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MARIESKLODOWSKA-CURIEACTIONS Co-fundingofregional,nationalandinternationalprogrammes(COFUND) DOC2AMUPROJECT2017CALLFORAPPLICATIONS Cellmechanicsregulationbycytoskeletondynamics: Combinedatomicforceandopticalmicroscopy (CellMechDyn) 1.DESCRIPTIONOFTHEPHDTHESISPROJECT 1.1OBJECTIVESOFTHEPROJECTBASEDONTHECURRENTSTATEOFTHEART Mechanical forces are essential in almost all cellular processes. For example, stiffening occurs during cell division,forcegenerationallowscellmigration,andchangesinthemechanicalmicroenvironmentinducecell 1,2,3 differentiation .Thus,biologicalfunctiondeterminesthemechanicalpropertiesoflivingcells.Remodeling and organizing the cytoskeleton mediate these dynamic changes. The cytoskeleton is a complex network of filaments that forms the structural scaffold of the cell. Among other proteins, the cytoskeleton is mainly formed of actin, microtubules and intermediate filaments. While the mechanical properties of individual filamentsremainmostlyunchanged,itsorganization,assembly,crosslinkingandappliedstressallowthecellto modulateitsmechanicalresponse. Twoimportantexamplesinwhichcellmechanicsandcytoskeletonorganizationisessentialarecellmalignancy and cell migration. In the context of cancer, it has been shown that malignant tumor cells are softer but 4,5 generatehighermechanicalforcesthanbenigncells . Migratingcellsareknowntopolarizeandrestructure theiractincytoskeletondynamicallytoundergomovement,furthermoretheyaredirectedbytheelasticityof the microenvironment. However, the interplay between cytoskeleton’s dynamic structure and mechanics is poorlyunderstood,mainlyduetothelackofavailabletoolscombiningmechanicsandstructuralinformation. One of the most versatile nanotechniques is atomic force microscopy (AFM), as it allows topographical imagingwithnanometerresolutionandmechanicalinvestigationofthesamplesurfaceatpiconewtonforces. ForcemeasurementsoflivingcellsusingAFMconsistinapplyingacontrolledforcethatresultinmeasurable deformation.Combinedwithitsimagingcapabilities,AFMforcemappingprovidesamapoftheelasticityofthe cell.Therecentapplicationofhigh-speedAFM(HS-AFM)tocarryoutforcemeasurementsonsinglemolecules 6,7 andcellshaswidenedthedynamicrangeatwhichwecanobservebiologicalprocesses . Opticalmicroscopyplaysacentralroleincellbiologyasitprovidesapowerfultooltodeterminethestructure andsupramolecularorganizationofcellorganelles,membraneproteinsand,importantly,thecellcytoskeleton. Advanced optical methods such as super-resolution techniques provide images with tens of nanometers resolution,whilepolarizationmicroscopyallowstodeterminelocalorientationalorder. 1 Matzke,R.,etal.NatureCellBiology2001,3,(6) Discher,D.E.,etal.Science2005,310,(5751) 3 Dembo,M.;Wang,Y.-L.Biophysicaljournal1999,76,(4) 4 Lekka,M.,etal.EuropeanBiophysicsJournalwithBiophysicsLetters1999,28,(4) 5 Guo,M.,etal.Cell2014,158,(4) 6 Rico,F.,etal.Science2013,342,(6159) 7 Rigato,A.,AMiyagi,SScheuring,FRico.NaturePhysics2017,(inrevision) 2 1 Themaingoalofthisprojectistocorrelatethemechanicalresponseoflivingcellswiththestructureofthe cytoskeleton of tumor and migrating cells by combining advance atomic force and optical microscopy. The projectisdividedintwospecificaims: Specific aim 1. To determine the dynamic structure of the cortical cytoskeleton of benign and malignant