The Hippo Pathway: Methods and Protocols PDF
Preview The Hippo Pathway: Methods and Protocols
Methods in Molecular Biology 1893 Alexander Hergovich Editor The Hippo Pathway Methods and Protocols M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire,AL109AB,UK Forfurther volumes: http://www.springer.com/series/7651 The Hippo Pathway Methods and Protocols Edited by Alexander Hergovich Cancer Institute, University College London, London, UK Editor AlexanderHergovich CancerInstitute UniversityCollegeLondon London,UK ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-4939-8909-6 ISBN978-1-4939-8910-2 (eBook) https://doi.org/10.1007/978-1-4939-8910-2 LibraryofCongressControlNumber:2018963058 ©SpringerScience+BusinessMedia,LLC,partofSpringerNature2019 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeenmade. Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC part of SpringerNature. Theregisteredcompanyaddressis:233SpringStreet,NewYork,NY10013,U.S.A. Preface Over the past 15 years, the Hippo pathway has emerged as an essential regulator of organ growth and tissue homeostasis. Beginning with Drosophila geneticists, who laid much of the groundwork in Hippo signaling, we havenow cometo a greater understanding of this conservedandessentialpathwayacrossspecies.Theworkofmanyresearchteamssincethen hasuncoveredthecrucialrolesoftheHippopathwayincellproliferation,death,differenti- ation,stemness,andotheressentialprocesses.Equallyimportant,combinatorialapproaches ranging from structural biology to mouse genetics have helped to establish the Hippo pathway as a vital signal transduction network that integrates numerous intracellular and extracellularsignalingcues. Beforepresentingthecontentofthisbook,Iamveryfleetinglyprovidinganoverviewof our current understanding of the core of the Hippo pathway, as a kind of crash course for newcomers to the Hippo field. The intracellular core of the mammalian Hippo pathway is composed of scaffolding proteins (such as NF2/Merlin and MOB1), serine/threonine kinases (such as MST1/2 and LATS1/2; aka Hippo and Warts in flies), and the co-transcriptional regulators YAP/TAZ (aka Yorkie in flies). In the on state of the Hippo pathway, MST1/2 phosphorylate and thereby activate LATS1/2 in complex with MOB1. The activated LATS1/2-MOB1 complex then phosphorylates YAP/TAZ, hence resulting intheinactivationofYAP/TAZ(atleastregardingtheirco-transcriptionalactivities).Inthe off state, MST1/2-LATS1/2 signaling is inactive, thus allowing YAP/TAZ to drive pro-survival and pro-growth transcriptional programs in the nucleus. To date, YAP/TAZ are considered the main effectors of the Hippo pathway, and therefore quite a number of chaptersinthisbookaredevotedtostudiesofYAP/TAZ. This volume of the Methods in Molecular Biology series is a collection of expert lab protocols and commentaries for studies in the Hippo pathway. More specifically, this book documents the most common experimental approaches, recording methods ranging from single-moleculeanalysistocomplexgeneticsandimaginginmulticellularorganisms.Thus, this book covers numerous expert methodologies to examine Hippo signaling on the structural,molecular,cellular,andorganismallevel.Notably,allchaptershavebeenwritten by specialists in their fields who have repeatedly tested and validated these methods in the lab. On the one hand, this book introduces scientists from diverse research fields, such as genetics,biochemistry,structure,molecular,andcellbiology,totheHippopathway.Onthe otherhand,thisbookprovidesexperimentaltemplatesfornewcomerstoexcitingstudiesof theHippopathway.Inthisregard,wewouldliketoemphasizethateachmethodschapteris complementedbya“Notes”sectioninorder tofurtherequipresearcherswithinformation onhowtotackleanyproblemordifficultythatmightarisewhenusingagiventechnique. Thisbookhas26chaptersdistributedover fourmainparts.Thefirstpartisfocusedon fly genetics, containing chapters that present to newcomers how the Hippo pathway was initially discovered and subsequently dissected genetically. Thus, approaches regarding mosaic genetics and immunofluorescence microscopy in flies are presented in the first part, which is comprised of six chapters. The second part addresses procedures that are usefulforthemolecularandcellbiologicalanalysisoftheHippopathway.Morespecifically, the11chaptersofthesecondpartdescribestate-of-the-artmethodsincludingstudiesofthe subcellularlocalization,co-transcriptionalactivities,andCRISPR-mediatedmanipulationof