Nanodynamic Imaging of Leukemic Cell Adhesion

MARIESKLODOWSKA-CURIEACTIONS
Co-fundingofregional,nationalandinternationalprograms(COFUND)
DOC2AMUPROJECT2016CALLFORAPPLICATIONS
NanodynamicImagingofLeukemicCellAdhesion
GENERALINFORMATION
Call
2016
Topic
Imaging
Keywords
diffusion, single molecule, tracking, super-resolution, nanoscopy, membrane
dynamics,phaseimaging,stemcell,leukemia
THESISDIRECTOR(S),RESEARCHUNITSANDDOCTORALSCHOOLS
Thesisdirector
MichelAurrand-Lions,[email protected]
Researchunit CancerResearchCentreofMarseille(CRCM)InsermU1068
Doctoralschool
LifeSciencesandHealth
Thesisco-director
SergeMonneret,[email protected]
Researchunit FresnelInstitute,CNRSUMR7249
Doctoralschool
PhysicsandMatterSciences
1
DESCRIPTIONOFTHEPHDTHESISPROJECT
Celljunctionsplayakeyroleintheintegrityofbiologicaltissues,viaCellAdhesionMolecules(CAMs).
In particular, in the bone marrow, interactions between hematopoietic and stromal cells allow the
mutualtransmissionofsignalsinvolvedinthedevelopmentandhomeostasisofbothcelltypes.This
crosstalk also involves adhesion mechanisms, with a major impact on the physiology of
hematopoieticandstromalcells(development,maintenance,proliferation).PartnerI(A.Sergé&M.
Aurrand-Lions, Leuko/Stromal Interactions team, CRCM) is specialized in these interactions,
especiallyforintegrinsandJunctionalAdhesionMolecules(JAMs,DeGrandisetal.CellMolLifeSci
2015). In physiological context, the team reported contacts between JAM-C-expressing
hematopoietic stem cells and JAM-B-expressing stromal cells (Arcangeli et al. Blood 2011). These
interactions are deeply revised in tumor context. Preliminary data show that JAM-C-mediated
adhesion of leukemic stem cell (LSC) to the stroma is involved in Cell-Adhesion-Mediated Drug
Resistance(CAM-DR),suggestingthatJAM-Cconstitutesapotentialtherapeutictargetinleukemia.
Recent advances in optical nanoscopy completely revisited the classic pattern of static adhesion
structuresincells(Rossieretal.NatCellBiol2012;Paszeketal.Nature2014).Majorplayers,suchas
integrins and their adapters, are tightly regulated by association/dissociation mechanisms,
modulatedaccordingtopathophysiologicalconditionsandsignalsreceivedbythecell.Briefepisodes
ofconfinementorcolocalizationcanrevealmoleculareventsleadingtocellularpathwayactivation.
Hence, discrete events can lead to critical outcomes, thanks to non-linear amplification, as often
reportedincellsignaling.Moreover,integrin-mediatedadhesiontocollagen,amajorcomponentof
the extracellular matrix (ECM), is profoundly implicated in tumor evolution. Analysis of molecular
trajectorieswithourhomemadesoftwareMTT(Sergeetal.NatMet2008;Rougeretal.JoVE2012)
in combination with simultaneous monitoring on the fine distribution of collagen in the close
environmentofthelivingcellwillidentifyinteractionsduringtheonsetandstabilizationofleukemic
cells/stroma contacts. Ultra-resolved imaging will document the role of CAMs in the dynamic
establishmentofcell/cellandcell/ECMadhesioninrealtime.Wewillnotablystudycellsweaklyor
stronglyexpressingJAM-C,toassesstheimpactofblockingantibodiesinLSC/stromainteraction.
