The Hippo Pathway: Methods and Protocols

371 Pages·2019·10.846 MB·English

by  Alexander Hergovich

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
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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