtwocatalyticdomains(kinaseandGTPase).MutationsinvariousdomainsofLRRK2mayleadtoParkinson’sDiseaseandhavebeen found to increase the kinase activity of LRRK2. All purified or co‐immunoprecipitated LRRK2 fragments are dimeric, and LRRK2 kinase activityseemstobedependentondimerization.RecentlyitwasshownthatinliveCHO‐K1cellsLRRK2ismonomericandinactiveinthecytosol,butattainspre‐dominantlyas an active dimer at the membrane. These results suggest that LRRK2 cyclesbetweenalowactivemonomericstateandhighactivedimericstate.However,theregulationofdimerizationandtranslocationarenotwellunderstood.IamusingFluorescence Correlation Spectroscopy (FCS) in Dictyostelium discoideum tomeasurediffusiontimesofRoco4,.HereIwilldiscusstheimplicationofthesedatainthecontextofthemolecularfunctionandactivationmechanismofRoco4.

83

Poster28

StructuralcharacterizationofphenylalaninehydroxylasefromDictyosteliumdiscoideum

NingningZhuang1,3,AsaithambiKillivalavan1,2,RuijieYu1,2,YoungShikPark4andKonHoLee1,2,3*

1DepartmentofMicrobiology,SchoolofMedicine,GyeongsangNationalUniversity,Jinju,660‐751,RepublicofKorea.

2DepartmentofConvergenceBiomedicalScience,GraduateSchool,GyeongsangNationalUniversity,Jinju,660‐751,RepublicofKorea.

3PlantMolecularBiologyandBiotechnologyResearchCenter,GyeongsangNationalUniversity,Jinju,660‐751,RepublicofKorea.

4DepartmentofHealthScienceandTechnology,GraduateSchool,InjeUniversity,Kimhae621‐749,RepublicofKorea

Abstract

Phenylalaninehydroxylase(PAH)catalyzesthehydroxylationoftheaminoacidL‐phenylalanine to L‐tyrosine in the presence of the specific cofactortetrahydrobiopterin (BH4) and O2. The PAH from Dictyostelium discoideum(DicPAH) has a different cofactor specificity and regulatory functions though ithighsequencesimilaritiestomammalianPAHs.IthasbeenreportedthatDicPAHis able to use both L‐erythro‐tetrahydrobiopterin (BH4) and its stereoisomer D‐threo‐BH4 (DH4) for its function in vitro. Moreover, DicPAH is devoid of thecharacteristic regulatory mechanisms of mammalian PAH such as positivecooperativityforPheandactivationbypre‐incubationwiththesubstrate.

TounderstandthedualcofactorspecificitiesanddifferentallostericregulationofDicPAH,wehavedeterminedthree‐dimensionalstructuresofanapo‐DicPAH_RC1‐415anditscomplexeswiththeBH2andbothBH2andL‐norleucine(NLE)at2.07,2.07and2.39Å,respectively.Theoverallstructuresofthecatalyticdomainshowsignificant similarities to other mammalian PAHs and bacteria PAHs with well‐conservedresiduesaround theBH2andFe(III)bindingsites. It is found that theNLEbindingofDicPAH_RC1‐4151 caused local conformational changes in residues262‐269andsimilar largeglobalconformationalchanges like in thehumanPAHstructure. Comparing DicPAH_RC1‐415 with the rat PAH_RC structure, the N‐terminalregulatorydomainofDicPAHhasashorterN‐terminal loopandrotatedabout9°awayrelatively to thecatalyticdomain,producingmoreexposedactivesite than that in the rat PAH_RC structure. These structural differences at theregulatorydomainseemtobeassociatedwiththedifferentregulationofDicPAH.

84

Poster29

GflB,aGα2stimulatedRap1‐specificGEFisimportantforDictyosteliumchemotaxisanddevelopment

YoutaoLiu,1JesusLacalRomero,2DouweM.Veltman,3InekeKeizer‐Gunnink,1PeterJ.M.vanHaastert,1RichardA.Firtel,2andArjanKortholt,1

1DepartmentofCellBiochemistry,UniversityofGroningen,Nijenborgh7,9747AGGroningen,TheNetherlands.