v vi Preface YAP/TAZ; the regulation of the Hippo pathway by phosphorylation events; and three- dimensionaltissueculturemodelsandmechano-sensing.Thethirdpartconsistsof7chap- ters, covering experimental applications to study key components of the Hippo pathway usingstructuralbiologyandbiochemistry.Theseprovideinsightsintostate-of-the-artassays to study protein–protein interactions of Hippo core components both structurally and biochemically, as well as measurements of the phosphorylation and activity status of Hippocorecomponents.Thefourthandfinalpartofthisbookgivesinformationonstudies of the Hippo pathway using mouse genetics. Given the present availability of excellent reviews of the Hippo pathway in cancer mouse models, we include here only two chapters that are focused on Hippo components in early embryonic development and regenerative medicineinmice,respectively. Collectively,Ihopethatthisbookwillbeanessentialpartofmanylaboratorylibrariesto assistnewcomersaswellasexperts,althoughIwouldbemostpleasedifthisbookwouldbe mainlyusedonthelabbenchrather thanbeingstoredawayonabookshelf.Iamconfident thatthisbookequipsnewcomersandspecialistsalikewithkeymethodologiestoaccurately define the status of Hippo signaling in their experimental settings. I sincerely believe that this book will encourage the scientific community to embrace coherent standards when studyingtheHippopathway. I extend my thanks and appreciation to all the contributing authors and Prof. John Walker (University ofHertfordshire, UK) for their invaluable efforts during the process of assemblingthisbook.VeryspecialthanksgotoJoannaHergovichLisztwanforherediting efforts. I also thank University College London and Springer Nature for their support required to produce this book. Last but not least, I would like to thank God for continu- ouslyencouragingmeinthecourseofthisbookjourney. London,UK AlexanderHergovich Contents Preface ..................................................................... v Contributors................................................................. xi PART I THE HIPPO PATHWAY: DROSOPHILA GENETICS LEAD THE WAY 1 ThePowerofDrosophilaGenetics:TheDiscoveryoftheHippoPathway...... 3 RewateeGokhaleandCathieM.Pfleger 2 DrosophilaGenetics:ThePowerofGeneticMosaicApproaches............... 27 MardelleAtkins 3 DrosophilaGenetics:AnalysisofTissueGrowthinAdultTissues.............. 43 AlexanderD.FulfordandPauloS.Ribeiro 4 LiveImagingofHippoPathwayComponentsinDrosophilaImaginalDiscs ... 53 JiajieXu,TingSu,SherzodA.Tokamov,andRichardG.Fehon 5 LocalizationofHippoSignalingComponentsinDrosophila byFluorescenceandImmunofluorescence ................................. 61 CordeliaRauskolbandKennethD.Irvine 6 BimolecularFluorescenceComplementation(BiFC)inTissueCulture andinDevelopingTissuesofDrosophilatoStudyProtein-Protein Interactions ............................................................ 75 YurikaMatsuiandZhi-ChunLai PART II MOLECULAR AND CELL BIOLOGY STUDIES OF THE HIPPO PATHWAY 7 ImmunohistochemistrytoStudyYAPinHumanTissueSamples ............. 89 FranziskaHaderk,VictorOlivas,andTreverG.Bivona 8 ImmunofluorescenceStudyofEndogenousYAPinMammalianCells ......... 97 ValentinaRauschandCarstenG.Hansen 9 ImmunofluorescenceMicroscopytoStudyEndogenousTAZ inMammalianCells..................................................... 107 NathanM.Kingston,AndrewM.Tilston-Lunel,JuliaHicks-Berthet, andXaralabosVarelas 10 Nuclear/CytoplasmicFractionationtoStudyHippoEffectors................ 115 MariaChatzifrangkeskouandEricO’Neill 11 LuciferaseReporterAssaystoDetermineYAP/TAZActivity inMammalianCells..................................................... 121 SirioDupont vii viii Contents 12 QuantitativeReal-TimePCRtoMeasureYAP/TAZActivity inHumanCells......................................................... 137 XiaoleiCaoandBinZhao ® 13 HTRF TotalandPhospho-YAP(Ser127)CellularAssays ................... 153 DianaZindel,ClaireVol,OdileLecha,IsabelleBequignon, MerveBilgic,MarionVereecke,FabienneCharrier-Savournin, Ma¨ıte´Romier,EricTrinquet,Jean-PhilippePin,JuliePannequin, ThomasRoux,ElodieDupuis,andLaurentPre´zeau 14 StudyingYAP-Mediated3DMorphogenesisUsingFishEmbryos andHumanSpheroids................................................... 167 YoichiAsaoka,HitoshiMorita,HirokoFurumoto,Carl-PhilippHeisenberg, andMakotoFurutani-Seiki 15 RegulationofYAP/TAZActivitybyMechanicalCues:AnExperimental Overview .............................................................. 