tumor cells and its relation with cells’ viscoelasticity. Our group (BioAFMLab) has recently shown that the viscoelastic response at high frequencies using high-speed AFM provides a signature of the dynamics of the 7 cytoskeleton .Moreover,wehaveshownremarkablydifferentviscoelasticresponseofbenignandmalignant tumorcellsatshorttimescales.However,thecorrelationbetweenthismechanicalsignatureandthestructure of the cytoskeleton at high resolution is still unknown. In this specific aim we will use HS-AFM to image the dynamical structure of the cortical cytoskeleton and to probe the viscoelasticity of tumor cells at different levels of malignancy. The same cells will be fixed for being analyzed using super-resolution microscopy (Fresnel).Therequiredequipmentandmethodsarereadilyavailableintheimplicatedlabs. Specific aim 2. To implement a new adaptive microscope that combines AFM and confocal fluorescence imaging to simultaneously determine the structure and mechanics of migrating cells. Cellmigration,which playsaprominentroleincancerinvasion,iselusivetoaconcomitantprobingofmechanicsandcytoskeleton structure,asmigratingcellsconstantlymoveoutofthefieldofviewoftheAFMorfluorescentmicroscope.To solve this issue, we will couple an AFM to a custom confocal microscope (already operational at Institut Fresnel)andusecodesign(optics/AFM/dataprocessing)toadapttheAFMandopticalscanningschemeinreal time to the trajectory of cells so as to keep them in the center of the imaging field. This will allow simultaneousmechanicalmappingandcytoskeletalimagingoftheentirecellorjustregionsofinterest.The 88 migratingcellishighlypolarized .Thus,knowingthedifferentmechanicalpropertiesofthefrontandrearof the cell is important to understand this complex and essential biological process. Dr. Rico has built multiple AFMsystemsduringhisPhDandpostdoctenures.Dr.LeGoffhaslongexperienceinthebiologyofmigrating cellsandontheuseandimplementationofadvancedopticalmicroscopysystems. ThisprojectinvolvescollaborationwiththetechnologytransferagencySATTSudEst,whichwillprovideadvice andexpertiseontheprivatesector.ThestudentwillbeinconstantinteractionwiththeSATTSudEstandwill perform an internship during the last year of PhD. In addition, Dr. Charafe-Jauffret from the Institut PaoliCalmettesforCancerology,MDpractitionerandresearcherandexpertincancerstemcells,willprovideadvice andsupervisionregardingthebiologicalandclinicalcontexts. An internship in the laboratory of Dr. Xavier Trepat from the Institut of Bioengineering of Catalonia in Barcelona(Spain)willallowthestudenttolearn traction microscopy onlivingcellsandtobeexposedtoan 9 internationalenvironment.Dr.Trepatisexpertinthestudyofphysicalforcesduringcollectivecellmigration . Thisprojectcombinesstate-of-the-arttechnologiessuchashigh-speedatomicforcemicroscopy(onlyavailable in a few labs worldwide) and polarization confocal and super-resolution microscopy (Pioneered at Fresnel). 6 Indeed,theBioAFMLabispioneerintheapplicationofHS-AFMinforcespectroscopymode .Theconceptof adaptivemicroscopesthatusecodesigntooptimizeimaginginrealtimetothechangingbiologicalsystemisa new field about to explode due to increasing demands in resolution. The project is interdisciplinary, as it addresses biological processes (cancer malignancy and migration) from a physics perspective. The project is feasiblesincealltechniquesareavailableintheinvolvedlabsandthesupervisorshavelongexperienceintheir respective fields. The expected outcomes of the project will provide a long-term collaboration between the twogroupsandarepronetoresultin industrial patents.Theexposuretothreedifferent sectors:academic, industrialandclinicalwillresultinstrongrecruitmentperspectivesofthestudentafterPhDcompletion. 