2SectionofCellandDevelopmentalBiology,DivisionofBiologicalSciences,UniversityofCalifornia,SanDiego,LaJolla,CA92093,USA

3MRCLaboratoryofMolecularBiology,CambridgeCB20QH,England,UK

Abstract

Bindingof chemoattractant toGprotein‐coupled receptors (GPCRs) leads to theactivation and dissociation of the heterotrimeric G‐protein complex. ThedissociatedGα‐GTPandGβγdimerinteractwiththeirrespectiveeffectorproteinsto induce complex rearrangements of the actin/myosin network whichsubsequentlydrivescellmovementupthechemoattractantgradient.ItishoweverstillunclearhowheterotrimericGproteinstransducetheintracellularsignaltotheactin/myosinII‐basedmovingapparatus.Withapull‐downproteomicscreenweidentified a novel RasGEF and RhoGAP containing protein, GEF‐Like Protein B(GflB),asGα2bindingpartner.GflBlocalizestotheleadingedgeandfunctionsasaGα stimulated,Rap1‐specific GEF that is important for the balance betweenRasandRapsignalingduringchemotaxis.ThekineticsofGflB translocationare fine‐tuned by GSK‐3 mediated phosphorylation. Cells lacking gflB display impairedactin andmyosin dynamics, resulting in defective chemotaxis. Our observationsdemonstrate that GflB is a central, upstream regulator of chemoattractant‐mediated cell polarity and cytoskeletal reorganizations by directly linking GαactivationtomonomericG‐proteinsignaling.

85

Poster30

DictyosteliumRoco4asmodelforLRRK2‐mediatedParkinson’sDisease

KatharinaE.Rosenbusch,BerndK.GilsbachandArjanKortholt

DepartmentofCellBiochemistry,UniversityofGroningen,Groningen,TheNetherlands

Abstract

DictyosteliumRoco4belongs to the conserved familyofRocoproteins.Themostprominent family member, human LRRK2 (Leucine Rich Repeat Kinase 2), hasbeen associated with both familial and idiopathic Parkinson’s Disease (PD).Although several potential LRRK2 mediated pathways and interaction partnershave been identified, yet much about the cellular functions remain unknown,including the underlying molecular mechanisms of enhanced LRRK2 catalyticactivity of several PD associatedmutations. Important understanding of LRRK2has come from our work with Dictyostelium Roco family proteins wherebyespecially Roco4 provides a genetic and structural homology. Here I show thatdisruptionofroco4results in impaireddevelopment,phototaxis,stalkformation,andATPmetabolism,phenotypeswhichareknowntobeaffectedbydysregulatedmitochondrial homoeostasis. The LRR (Leucine Rich Repeat) containing N‐terminus of both LRRK2 and Roco4 is essential for their function in vivo,suggesting intramolecularregulationofproteinactivity.Theroco4‐nullcellsalsoprovided an excellent tool to investigate this complex activation mechanism inmore detail. Studies with chimera proteins of Roco4 and LRRK2 in vivo haveshownthattheLRRdeterminesthespecificityofthechimeraandthatthekinasepart is freely exchangeable; thus the kinase of human LRRK2 in the context ofRoco4 restores the roco4‐ phenotype. Importantly, the N‐terminus of Roco4directlybindsandinhibitskinaseactivityinvitro.TogetherthisstronglysuggeststhattheN‐terminusofLRRK2/Roco4isessentialfortheintramolecularregulationofkinaseactivityandmostlikelydeterminestheinputand/oroutputspecificityoftheproteinsbybindingtoupstreamand/ordownstreamregulators.

86

Poster31

ErkBisamasterproteinkinasethatregulatesacoresetofchemotaxisproteins

JohnNichols1,SewPeak‐Chew2,GianlucaDegliesposti2,ElaineStephens3,MarkSkehel2,RobKay2

1MRCLaboratoryforMolecularCellBiology,UniversityCollegeLondon,GowerStreet,London,WC1E6BT

2MRCLaboratoryofMolecularBiology,FrancisCrickAvenue,Cambridge,CB20QH3BlueStreamLaboratories,763ConcordAve,CambridgeMA

Abstract

Wehave takena globalphosphoproteomic approach to expand the inventoryofproteinsinvolvedinchemotaxis.ByusingSILACandphosphopeptideenrichment,we have quantified changes in protein phosphorylation in response tochemoattractantsatspecificsitesacrossseveralthousandproteins.Amongthese,wehaveidentifiedseveralhundredproteinswhosephosphorylationischangedatleast 3‐fold following cAMP stimulation. These phosphorylation changes can begroupedintospecifickineticclasses,withbothincreasesanddecreasesobserved.However, responses to cAMP stimulation are complex and not limited tochemotaxis; other processes, including signal relay and developmental geneexpression,alsoresidedownstream.

BycomparingcAMPdatawithresponsestothevegetativechemoattractantfolate,wefoundasmallsetofproteinswhosephosphorylationisincreasedinbothcases.We propose that this set represents a group of core chemotactic proteins. Themajorityoftheseproteinsarephosphorylatedatasinglenovelpeptideconsensussequence,suggestingthatasinglemasterkinaseisresponsible.Amutantscreen,confirmed by further phosphoproteomic experiments, demonstrated that theproteinkinaseErkBregulatesthesephosphorylationsites.WehaverevisitedthechemotaxisdefectsoftheerkB‐null,andinvestigatedthechemotaxisfunctionsofcandidateproteinsfromthe‘core’chemotacticset.