Simultaneous visualization of collagen fibers and CAM dynamics requires developing a specific,
multi-modal imaging system on live cells. Partner II (S. Monneret, biophotonics group, Fresnel
Institute)willconductsuchaninstrumentaldevelopmentthankstoitsexpertisebothinnanoscopy
(Bonetal.NatureComm.2015)andinphaseimaging(developmentofanowcommerciallyavailable
phaseimagingsysteminclosecollaborationwithaSME).PartnerIIalreadyproposedafluorescence/
phasebimodalmicroscope(Bonetal.JBO2012)andmorerecentlyamodalityparticularlyadapted
toenhancereal-timevisualizationofcollagenfibers(Aknounetal.OptExp2014).Inthisproject,we
proposetoimprovethesystemsoastocombinesinglemoleculefluorescenceimaginginthevisible
wavelengthsrange,withbothphaseandintensityimagingintheinfrared(IR)range.Indeed,cancer
cellsexhibitremarkableIRsignaturesthroughcollagenandlipids,especiallyinIRwavelengths,which
couldbeexploited.PartnerIII,FirstLightImaging,isacompanythatprovidesworldfastestcameras
with single-molecule sensitivity, initially developed for astronomy. We plan to integrate this new
generation of cameras into the phase imaging system. EMCCD has already been integrated for
nonlinearphaseimaging(Bertoetal.PRL2012). Wewilluseiteitherin2D,platingcellsoncoated
coverslips, or in 3D, cultivating cells within collagen spheroids, to obtain more physiological cell
geometries.
2
3IDIMENSIONSANDOTHERASPECTSOFTHEPROJECT
This biophysical project is fundamentally interdisciplinary. Indeed, on one side, it concerns the
biology of cell adhesion molecules in leukemic context (expertise from Partner I). This includes
molecular biology, cell biology, cell culture, and possibly transgenic animal use, such as JAM-C KO
mice,availableintheteam.Ontheotherside,theprojectaddressesthephysicsofBrownianmotion
anditsrepercussionsontheorganizationofthecellmembrane(sharedexpertisebetweenPartnersI
& II), in combination with a need to develop optical instrumentation and innovative imaging
techniques (partners II & III). The project will also use different existing imaging modes to
compare/validate the new system, including fast videonanoscopy (based on spinning disc confocal
microscopy),TotalInternalReflectionFluorescence(TIRF,particularlysuitedtoobservethebasalcell
membrane coated on collagen) and Fluorescence Recovery After Photobleaching (FRAP, an
interestingalternativetomeasurediffusionwithincellmembranes).Thesetechnicsarealllimitedby
detection sensitivity and acquisition speed. This limit will be challenged by the potential of the
camerasofPartnerIII.Wewillalsodevelopdedicatedinnovativetoolsforautomatedsinglemolecule
tracking, analysis of molecular trajectories and collagen fibers in phase images. Such experimental
and analytical developments are expected to ultimately beneficiate to the whole academic
community. Notably, Partner II is member of France Bio Imaging and France Life Imaging French
nationalstructures,implyingthattechnologicalresultswillbesharedwithinatleastFrenchnational
bio-imagingfacilities.
Attheinternationallevel,wehavealong-standingcollaborationwiththeteamofS.Noursharghin
London (Leinster et al. FASEB J 2013; Scheiermann et al. ATVB 2009; Scheiermann et al. Science
2007). Together we have addressed questions related to the dynamic of CAMs during leukocyte
trans-endothelial migration in inflammatory conditions. The imaging system used in this particular
settingconsistsinfastspinningdiskconfocalvideo-microscopyandacquisitionoftime-lapsez-stacks
on exteriorized tissues of living animals (Woodfin et al. Nat Immunol 2011). Here again, the major
limitation is the acquisition speed since the transmigration event by itself may take less than one
minuteandsequentialremodelingofadhesivestructuresisintheframeofseconds.ThePhDstudent
willhavetheopportunitytojointheteamofS.Noursharghinordertotranslateobservationsmade
invitrousingourimagingmodalitytoinvivosituations.