8 Ridley,A.J.,etal.Science2003,302,(5651) Sunyer,R.,etal.Science2016,353,(6304) 9 2 1.2METHODOLOGY Cellculture.TumorcelllinesfrombreastcancerwillbegrownonHS-AFMglasssupportspresentingagridfor later localization of the same cells using super-resolution microscopy. Cells for migration experiments with fluorescentactincytoskeletonarealreadyavailableatFresnel.Micropatternsfeaturinglongrailswillbeuseto directcellmigrationalongasingledirection. HS-AFM.TheBioAFMLabhastwoHS-AFMsystems(Ando-typeRIBM,Japan)andaconventional/cellularAFM (JPK,Germany)coupledtoopticalmicroscopy.HS-AFMimagingwillbecarriedoutusingfastcantileversathigh amplitude tapping mode using a wide scanner. Viscoelastic measurements at high frequencies using HS-AFM will be carried out using cantilevers with a grown electron beam deposited tip featuring a sphere of ~1µm. Measurementswillconsistonapplyinglowamplitudesinusoidaloscillationsaroundaconstantindentationat frequencies between 1Hz and 100kHz. The HS-AFM systems are already operational for the proposed measurements. Optical microscopy. For correlative HS-AFM/Fluorescence microscopy, after mechanical measurements, cells willbefixedandstainedforopticalimaging.Toprobespatialorganizationoftheactincytoskeleton,cellswill beimagedwithsuperresolutionmicroscopy,firstonacommercialSTEDmicroscopeoftheFrancebioimaging platform at IBDM near the BioAFMLab, then using STORM at Institut Fresnel (higher resolution but more complexanalysisthanSTED).Furtherstructuralinformationontheorientationalorderoftheactinmeshwork willbegainedusingpolarizationconfocalmicroscopy,andpolarizationSTORMmicroscopyatFresnel. AFM-basedsystemcoupledtoconfocalmicroscopy.TheAFMsystemwillbebasedonadesignpreviouslybuilt 10 byDr.Rico .Thesystemwillfeaturez-piezoforcantilevermovementandxy-piezosforsamplemovement,all mountedonthestageofamotorizedopticalmicroscope.PrototypingwillbedoneattheBio-AFMlab,while thefinaldesignwillbebuiltattheworkshopofInstitutFresnelandcoupledtoaconfocalmicroscope(already operational). As force mapping is slower than confocal imaging, an optical image-based feedback will ensure repositioningofcellsinthefieldofviewoftheAFMand“guide”themechanicalmappingtospecificregionsof interestsuchasthefrontandrearofmovingcellsidentifiedbyimageanalysis.Softwarewillbedevelopedin LabView(NI,France)tocombinetheAFM-basedsysteminforcemappingmodeandthetrackingofmigrating cellsusingconfocalmicroscopy.ThegroupatFresnelinstitutehasalreadydevelopeddifferentscanningcontrol softwarepackagesthatwillbeeasilymodifiedtoallowforcemapping.Inparticulartherecentimplementation of“smartscans”byDr.LeGoffthatusesimageanalysistofocuslaserscanningtoregionsofinterestofhigh informationcontentwillbeeasilyconvertedforthe“smart”mechanicalmappingaforementioned. 