87

Poster32

EpigallocatechingallatealtersgrowthandPIP3signalinginDictyosteliumdiscoideum

AprilN.Ilacqua,KathrynF.Hawley,JessahK.Skalla,KyleJ.McQuade

DepartmentofBiologicalSciences.ColoradoMesaUniversity.GrandJunction,CO.USA.

Abstract

Epigallocatechingallate(EGCG),aflavonoidfoundathighlevelsingreentea,hasreceived significant recent attention due to its potential health‐promotingactivities.EGCGappearstoexertitseffectsviaavarietyofmechanisms,includingregulating the activities of cell surface and intracellular signaling proteins. Wehave previously shown that EGCG blocks the Dictyostelium life cycle ataggregation, a defect that likely arises from reduced cellmotility and resemblesdefectsseenwithalossofPI3Kactivity.Here,weshowthattreatmentwithEGCGaffectsgrowthandPIP3signalinginDictyostelium.Axenicgrowthisinhibitedbyaslittle as 100µM EGCG, a concentration that exhibits low cytotoxicity. EGCG alsoblocksmembraneassociationofCRAC‐GFPincellstreatedwithcAMP,suggestingthat PIP3 does not accumulate in the presence of the catechin. EGCG has nodramaticeffectsonmembraneassociationofPTEN‐GFPoronitsredistributioninresponse to cAMP. Given the important role of PIP3 signaling in cancer andinflammation, these results suggest a possible mechanism by which EGCG mayimpacthumanhealth.

88

Poster33

Dictyosteliumasanon‐animalmodeltoidentifypathwaysthatdriveresistanceofhomologousrecombination

defectivetumourstoPARPinhibitors

Anna‐LenaKolb1,NicholasD.Lakin1

1DepartmentofBiochemistry,UniversityofOxford,SouthParksRoad,OxfordOX13Q,UK

Abstract

TumourcellsareoftendefectiveinDNArepairandareconsequentlysensitivetoDNA damaging agents used in chemo‐ and radio‐therapy. The inherent repairdefects in these cells can be exploited in synthetic lethal strategies to targettumourcells.Forexample,familieswithhighriskofbreastandovariancancerlackBRCA2, a protein essential for DNA double‐strand break (DSB) repair byhomologousrecombination(HR).Survivalofthesetumourcellsisdependentonapathway involvingPoly (ADP‐Ribosyl)Transferases (PARPs)and treatmentwithPARP inhibitors (PARPi) results in cell death. However, after an initial clinicalresponse, some tumours develop resistance to PARPi. Identifying pathwaysdriving the desensitization of HR‐defective cells to PARPi will provide noveltherapeutictargetsthat,wheninhibited,willovercomeresistance.Previously,wereported that DNA repair pathways, including PARPs, are conserved inDictyostelium.HereweextendthesefindingstoestablishDictyosteliumasamodelto identify pathways that drive resistance of tumours to PARPi. We screenedseveral clinical relevant PARPi for effectiveness inDictyostelium and show thatsyntheticlethalitybetweenPARPiandHR‐deficiencyisconservedinDictyostelium,illustratingthissystemisatractablemodelforthesestudies.Finally,disruptionofthe non‐homologous end‐joining (NHEJ) DSB repair pathway suppressessensitivityofHR‐defectivestrainstoPARPi.ThesedataindicateDictyosteliumisaneffectivemodeltoidentifypathwaysthatdrivesensitivityofHR‐defectivecellstoPARPinhibitorsanddisruptingtheNHEJpathwaycancounteractthiseffect.

89

Poster34

REMI‐seq–generationofagenome‐widemutantresourceforDictyosteliumfunctionalgenomics

SarahJaques1,AmyBaldwin1,NicoleGruenheit2,ThomasKeller2,RexChisholm3,ChristopherThompson2andAdrianHarwood1

1CardiffSchoolofBiosciences,HadynEllisBuilding,MaindyRoad,CardiffUniversity,Cardiff,UK,CF244HQ

2FacultyofLifeSciences,MichaelSmithBuilding,OxfordRoad,TheUniversityofManchester,Manchester,UK,M139PT

3FeinbergSchoolofMedicine,NorthwesternUniversity,Chicago,Illinois,USA,60611

Abstract

REMI‐seq combines Restriction EnzymeMediated Integrationmutagenesis withNGStechnologytorapidlyidentifygeneknockoutmutantsonalargescale.Weaimto use thismethod to generate a near total collection ofDictyostelium mutants.Thiswill create a community resource that comprises individual, sets and largepoolsofmutants,withthelociofREMImutationssearchableondictyBase.