Asdescribedabove,theimagingsystemweareproposingneedstobeveryefficientinthevisibleas
intheIRspectraldomains,inordertoreachsinglemoleculeaswellassinglecollagenfiberimaging
and cancer signature, respectively. It must also be very rapid to allow large averaging in order to
improve phase sensitivity. Both limits are technologically challenging, calling for intersectoral
collaborationwithindustrytogainaccesstotop-of-the-artcameras.PartnerIIIdevelopshigh-speed
low-noise EMCCD cameras (able to run up to 3500 frames/s with sub-electron readout noise), and
starts to extend their applications to biology. They agree to borrow cameras so as to reach both
required sensitivity and rapidity that should simultaneously allow exhibiting cancer signatures and
resolving collagen fibers in phase microscopy. Access to such outstanding cameras will be a key
advantageformonitoringadhesiondynamicsofmoleculesintheircomplexenvironment.
Ofcourse,weplantocommunicateourmajorresultsatinternationalconferences,beforeorupon
publicationinpeer-reviewjournals.WenotablyplantoattendtheEuropeanorAmericanBiophysical
Meetings.ProjectvaluationisalsoclearlyexpectedbyindustrialPartnerIII.
3
RECENTPUBLICATIONS
M.Aurrand-Lions
1. Cartier-MichaudA,BetziS,ShiX,LissitzkyJC,ZarubicaA,BaillyAL,RocheP,LugariA,Hamon
V,SergéA,BardinF,DerviauxC,LemboF,AudebertS,MarchettoS,BorgJP,ShiN,MorelliX,
Aurrand-Lions M. Structural, genetic and chemical insights into the function of
GRASP55/JAM-Cpathwayinspermatogenesis.Submitted.
2. DeGrandisM,LhoumeauAC,ManciniSJ, Aurrand-LionsM.Adhesionreceptorsinvolvedin
HSC and early-B cell interactions with bone marrow microenvironment. Cell Mol Life Sci.
2015Oct22.
3. MeguenaniM,Miljkovic-LicinaM,FagianiE,RoprazP,HammelP,Aurrand-LionsM,Adams
RH,ChristoforiG,ImhofBA,Garrido-UrbaniS.JunctionaladhesionmoleculeBinterfereswith
angiogenicVEGF/VEGFR2signaling.FASEBJ.2015Aug;29(8):3411-25.
4. Jouve N, Bachelier R, Despoix N, Blin MG, Matinzadeh MK, Poitevin S, Aurrand-Lions M,
FallagueK,BardinN,Blot-ChabaudM,VelyF,Dignat-GeorgeF,LeroyerAS.CD146mediates
VEGF-induced melanoma cell extravasation through FAK activation. Int J Cancer. 2015 Jul
1;137(1):50-60.
5. ArcangeliML,BardinF,FronteraV,BidautG,ObradosE,AdamsRH,ChabannonC,AurrandLions M. Function of Jam-B/Jam-C interaction in homing and mobilization of human and
mousehematopoieticstemandprogenitorcells.StemCells.2014Apr;32(4):1043-54.
6. Leinster DA, Colom B, Whiteford JR, Ennis DP, Lockley M, McNeish IA, Aurrand-Lions M,
Chavakis T, Imhof BA, Balkwill FR, Nourshargh S. Endothelial cell junctional adhesion
molecule C plays a key role in the development of tumors in a murine model of ovarian
cancer.FASEBJ.2013Oct;27(10):4244-53.
7. ArcangeliML,FronteraV,BardinF,ThomassinJ,ChetailleB,AdamsS,AdamsRH,AurrandLions M. The Junctional Adhesion Molecule-B regulates JAM-C-dependent melanoma cell
metastasis.FEBSLett.2012Nov16;586(22):4046-51.
8. ArcangeliML,FronteraV,BardinF,ObradosE,AdamsS,ChabannonC,SchiffC,ManciniSJ,
Adams RH, Aurrand-Lions M. JAM-B regulates maintenance of hematopoietic stem cells in
thebonemarrow.Blood.2011Oct27;118(17):4609-19.
9. FronteraV,ArcangeliML,ZimmerliC,BardinF,ObradosE,AudebertS,BajenoffM,BorgJP,
Aurrand-Lions M. Cutting edge: JAM-C controls homeostatic chemokine secretion in lymph
node fibroblastic reticular cells expressing thrombomodulin. J Immunol. 2011 Jul
15;187(2):603-7.