1.3WORKPLAN Theprojectwillbedividedintotwomainpartsrelatedtothespecificaims.Thefirststagewillbedevotedto theapplicationofHS-AFMforimagingandviscoelasticmeasurementsofthecorticalcytoskeletonoftumor cells(BioAFMLab)andtheposteriorimagingoffixedcellsusingsuper-resolutionimaging(Fresnel).Thiswill allowthestudenttomastertheuseofHS-AFMtechnologybothinimagingandforcemodesandfamiliarize withadvancedopticalmicroscopymethodsandinstrumentation.Thestageintheinternationalpartnerwillbe partofthisspecificaim.Thisstageisdividedinto6tasks: Task1.1 Task1.2 Task1.3 Task1.4 Task1.5 EstablishingcellcultureconditionsforHS-AFMandposteriorsuper-resolution(months1-9) Microrheologymeasurementsontumourcells(months3-9) HS-AFMimagingofcorticalcytoskeletonoftumourcells(months3-12) Imagingoffixedtumourcellswithsuper-resolutionopticalmicroscopy(months6-12) Internationalinternship(months12-15) 10 Chu,C.,Celik,E.;Rico,F.;Moy,V.Tetal.PLoSONE2013,8,(5) 3 Task1.6 Dataprocessing,correlationandanalysis.Manuscriptwriting(months12-18) Theexpertisegainedduringthefirststagewillbeessentialforthesecondstageoftheprojectdedicatedto implementandAFM-basedsystemonaconfocalmicroscopetostudymigratingcells.Thissecondstagewillbe devotedtothedesignandconstructionoftheAFM-basedsystemanditstestingonlivingcells,itscouplingon thestageofthespinningdiskconfocalmicroscopeandfinallyapplicationtothestudyoflivingmigratingcells. Theinternshipintheprivatesectorpartnerwillbepartofthisstage.Itisdividedinto5tasksplusthesiswriting: Task2.1 Design,constructionandtestingoftheAFM-basedsystem(months15-21) Task2.2 CouplingoftheAFM-basedsystemontheconfocalmicroscope(months18-21) Task2.3 Simultaneousmechanicalmappingandconfocalimagingofmigratingcells(months21-27) Task2.4 Dataprocessing,correlationandanalysis.Manuscriptwriting(months28-33) Task2.5 Thesiswriting(months30-36) Tasks1.1-1.3and2.1willbecarriedoutintheBioAFMlab,tasks1.4,2.2and2.3willbecarriedoutatthe Fresnelinstitute.Tasks1.6and2.4willbecarriedoutbetweenthetwolabs.Anintersectorialinternshipat SATTSudEstwillalsotakeplaceinthecourseofthelastyearofthePhD. 1.4SUPERVISORSANDRESEARCHGROUPSDESCRIPTION ThestudentwillbecosupervisedbyFelixRico(Bio-AFM-Lab)andLoicLeGoff(Fresnel) Mainsupervisor:Dr.FelixRICOismaîtredeconférenceswithChaireMixteattheBio-AFM-Lab,U1006INSERM &AMU.SinceJanuary2017,heisinterimdirectoroftheunit.HewilldefendhisHDRon17January,2017.He holdsaPhDinbiophysicsfromtheSchoolofMedicineoftheUniversityofBarcelona,Spain.Hehaspublished 35articlesandreviewsinjournalslikeScience,NanoLettersandtheBiophysicalJournal.Hehascontributedto about36conferences(13asspeaker)andhasbeeninvitedtoabout24seminars.Hehassupervisedmasterand PhDstudents,postdocsandvisitorresearchers.HeteachesatAMU(atundergraduateandgraduatelevels)and isregularlectureratinternationalschools(e.g.AFMBioMed2011-2016,CNRSthematicschools).Dr.Ricohas been working in force spectroscopy with AFM since 2001 when he started his PhD on cell mechanics. His postdocinthelaboratoryofVincentT.Moy(Miami,FL)focusedonthebiophysicaldeterminantsofadhesion molecules. At Institut Curie, Dr. Rico gained further expertise in force spectroscopy to probe cellular and molecular mechanics, and high-resolution imaging and force mapping on native membranes and cells. He recentlypioneeredtheimplementationofthefirsthigh-speedforcespectroscopysystem,appliedittounfold singleproteins.Relevanttothisproject,asco-directorofA.Rigato’sthesis,hedevelopedmechanicalmapping on micropatterned cells and pioneered the implementation of high-speed microrheology measurements on