Insertionsitesareidentifiedbysequencingtheinsert/genomicDNAjunctions.Thejunctions are captured usingMmeI, a type IIC restriction enzyme, indexedwithsequencing adapters and amplified by PCR. Following size selection, the targetDNA is sequenced using an Illumina MiSeq. A bioinformatic pipeline has beendesigned todeconvolve thesequencingdataandcatalogue themutants.Todate,we have generated and stored more than 11,000 insertion mutants and storedthem in an individual and accessible format. So far,we have sequenced~1,000mutantinsertionsitesandidentified280mutantswith‘in‐gene’insertionsthatarenotcurrentlycataloguedinthedictyBasestockcentre.

We believe that this new resource will produce a step change inDictyosteliumgenetics,offeringon‐lineavailabilityofindependentandmulti‐allelicmutantsfornearly all Dictyostelium genes. Providing a capacity to conduct complexphenotypingofproteinfamiliesandwholegenomephenotypicscreensspecifictotheuser’sneed.

90

Poster35

Evidencethatchromatinmodificationsregulatefluctuatinggeneexpressionandcontrol‘salt‐and‐pepper’

differentiation

WilliamSalvidge1,NicoleGruenheit1,ChrisThompson1

FacultyofLifeSciences,UniversityofManchester,MichaelSmithBuilding,Manchester,M139PT

Abstract

How cells are able to make robust cell fate decisions that lead to the correctnumbers of different cell types is a fundamental question in developmentalbiology. Inmany cases cell communication and positional signaling provides anexplanation. However, the mechanism underpinning ‘salt‐and‐pepper’differentiation,which is seen in examples raging fromcompetence inB. subtilis,lineagespecificationinthemouseblastocysttostalkandsporedifferentiationinDictyosteliumisstillpoorlyunderstood.Onepossibilityisthatfatedecisionsmadein this way reflect fluctuating gene expression patterns within a population,ensuringthatonlysomearecompetentorprimedtoadoptaparticularfateatanyonemoment.

Modificationstochromatinareknowntoinfluencedynamicgeneexpression.Wethereforehypothesized that lineage choice couldbe affected through removalofimportantchromatinmarks.TotestthispredictionwecreatedmutantsinseveralchromatinmodificationenzymesinD.discoideum.Consistentwiththisidea,foundthat knockoutof thehistonemethyl‐transferase set1, affects this cell fate choiceand strongly biases cells towards a pre‐stalk fate. BasedonRNA‐seq expressionwehavediscoveredthatset1isrequiredtorepressaspecificsetofdevelopmentalgenesduringgrowth,whenlineageprimingtakespace.Moreover,wefindthatthepatternofmisexpressionsuggestsakeyrole inacrossrepression feedback looprequiredforbufferingagainst fluctuations incellextrinsicnoise,a feedbackloopwehavediscoveredtobeessential toensurethecorrectpre‐stalkproportioningbasedondifferentpositivepre‐stalkinputs.

91

Poster36

Understandingcelltypesortingoutbydifferentialmigrationandadhesion

YuanWang1,NicoleGruenheit1,SuzanneBattom1,ThomasKeller1andChrisThompson1

FacultyofLifeSciences,UniversityofManchester,MichaelSmithBuilding,Manchester,M139PT

Abstract

Saltandpepperdifferentiationfollowedbysortingoutrepresentsafundamentalmechanismofpattern formationduringdevelopment. Cells initially differentiatescattered due to heterogeneity in their response thresholds to a uniformconcentration of differentiation inducing signals. These cells then sort out fromone another, a process thought to involve differential cellmigration/and or celladhesion.AgoodmodeltostudythisprocessisDictyostelium,whichonlycontainstwo main cell types, prespore and prestalk, which differentiate intermingledbefore sorting into discrete tissues. It has been proposed that that cell typedifferentiationendowscellswithdifferentgeneexpressionprofiles,whichinturncauses cells to acquire a different cell properties such as cell shape, rigidity,motilityandadhesionwhichcanbeusedtodrivethedifferentialbehaviourofthecell types. To test this idea, we first determined the cell type specific geneexpressionprofileofprestalkandpresporecells.RNAsequencingwasperformedonFACS sortedprespore (psA‐GFP) andprestalk (ecmAO‐RFP) cells. A cell typeindexofpst/(pst+psA)wasusedtoidentifypresporeandprestalkspecificgenes.By classifying cell type specific genes, a group of myosin II structural andregulatorygeneswerefirstidentifiedaspre‐stalkspecificandwedecidedtostudytheir functions in sorting andmorphogenesis. The developmental phenotype ofthese myosin II structural and regulatory gene mutant cells showed differentlevelsofdefects inmorphogenesis.Thechimericdevelopmentofmutantcellsvsactin::15 labelled Ax4 cells revealed that cells lacking myosin II structural andregulatory genes failed to sort to their given region. Finally,we have developednovel assays to directlymeasure the cellmotility ofwild type andmutant cellsduringcellsortingandcAMPmediatedchemotaxisina3Denvironment.