183 SirioDupont 16 CRISPR-MediatedApproachestoRegulateYAP/TAZLevels................ 203 RyanJ.QuintonandNeilJ.Ganem 17 HippoPathwayRegulationbyTyrosineKinases ............................ 215 NinaReuven,MatanShanzer,andYosefShaul PART III STRUCTURE BIOLOGYAND BIOCHEMISTRY TO STUDY THE HIPPO PATHWAY 18 StructuralandBiochemicalAnalysesoftheCoreComponents oftheHippoPathway................................................... 239 LishengNiandXuelianLuo 19 IsothermalTitrationCalorimetryAssaystoMeasureBinding AffinitiesInVitro....................................................... 257 KuiLinandGengWu 20 GSTPull-DownAssaytoMeasureComplexFormations..................... 273 Sun-YongKimandToshioHakoshima 21 DeterminingthePhosphorylationStatusofHippoComponents YAPandTAZUsingPhos-tag............................................ 281 RuiChen,StevenW.Plouffe,andKun-LiangGuan 22 QuantifyingtheKinaseActivitiesofMST1/2 .............................. 289 NiamhA.O’DriscollandDavidMatallanas 23 MeasuringtheKinaseActivitiesoftheLATS/NDRProteinKinases........... 305 AlexanderHergovich 24 MST1/2KinaseAssaysUsingRecombinantProteins ....................... 319 MartaGomez,YavuzKulaberoglu, andAlexanderHergovich Contents ix PART IV THE HIPPO PATHWAYAND MOUSE MODELS 25 VisualizingHIPPOSignalingComponentsinMouseEarly EmbryonicDevelopment ................................................ 335 TristanFrumandAmyRalston 26 TheHippoSignalingPathwayinRegenerativeMedicine..................... 353 LixinHong,YuxiLi,QingxuLiu,QinghuaChen,LanfenChen, andDawangZhou Index ...................................................................... 371 Contributors YOICHIASAOKA (cid:1) DepartmentofSystemsBiochemistryinPathologyandRegeneration, YamaguchiUniversityGraduateSchoolofMedicine,Yamaguchi,Japan MARDELLEATKINS (cid:1) DepartmentofBiologicalSciences,SamHoustonStateUniversity, Huntsville,TX,USA ISABELLEBEQUIGNON (cid:1) IGF,UnivMontpellier,CNRS,INSERM,Montpellier,France MERVEBILGIC (cid:1) IGF,UnivMontpellier,CNRS,INSERM,Montpellier,France TREVERG.BIVONA (cid:1) DepartmentofMedicine,UniversityofCalifornia,SanFrancisco, SanFrancisco,CA,USA;DepartmentofCellularandMolecularPharmacology, UniversityofCalifornia,SanFrancisco,SanFrancisco,CA,USA;HelenDillerFamily ComprehensiveCancerCenter,UniversityofCalifornia,SanFrancisco,SanFrancisco, CA,USA XIAOLEICAO (cid:1) LifeSciencesInstituteandInnovationCenter forCellSignalingNetwork, ZhejiangUniversity,Hangzhou,Zhejiang,China FABIENNECHARRIER-SAVOURNIN (cid:1) CisbioBioassays,F-30200,Codolet,France MARIACHATZIFRANGKESKOU (cid:1) DepartmentofOncology,UniversityofOxford,Oxford,UK LANFENCHEN (cid:1) StateKeyLaboratoryofCellularStressBiology,InnovationCenter forCell SignalingNetwork,SchoolofLifeSciences,XiamenUniversity,Xiamen,Fujian,China QINGHUACHEN (cid:1) StateKeyLaboratoryofCellularStressBiology,InnovationCenterforCell SignalingNetwork,SchoolofLifeSciences,XiamenUniversity,Xiamen,Fujian,China RUICHEN (cid:1) DepartmentofPharmacologyandMooresCancerCenter,Universityof California,LaJolla,CA,USA SIRIO DUPONT (cid:1) DepartmentofMolecularMedicine,SchoolofMedicine,Universityof Padova,Padova,Italy ELODIE DUPUIS (cid:1) CisbioBioassays,F-30200,Codolet,France RICHARDG.FEHON (cid:1) DepartmentofMolecularGeneticsandCellBiology,TheUniversityof Chicago,Chicago,IL,USA;CommitteeonDevelopment,RegenerationandStemCell Biology,TheUniversityofChicago,Chicago,IL,USA TRISTANFRUM (cid:1) DepartmentofBiochemistryandMolecularBiology,MichiganState University,EastLansing,MI,USA ALEXANDERD.FULFORD (cid:1) CentreforTumourBiology,BartsCancerInstitute,QueenMary UniversityofLondon,London,UK HIROKOFURUMOTO (cid:1) DepartmentofSystemsBiochemistryinPathologyandRegeneration, YamaguchiUniversityGraduateSchoolofMedicine,Yamaguchi,Japan MAKOTO FURUTANI-SEIKI (cid:1) DepartmentofSystemsBiochemistryinPathologyand Regeneration,YamaguchiUniversityGraduateSchoolofMedicine,Yamaguchi,Japan NEILJ.GANEM (cid:1) DepartmentofPharmacologyandExperimentalTherapeutics,TheCancer Center,BostonUniversitySchoolofMedicine,Boston,MA,USA;DivisionofHematology andOncology,DepartmentofMedicine,BostonUniversitySchoolofMedicine,Boston,MA, USA REWATEEGOKHALE (cid:1) DepartmentofOncologicalSciences,TheIcahnSchoolofMedicineat MountSinai,NewYork,NY,USA MARTAGOMEZ (cid:1) UniversityCollegeLondonCancerInstitute,London,UK xi
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