livingcells(inrevision).HewasawardedanANRJCJCgrant(260k€)in2015andhasanannualassignmentof 10k€/yearlinkedtohischairemixteposition.Partofthesefundswillbeavailabletosupportthisproject. Co-supervisor: Dr. Loic Le Goff: DrLoïcLeGoffischargéderecherchéatCNRS.AfteraPhDatInstitutCurie (Paris) on actin filaments dynamics and a postdoc at Rockefeller University (New York) on the biophysics of hearing,hegainedinterestindevelopmentalbiologyuponjoiningtheInstitutdeBiologieduDévelopementde Marseille.Herecentlyuncoveredtheimportanceofglobalpatternsofmechanicalstressinshapingthegrowing D. melanogaster wing. He also pioneered in vivo imaging for D. melanogaster wings and developed tools to quantify deformations therein. At Institut Fresnel, in the MOSAIC team, he further develops new imaging endeavors on tissues while pursuing his interest on cell and developmental biology. Of particular interest to him are processes of guided cell migration in embryos, as well as the development of new adaptive microscopes that can track and optimally image migrating cells over large distances. The development of adaptive microscopes, upon which aim 2 builds, is financially supported by the Institut Carnot Star and the transfer agency SATT SudEst. The onsite expertise in super resolution microscopy, polarization resolved microscopyandimageanalysiswillbeinstrumentalfortheachievementofthisPhD. 4 2.3IDIMENSIONSANDOTHERASPECTSOFTHEPROJECT 2.1 INTERDISCIPLINARYDIMENSION Theinterdisciplinaritydimensionwillbefirstlyassuredbythecandidatebeingenrolledintwodoctoralschools: ED062(SciencesdelaVieetdelaSanté)andED352(PhysiqueetSciencesdelaMatière).Theprojectcovers two of the priority thematic axes of the DOC2AMU program: nano-health and imaging. The supervisors are formedunderaninterdisciplinaryperspective,asbothusephysicalapproachestoaddressbiologicalquestions. The biological experience is however different, Rico has a molecular to cellular level expertise, while Le Goff has been devoted to cells and organisms. The PhD project clearly combines various disciplines. First, physics andcellbiology:thesystemsofstudyinvolvetwoimportantbiologicalprocessesmalignancyincancerandcell migration,whilethemainapproachisphysical,beingthusoriginal.Andsecond,thestudentwillgainexpertise in two different techniques, advanced optical microscopy and imaging and mechanical measurements using high-speed andconventional atomic force microscopy.Masteringdifferenttechniquesisessentialforfuture research in the field of biophysics. The experience and collaboration established from the PhD will have two mainbenefits:theFresnelinstitute,anadvancedopticscenter,willgaininsightintoatomicforcemicroscopy and biophysics of cancer cells. The U1006, an AFM lab, will gain access and knowhow on advanced optical microscopyandoncellmigration.Itisexpectedthatthiscombinationofmutualbenefitwillallowestablishing along-termcollaborationbetweenthetwogroups. 