92

Poster37

TheroleoftheDictyosteliumAkthomologsinmacropinocytosis

ThomasDWilliams1,RobertRKay1

1MRC‐LMB,FrancisCrickAvenue,CambridgeBiomedicalCampus,Cambridge,CB20QH

Abstract

Macropinocytosis is an actin driven process whereby cells can engulf relativelylarge volumes ofmedia, for example in immune sampling, growth of someRas‐drivencancersandentryofcertainpathogenssuchasSalmonellaintohostcells.

InDictyosteliumapatchofactiveRas isproducedat themembrane,stimulatingtheclassIPI3K’stomakeaPIP3patch.TheSCAR/WAVEcomplexisrecruitedtothe edge of the PIP3 patch, driving actin polymerisation and forming amacropinocytic cup which closes, delivering medium into the cell. Increasedmacropinocytosis has been established as a way in which axenic cells candrastically increase their fluid uptake compared to wild strains, allowingpropagationinnutrientbroth.ThishasbeenachievedbytheinactivationofNF1,whichactstolimitthesizeofpatchesofactiveRas.Initsabsence,theactiveRasandPIP3patchescangrowlargergivingmorevoluminousmacropinosomes.

The Dictyostelium homologs of Akt, PkbA and PkbR1 have long been known asbeingimportantforchemotaxis,aswelltherebeinganotableaxenicgrowthdefectin the double mutant. Here we explored this growth defect further, finding areductioninthevolumeofmediumtakenupbythemutantanddeterminedthatthis was due to the volume of the macropinosomes being smaller than in theparental strain. I propose that PkbA/PkbR1 act to increase the size of themacropinosomes, most likely by affecting the size of the active Ras or PIP3patches.

93

Poster38

AnalysisofIntracellularCalciumChannelsinDictyosteliumDevelopment.

Fu‐ShengChang1,EleanorWarren1,JulianD.Gross2,AntonyGalione2,CatherinePears1

1DepartmentofBiochemistry,UniversityofOxford,Oxford,UK

2DepartmentofPharmacology,UniversityofOxford,Oxford,UK

Abstract

Calcium isaubiquitous intracellular signal responsible forcontrollingnumerouscellular responses including development, differentiation and proliferation.Calcium is stored in both neutral and acidic stores and its release though gatedchannels has been implicated in regulating development and cell fate choice inDictyostelium.Thisresearchaims tounderstandtherolesof thecalciumchannelproteinsfoundonacidic(two‐porechannelproteins(TPCs),mucolipin(TRP‐ML)),andneutral(IplA)storesincalciumsignalingduringDictyosteliumdevelopment.

Aseriesofstrainsthataredisruptedintheoneormoreofgenesencodingcalciumchannels have been generated. All disrupted mutants (including one lacking allthreeofthesechannels)areabletoformfruitingbodies.However,strainslackingthe TPC channel show a delay in early development. tpc‐null cells also showpreference forastalkcell fate, consistentwith theTPCchannelplayingarole indevelopment. In vitro vesicles derived from intracellular organelles can take upcalciumandreleaseituponstimulationwitharachidonicacid.Invesiclesfromtpc‐null cells, the rate of uptake of Ca2+ is increased, suggesting Ca2+ homeostasismightbealteredintheabsenceofthechannel.InvivoanalysisofintracellularCa2+responsesisbeingcarriedoutinstrainsexpressingthesensitiveCa2+indicatorYC‐Nano.

Thesestrainsdeficientinoneormoreofthecalciumchannelscontrollingreleasefrom both neutral and acidic Ca2+ stores, in conjunction with real‐time Ca2+detection,willfacilitateanalysisoftherolesoftheindividualchannelsandstoresinDictyosteliumdevelopment.

94

Poster39

Identifyingthemoleculartargetsofcannabisderivativesusingasimplemodelsystem

ChristopherJPerry1,GrantOtto1,BalintStewart2,ChrisThompon2,RobinSBWilliams1

1CentreforBiomedicalSciences,SchoolofBiologicalSciences,RoyalHollowayUniversityofLondonEgham,Surrey,TW200EX;2FacultyofLifeSciences,