10. Tenan M, Aurrand-Lions M, Widmer V, Alimenti A, Burkhardt K, Lazeyras F, Belkouch MC,
Hammel P, Walker PR, Duchosal MA, Imhof BA, Dietrich PY. Cooperative expression of
junctional adhesion molecule-C and -B supports growth and invasion of glioma. Glia. 2010
Apr;58(5):524-37.
11. LiX,StankovicM,LeeBP,Aurrand-LionsM,HahnCN,LuY,ImhofBA,VadasMA,GambleJR.
JAM-Cinducesendothelialcellpermeabilitythroughitsassociationandregulationof{beta}3
4
integrins.ArteriosclerThrombVascBiol.2009Aug;29(8):1200-6.
12. Scheiermann C, Colom B, Meda P, Patel NS, Voisin MB, Marrelli A, Woodfin A, Pitzalis C,
ThiemermannC,Aurrand-LionsM,ImhofBA,NoursharghS.Junctionaladhesionmolecule-C
mediates leukocyte infiltration in response to ischemia reperfusion injury. Arterioscler
ThrombVascBiol.2009Oct;29(10):1509-15.
13. ScheiermannC,MedaP,Aurrand-LionsM,MadaniR,YiangouY,CoffeyP,SaltTE,DucrestGay D, Caille D, Howell O, Reynolds R, Lobrinus A, Adams RH, Yu AS, Anand P, Imhof BA,
Nourshargh S. Expression and function of junctional adhesion molecule-C in myelinated
peripheralnerves.Science.2007Nov30;318(5855):1472-5.
A.Sergé
14. SergéA,BaillyAL,Aurrand-LionsM,ImhofBA,IrlaM.For3D:Fullorganreconstructionin3D,
anautomatizedtoolfordecipheringthecomplexityoflymphoidorgans.JImmunolMethods.
2015Sep;424:32-42.
15. Salles A, Billaudeau C, Sergé A, Bernard AM, Phélipot MC, Bertaux N, Fallet M, Grenot P,
Marguet D, He HT, Hamon Y. Barcoding T cell calcium response diversity with Methods for
Automated and Accurate Analysis of Cell Signals (MAAACS), PLOS Comput Biol. 2013.
Sep;9(9):e1003245.
16. Sergé A, Irla M. Cellular and molecular motions: order and disorder. Med Sci (Paris). 2013
Mar;29(3):317-23.
17. Rouger V, Bertaux N, Trombik T, Mailfert S, Billaudeau C, Marguet D, Sergé A. Mapping
molecular diffusion in the plasma membrane by Multiple-Target Tracing (MTT). J Vis Exp.
2012May27;(63):e3599.
18. Sergé A, de Keijzer S, Van Hemert F, Hickman MR, Hereld D, Spaink HP, Schmidt T, SnaarJagalska BE. Quantification of GPCR internalization by single-molecule microscopy in living
cells.IntegrBiol(Camb).2011Jun;3(6):675-83.
19. Cebecauer M, Spitaler M, Sergé A, Magee AI. Signalling complexes and clusters: functional
advantagesandmethodologicalhurdles.J.CellScience,2010Feb1;123(Pt3):309-20.
20. Sergé A, Bertaux N, Rigneault H, Marguet D. Dynamic multiple-target tracing to probe
spatiotemporalcartographyofcellmembranes.NatureMethods,2008Aug;5(8):687-94.
S.Monneret
21. P.Bon,G.Maucort,B.Wattellier,S.Monneret,"Quadriwavelateralshearinginterferometry
for quantitative phase microscopy of living cells", Optics Express 17 (15), 13080-13094
(2009).
22. P. Berto, D. Gachet, P. Bon, S. Monneret, H. Rigneault, «Wide-field vibrational phase
imaging»,Phys.Rev.Lett.109,093902(2012).