2.2INTERSECTORIALDIMENSION: TheintersectorialdimensionissecuredbyourcollaborationwiththetechnologytransferagencySATTSudEst. The SATT SudEst has already supported us financially for the development of codesign optical microscopy, whereimageanalysisanddataacquisitionareintermixedinordertooptimizetheimagingprocess.Thecurrent projectextendstheconceptofcodesigntotheAFM/optics/dataanalysismodality.Itbearsgreatpromisesfor thedevelopmentofanewcuttingedgetechnologywithastrongpotentialityforcommercialization.Thetwo supervisors and the PhD student will be in constant interaction with the SATT SudEst for advise and support regarding intellectual property, patents and contacts with companies interested in this development. The studentwillperformaninternshipattheSATTSudEst,duringwhichhe/shewilllearnmoredeeplyconcepts related to intellectual property and make contacts with other technology actors from the private sector (Optitec,smallbusinessandlargermicroscopycompanies,seetheletterofsupportfromSATTSudEst). Besidesitsrootsinfundamentalbioscience,connectionofthePhDworktohealthsectorwillbeensuredbyour regional collaboration with a practitioner in cancerology, Dr. Charafe-Jauffret from Insitut Paoli-Calmettes. The student will interact with hospital technicians to learn the conventional cancer diagnosis and prognosis toolsusedintheclinic,providingexperienceinthecommonprocedures,possibleproblemsandcurrentneeds. 2.2 INTERNATIONALDIMENSION: Theinternationaldimensionwillbefulfilledbyatwo-monthsecondmentduringthesecondyearofPhDinthe laboratory of Dr. Xavier Trepat at the Institut of Bioengineering of Catalonia in Barcelona, Spain. Dr. Trepat expert in traction microscopy applied to collective cell migration. This secondment will complement the mechanical AFM data by measuring traction forces generated by the tumor cells studied in aim 1. Traction microscopyconsistsofanelasticsubstrateofknownstiffnesswithembeddedfluorescentbeads.Cellsgrowing onthegelgeneratetractionforcesthatdeformthegel.Trackingthepositionofthebeadsprovidesameasure ofthedeformationofthegel,whichtranslatesintocellgeneratedtractionforces.Tractionmicroscopywillbe laterappliedtomigratingcellsinthecontextofaim2.Apartfromtheinternship,thestudentwillattendone internationalconferenceperyear(BiophysicalSocietyMeeting,InternationalMicroscopycongress…). 5 3.RECENTPUBLICATIONS High-speedforcespectroscopyunfoldstitinatthevelocityofmoleculardynamicssimulations.Science,2013, 342(6159):741-743.FelixRico,LauraGonzalez,IgnacioCasuso,ManelPuig&SimonScheuring Ahybridhigh-speedatomicforce-opticalmicroscopeforvisualizingsinglemembraneproteinsoneukaryotic cells.NatureCommunications,2013,4:2155,AdaiColom#,IgnacioCasuso#,FelixRico&SimonScheuring AglobalpatternofmechanicalstresspolarizescelldivisionsandcellshapeinthegrowingDrosophilawing disc.LeGoff,L.,Rouault,H.&Lecuit,T.Development(2013). Dynamicclonalanalysisbasedonchronicinvivoimagingallowsmultiscalequantificationofgrowthinthe DrosophilawingdiscHeemskerk,I.,Lecuit,T.&LeGoff,L..Development.(2014). Atomic Force Microscopy Mechanical Mapping of Micro patterned Cells Shows Adhesion Geometry- DependentMechanicalResponseonLocalandGlobalScales.RigatoA,RicoF,EghiaianF,PielM,ScheuringS. ACSNano.2015Jun23;9(6):5846-56. Structural, mechanical, and dynamical variability of the actin cortex in living cells. Eghiaian F, Rigato A, ScheuringS.BiophysJ.2015Mar24;108(6):1330-40. BookChapter Atomic force microscopy studies of the mechanical properties of living cells.RicoF,EPWojcikieviczandVT Moy,inAppliedScanningProbeMethodsIX,B.Bhushan,H.Fuchs,M.TomitoriEds.,Springer,NewYork,2008 Mechanicalforcesandgrowthinanimaltissues.LeGoff,L.&Lecuit,T.ColdSpringHarb.Perspect.Biol.(2016). 