ManchesterUniversity,Manchester,M139PL

Abstract

Cannabis has been used for the treatment of medical conditions such asrheumatism, inflammation, seizures and pain management for many centuries.Cannabinoids, compounds derived from cannabis are now also licensed aspharmaceuticalsinthetreatmentofmultiplesclerosisandcannabidiol(CBD)themost abundant non‐psychotropic cannabinoid is undergoing phase III clinicaltrialsasatreatmentforrefractoryepilepsy.Exactlyhowcannabinoidsexerttheirtherapeutic affects remains unknown. Identifying the molecular targets ofcannabinoidswouldallowthedesignofpharmaceuticalswithgreaterefficacyandwithmoretolerablesideeffectprofiles.Thisstudyaimstoidentifythemoleculartargets of CBD, cannabidivarin (CBDV) and cannabidiolic acid (CBDA) usingD.discoideum as a model system. We have shown that D.discoideum growth isblocked by all three cannabinoids in a dose dependant manner. Screening arestriction enzyme mediated integration (REMI) library with each of thesecannabinoidshas identified25mutantsshowingresistance to theeffectof thesecannabinoids on growth, with some mutants showing resistance to multiplecompounds. From thesemutants four genes have been independently identifiedthatarelikelytoconferresistancetocannabinoids.Oneofthesegenesisinvolvedinmitochondrialelectron transportwhileanother ispotentially linkedtomyelinsheath maintenance and neurotransmitter regulation. Thus, this project willidentifynewmechanismsofcannabinoidsthatarelikelytorelatetothetreatmentofarangeofhumandisorders.

95

Poster40

Calciumsignallingisessentialforarrestinrecruitmenttotheplasmamembrane

DavidTraynor&RobertKay

MRCLaboratoryofMolecularBiology,FrancisCrickAve,Cambridge,UK

Arrestins in mammalian cells form an important part of G‐protein coupledreceptorsignalling: theybind to thephosphorylated tailsofGPCRsandcanbothcause endocytic down‐regulation of the receptor and act as signalling platformsfordownstreameffectors.

Caoetal(MBC25,3210,2014)describedapairofrelatedDictyosteliumproteinswitharrestindomains,whichappeartocontrolthefrequencyofcAMPoscillationsin aggregation, as their double mutant produces cAMP waves at twice thefrequency of wild‐type. They also showed that a reporter for one of theseproteins,AdcC‐GFP,istransientlyrecruitedtotheplasmamembraneinresponseto cAMP stimulation, and that (surprisingly) this depends on the presence ofextracellularcalcium.

WehavefollowedupthecalciumdependenceofAdcCtranslocation.WefindthattranslocationisabrogatedbymutationofthepresumedIP3receptor(IplA),whichabolishescalciumsignallinginresponsetocAMP.LikecAMP,ATPcausesarapidcalciuminfluxintocellsandtransientrecruitmentofAdcC‐GFPtothemembrane,but in this case both responses are independent of IplA. We have separatelyidentified the channel essential for the calcium response to ATP, and find thatmutation of this channel blocks AdcC translocation. Treatment of cells with acalcium ionophore, to elevate intra‐cellular calcium independent of receptoractivation,alsocausesAdcCtranslocation.

Our results show that coincident calcium signalling is required for AdcCrecruitmenttotheplasmamembraneafterligandstimulationofcells,andthatthiscalciumsignalmaybesufficient.

96

Participants

97

MurtakabAl‐HejjajSheffieldUniversity,UnitedKingdommyalhejjaj2@sheffield.ac.ukVlatkaAntolovicUniversityCollegeLondon,UnitedKingdomv.antolovic@ucl.ac.ukMonikaAntoniewiczAdamMickiewiczUniversity,Polandmonika.a@amu.edu.plTsuyoshiArakiUniverisityofDundee,UnitedKingdomt.araki@dundee.ac.ukAmyBaldwinCardiffUniversity,UnitedKingdombaldwinAJ@cardiff.ac.ukPetrosBatsiosUniversitätPotsdam,Germanypetros.batsios@uni‐potsdam.deGarethBloomfieldMRCLaboratoryofMolecularBiology,UnitedKingdomgarethb@mrc‐lmb.cam.ac.ukCristinaBosmaniUniversityofGeneva,Switzerlandcristina.bosmani@unige.chSalvatoreBozzaroUniversityofTurin,Italysalvatore.bozzaro@unito.itDerrickBrazillHunterCollege,USAbrazill@genectr.hunter.cuny.edu

CatherineBuckleyUniversityofSheffield,UnitedKingdomc.buckley@sheffield.ac.ukElenaCardenal‐MuñozUniversityofGeneva,SwitzerlandElena.Cardenal@unige.chFu‐ShengChangUniversityofOxford,UnitedKingdomfu‐sheng.chang@bioch.ox.ac.ukZhi‐HuiChenUniversityofDundee,UnitedKingdomz.y.chen@dundee.ac.ukJonathanChubbUniversityCollegeLondon,UnitedKingdomj.chubb@ucl.ac.ukMarcoCocorocchioRoyalHollowayUniversityofLondon,UnitedKingdommarco.cocorocchio@rhul.ac.ukSharonCollierUniversityofWarwick,UnitedKingdomS.Molyneux@warwick.ac.ukAdamCorriganUniversityCollegeLondon,UnitedKingdomadam.corrigan@ucl.ac.ukPierreCossonGenevaFacultyofMedicine,SwitzerlandPierre.Cosson@unige.ch