23. S.Aknoun,P.Bon,J.Savatier,B.Wattellier,S.Monneret,"QuantitativeRetardanceImaging
ofbiologicalsamplesusingQuadri-WaveLateralShearingInterferometry",OpticsExpress23
(12),16383-16406(2015).
24. P. Bon, N. Bourg, S. Lécart, S. Monneret, E. Fort, J. Wenger, S. Lévêque-Fort, "Three
dimensional nanometer localization of nanoparticles to stabilize super-resolution
microscopy",NatureComm.6,7764(2015).
5
FLI
25. FeautrierP.etal.Revolutionaryvisibleandinfraredsensordetectorsforthemostadvanced
astronomicalA0systems.ProcSPIE,2014jul;(9148):11110-17
26. FeautrierP.etal.OCamwithCCD220,theFastestandMostSensitiveCameratoDateforAO
WavefrontSensing.TheAstronomicalSocietyofthePacific,2011mar;123:263-274
EXPECTEDPROFILEOFTHECANDIDATE
We are seeking for an interdisciplinary profile in biophysics, with a strong background in biology,
mostlycellbiologyandcellculture.Knowledgeinmolecularbiology,cancerbiologyandhaematology
would be a bonus. Concerning the physics side of the project, we are looking for a candidate with
basicknowledgeinoptics,andgoodexperienceincomputing,inMatlaborequivalent,essentiallyfor
image analysis. Background in statistics would also be appreciated. The PhD student will share
her/histimebetweenthetwoinstitutes,CRCMforPartnerIandFresnelforPartnerII,accordingto
the tasks successively addressed, and notably following new instrumental developments in the
imaging systems (single-molecule tracking/collagen phase imaging) as allowed by integration of
visible and IR cameras from Partner III, First Light Imaging. In this framework, she/he will have to
spendtimeintheFirstLightImagingCompanyforanon-sitetrainingontheuseoftheirinnovative
camerasbeforeintegratingitinher/histhesisproject.
Language:goodlevelinEnglish.
6
SUPERVISORPROFILE
MichelAurrand-Lions
BornonFebruary9th1967inMarseille,France
Married,2children
Currentposition:CR1Inserm,HDR
Groupleaderteam«Leuko-stromalinteractionsinnormalandpathologicalhematopoiesis»,CRCM,
http://crcm.marseille.inserm.fr/equipesde-recherche/michel-aurrand-lions/
Education
-1992:MasterinImmunology,CIML,UniversitéAix-MarseilleII,France.
-1996:PhDinImmunologyinPrPhilippeNaquet'slaboratory,UniversitéAix-MarseilleII,France.
-2011:Abilitytomanageresearch(HDR)
Additionaltraining
-1997-2000:HumanFrontierScienceProgramOrganization(HFSPO)fellowinthegroupofPrB.A.
Imhof.CentreMédicalUniversitaire,Departmentoffundamentalpathology,GenevaUniversity,
Switzerland
-2000-2005:AssistantProfessorinthegroupofPrB.A.Imhof.CentreMédicalUniversitaire,
Departmentoffundamentalpathology,GenevaUniversity,Switzerland
-2005-2007:Juniorgroupleader(MER),DepartmentofPathologyandImmunology,Geneva
University,Switzerland
-2007-2012:AvenirInsermGroupLeader,Team«Adhesionmoleculesintumor/hostinteraction»,
CRCM,Inserm,UMR1068,Marseille
Publications:67articlesindexedinPubmed.H-index:28
Studentsupervision
-2001-2005:Co-supervisionThesisC.Lamagna(nowatRigelPharmaceuticals,SanFrancisco)–6
articles
-2003-2006:Co-supervisionThesisG.Mandicourt(publicrelations,Geneva)–2articles
-2004-2008:Co-supervisionThesisC.Zimmerli(nowatAbcam,Cambridge)–4articles
-2008-2011:SupervisionThesisV.Frontera(Post-docinBirmingham)–5articles
-SinceJanuary2013:SupervisionThesisA-L.Bailly
7