4.EXPECTEDPROFILEOFTHECANDIDATE ThecandidateshouldholdaMasterdegree(orequivalent)inafieldrelatedtothesubjectofstudy(biophysics, biomedicalengineering,biologyorsimilar).Experienceinopticalmicroscopyand/oratomicforcemicroscopy appliedtobiologicalsystemsandcellcultureskillswillbeuseful.Theprimarymethodofresearchusedinthis project(HS-AFM)requiresdetailedknowledgeofphysicstooperateit,sothecandidateshouldbefamiliarwith physicalconcepts related to scanning probe microscopies. Transnational mobility is an eligibility criterion for receivingMarieSkłodowska-CurieCOFUNDfunding:thecandidatemustnothaveresidedorcarriedouttheir main activity (work, studies, etc.) in France for more than 12 months in the 3 years immediately prior to thereferencedate.GoodknowledgeofEnglishisessential.Thecandidateshouldbeopen-minded,analytical, dedicatedandmotivated.Thecandidatemustalsobereadytospendlongerperiodsoftimeintheinstitutions oftheinternationalandintersectoralpartners. 5.SUPERVISORS’PROFILES FelixRicoprofessionalprofile 2001-2006 PhDinBiophysics,UniversityofBarcelonaSchoolofMedicine,Spain 2006-2008 FulbrightPostdoctoralFellow,VincentT.Moy’slab,,UniversityofMiami,FL,USA 2009-2011 MarieCuriePostdoctoralFellow,UMR168,InstitutCurie,Paris 2012-2013 ResearchAssociate,U1006INSERM&Aix-MarseilleUniversité,Marseille 2013-now Maîtredeconférences/Interimdirector,U1006INSERM&Aix-MarseilleUniversité,Marseille FormerPhDstudentsofthemainsupervisorFelixRICOandpublications •AnnafrancescaRIGATO,Marseille,France(03/2013-11/2015PhDthesis,12/2015–nowPostdoctobe) 6 High-frequencymicrorheologyrevealscytoskeletondynamicsinlivingcells RigatoA,AMiyagi,SScheuring,andFRico*.NaturePhysics,inrevision AtomicForceMicroscopyMechanicalMappingofMicropatternedCellsShowsAdhesionGeometry- DependentMechanicalResponseonLocalandGlobalScales RigatoA,RicoF,EghiaianF,PielM,ScheuringS.ACSNano.9(6):5846-56.2015 Structural,mechanical,anddynamicalvariabilityoftheactincortexinlivingcells EghiaianF,RigatoA,ScheuringS.BiophysJ.Mar24;108(6):1330-40.2015 Mechanicsofproteinswithafocusonatomicforcemicroscopy RicoF,RigatoA,PicasL,ScheuringS.JNanobiotechnology.11Suppl1:S3.2013 •CalvinCHU,Miami,FL,USA(07/2006-12/2008PhDthesis,03/2011–nowinindustry) Elongatedmembranetethers,individuallyanchoredbyhighaffinityα4β1/VCAM-1complexes,arethe quantalunitsofmonocytearrests ChuC,FRico,ECelik,andVTMoy.PLOSOne.8(5)e641872013 Temperaturedependenceofintegrin-mediatedcelladhesion RicoF*,CChu,MHAbdulreda,YQinandVTMoy.BiophysJ99(5)1387-13962010 Force-clampmeasurementsofreceptor-ligandinteractions RicoF,CChuandVTMoy.MethodsMolecularBiology.736331-532011 LoicLeGoffprofessionalprofile 2002: PhD,InstitutCurie,Paris 2002-2006:Post-doc,HearingResearch-Supervisor:AJHudspethTheRockefellerUniversity,USA 2007-2015:CNRSResearcher(section22)InstitutdeBiologieduDéveloppement,Marseille 2015- :CNRSResearcher(section22:cellanddevelopmentalbiology)InstitutFresnel,Marseille Mentoredpostdocs •HervéRouault,IBDM,Marseille12monthsofco-supervisionin2011 AglobalpatternofmechanicalstresspolarizescelldivisionsandcellshapeinthegrowingDrosophilawing disc.LeGoff,L.,Rouault,H.&Lecuit,T.Development(2013). •IdseHeeskerk,KITP,SantaBarbara,USA.9monthsofsupervisionin2013 Dynamic clonal analysis based on chronic in vivo imaging allows multiscalequantification of growth in the DrosophilawingdiscHeemskerk,I.,Lecuit,T.&LeGoff,L..Development.(2014). OnecurrentPhDStudent:HaiNguyenPhDStartedon6/01/2017 7