98

AlixDenoncourtUniversitéLaval,Canadaalix.denoncourt.1@ulaval.caSusanneDiSalvoWashingtonUniversityatSt.Louis,USAsdisalvo@wustl.eduJoeDanDunnUniversityofGeneva,Switzerlandjoedan.dunn@unige.chJanFaixHannoverMedicalSchool,Germanyfaix.jan@mh‐hannover.dePetraFeyNorthwesternUniversity,USApfey@northwestern.eduAnnaFrejRoyalHollowayUniversity,UnitedKingdomanna.frej.2007@live.rhul.ac.ukMasashiFukuzawaHirosakiUniversity,Japanfukuzawa@hirosaki‐u.ac.jpLilliGerstenmaierBernhard‐Nocht‐Institutefortropicalmedicine,Germanygerstenmaier@bnitm.deBerndGilsbachUniversityofGroningen,TheNetherlandsb.k.gilsbach@rug.nlAlbertGoldbeterUniversitéLibredeBruxelles(ULB),Belgiumagoldbet@ulb.ac.be

PierreGolsteinCentred'ImmunologiedeMarseille‐Luminy,Francegolstein@ciml.univ‐mrs.frRichardGomerTexasA&MUniversity,USArgomer@tamu.eduRalphGraefUniversityofPotsdam,Germanyrgraef@uni‐potsdam.deNicoleGruenheitUniversityofManchester,UnitedKingdomnicole.gruenheit@manchester.ac.ukAdrianHarwoodCardiffUniversity,UnitedKingdomHarwoodAJ@cardiff.ac.ukRobertInsallBeatsonInstitute,UnitedKingdomr.insall@beatson.gla.ac.ukKeiInouyeKyotoUniversity,Japaninoue@cosmos.bot.kyoto‐u.ac.jpKeithJacobsNationalInstitutesofHealth,USAkjacobs00@gmail.comSarahJaquesCardiffUniversity,UnitedKingdomJaquesS2@cardiff.ac.ukDavidJimenez‐MoralesNorthwesternUniversityFeinbergSchoolofMedicine,USAdavid.jimenez‐morales@northwestern.edu

99

TianJinNIAID,NIH,USAtjin@niaid.nih.govAlexanderJunemannHannoverMedicalSchool,Germanyjunemann.alexander@mh‐hannover.deYoichiroKamimuraRIKEN,QBiC,Japanykamimur@riken.jpMarjonKampRijksuniversiteitGroningen,TheNetherlandsm.e.kamp@rug.nlYoshinoriKawabeUniversityofDundee,UnitedKingdomy.kawabe@dundee.ac.ukTakefumiKawataTohoUniversity,Japantkawata@bio.sci.toho‐u.ac.jpRobertKayMRCLaboratoryofMolecularBiology,UnitedKingdomrrk@mrc‐lmb.cam.ac.ukElizabethKellyRoyalHolloway,UniversityofLondon,UnitedKingdomLizzie.Kelly.2007@live.rhul.ac.ukAlanKimmelNIH,USAalank@helix.nih.govJasonKingUniversityofSheffield,UnitedKingdomjason.king@sheffield.ac.uk

Anna‐LenaKolbDepartmentofBiochemistry,UnitedKingdomanna‐lena.kolb@seh.ox.ac.ukAnnaKondoSophiagraduateschool,Japananna.k@sophia.ac.jpArjanKortholtUniversityofGroningen,TheNetherlandsA.Kortholt@rug.nlKirstenKrügerUniversitätPotsdam,Germanykkrueger@uni‐potsdam.deAdamKuspaBaylorCollegeofMedicine,USAakuspa@bcm.eduSatoshiKuwanaIwateUniversity,Japansatoshi.kuwana@hotmail.co.jpKonHoLeeGyeongsangNationalUniversitySchoolofMedicine,SouthKorealkh@gnu.ac.krYoutaoLiuTheUniversityofGroningen,TheNetherlandsyoutao.liu@rug.nlAnaTeresaLópezJiménezUniversityofGeneva,SwitzerlandAna.Lopez@unige.chAyakaMatsumotoSophiaUniversity,Japanpines‐f@sophia.ac.jp

100

KyleMcQuadeColoradoMesaUniversity,USAkmcquade@coloradomesa.eduAnnetteMüller‐TaubenbergerBiomedicalCenter,CellBiology(AnatomyIII),Germanyamueller@lrz.uni‐muenchen.deTetsuyaMuramotoTohoUniversity,Japantetsuya.muramoto@sci.toho‐u.ac.jpOliverNagelUniversityPotsdam,Germanyolnagel@uni‐potsdam.deBryanNaritaUniversityofDundee,UnitedKingdomb.narita@dundee.ac.ukLauraNederveen‐SchippersUniversityofGroningen,TheNetherlandsl.m.schippers@rug.nlMattNeilsonBeatsonInstitute,UnitedKingdomm.neilson@beatson.gla.ac.ukMargaretNelsonAlleghenyCollege,mnelson@allegheny.eduJohnNicholsUniversityCollegeLondon,UnitedKingdomj.nichols@ucl.ac.ukClementNizakCNRS‐ESPCIParisTech,Franceclement.nizak@espci.fr

HiroshiOchiaiHirosakiUniversity,Japanochiai3@me.comGrantOttoRoyalHollowayUniversityofLondon,UnitedKingdomgrant.otto@rhul.ac.ukValeriePaquetUniversiteLaval,Canadavalerie.paquet.6@ulaval.caPeggyPaschkeMRCLaboratoryofMolecularBiology,UnitedKingdomppaschke@mrc‐lmb.cam.ac.ukCatherinePearsUniversityofOxford,UnitedKingdomcatherine.pears@bioch.ox.ac.ukChrisPerryRoyalHolloway,UniversityofLondon,UnitedKingdomChristopher.Perry.2014@live.rhul.ac.ukHenderikusPotsUniversityofGroningen,TheNetherlandsh.pots@rug.nlDanielRaszkowskiFriedrich‐Schiller‐UniversitätJena,Germanydaniel.raszkowski@uni‐jena.deMarkRobinsonCardiffUniversity,UnitedKingdomRobinsonME2@cardiff.ac.uk

101

KatharinaElkeRosenbuschRijksuniversiteitGroningen,TheNetherlandsk.e.rosenbusch@rug.nlMichelleRubinBaylorCollegeofMedicine,USAMichelle.Rubin@bcm.eduTamaoSaitoSophiaUniversity,Japantasaito@sophia.ac.jpWilliamSalvidgeUniversityofManchester,UnitedKingdomwilliam.salvidge@manchester.ac.ukPaulineSchaapUniversityofDundee,UnitedKingdomp.schaap@dundee.ac.ukHiroshiSenooJohnsHopkinsUniversitySchoolofMedicine,USAhsenoo1@jhmi.eduDevduttSharmaRoyalHolloway,UniversityofLondon,UnitedKingdomDevdutt.sharma.2009@live.rhul.ac.ukMonicaSkogeUCSanDiego,USAmskoge@gmail.comThierrySoldatiUniversityofGeneva,Switzerlandthierry.soldati@unige.ch

MihaStajdoharGenialisd.o.o.,Sloveniamiha@genialis.comPatrickSuessTexasA&MUniversity,USApsuess@bio.tamu.eduWilliamSwatsonUniversityofMissouri,USAwss62f@mail.missouri.eduAmyTaheriRoyalHollowayUniversityofLondon,UnitedKingdomWVAZ061@live.rhul.ac.ukMarcoTarantolaMax‐Planck‐InstituteforDynamicsandSelf‐Organization(MPIDS),Germanymarco.tarantola@ds.mpg.deElinorThompsonUniversityofGreenwich,UnitedKingdomte30@gre.ac.uk

ChrisThompsonUniversityofManchester,UnitedKingdomchristopher.Thompson@manchester.ac.ukDavidTraynorMRCLaboratoryofMolecularBiology,UnitedKingdomdt101@mrc‐lmb.cam.ac.ukEdwardTunnacliffeUniversityCollegeLondon,UnitedKingdomedward.tunnacliffe@ucl.ac.uk

102

SeijiUraUniversityofOxford,UnitedKingdomseiji.ura@bioch.ox.ac.ukPeterVanHaastertUniversityofGroningen,TheNetherlandsp.j.m.van.haastert@rug.nlDouweVeltmanMRCLaboratoryofMolecularBiology,UnitedKingdomdveltman@mrc‐lmb.cam.ac.ukHong‐YuWangNanjingUniversity,Chinayaqind@sina.comYuanWangUniversityofManchester,UnitedKingdomyuan.wang‐9@postgrad.manchester.ac.ukAmandaWebbBaylorCollegeofMedicine,USAanwebb@bcm.eduThomasWilliamsMRCLaboratoryofMolecularBiology,UnitedKingdomthomasw@mrc‐lmb.cam.ac.ukRobinWilliamsRoyalHolloway,UniversityofLondon,UnitedKingdomRobin.Williams@rhul.ac.uk

ThomasWincklerUniversityofJena,Germanyt.winckler@uni‐jena.deXuehuaXuNIH,USAxxu@niaid.nih.govYokoYamadaUniversityofDundee,UnitedKingdomy.yamada@dundee.ac.ukSagaYukikaTohouniversity,Japan6215005s@nc.toho‐u.ac.jpBlazZupanUniversityofLjubljana,Sloveniablaz.zupan@fri.uni‐lj.si