Signal processing by the endosomal system

Signal

processing

by

the

endosomal

system

Roberto

Villasen˜or

,

Yannis

Kalaidzidis

and

Marino

Zerial

Cellsneedtodecodechemicalorphysicalsignalsfromtheir

environmentinordertomakedecisionsontheirfate.Inthe

caseofsignallingreceptors,ligandbindingtriggersacascade

ofchemicalreactionsbutalsotheinternalizationofthe

activatedreceptorsintheendocyticpathway.Here,we

highlightrecentstudiesrevealinganewroleoftheendosomal

networkinsignalprocessing.Thediversityofentrypathways

andendosomalcompartmentsisexploitedtoregulatethe

kineticsofreceptortrafficking,andinteractionswithspecific

signallingadaptorsandeffectors.Bygoverningthe

spatio-temporaldistributionofsignallingmolecules,theendosomal

systemfunctionsanalogouslytoadigital-analoguecomputer

thatregulatesthespecificityandrobustnessofthesignalling

response.

Addresses

1_{RocheInnovationCenterBasel,Grenzacherstrasse,CH-4070Basel,}

Switzerland

2_{Max-Planck-InstituteofMolecularCellBiologyandGenetics,}

Pfotenhauerstr.108,01307Dresden,Germany

Correspondingauthors:Villasen˜or,Roberto

([email protected]),Kalaidzidis,Yannis ([email protected])andZerial,Marino([email protected])

CurrentOpinioninCellBiology2016,39:53–60

ThisreviewcomesfromathemedissueonCellregulation EditedbyManuelaBaccariniandIvanDikic

ForacompleteoverviewseetheIssueandtheEditorial Availableonline24thFebruary2016

http://dx.doi.org/10.1016/j.ceb.2016.02.002

0955-0674/#2016TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons. org/licenses/by/4.0/).

Introduction

to

signalling

and

endocytosis

Cellsconstantlyprocessinformationintheformof

chemi-cal or physical signals from the environment to make

decisionsontheirfate.Despiteagreatdealofmolecular

diversity, the communication of information based on

chemical signals follows general design principles that

canbedescribedwiththelanguageofinformationtheory

assource,channel,receiver,decoderanddestination[1,2]: (1)thesourceofsignalsarethecellsthatsecretesignalling

factors which are propagated by (2) the channel, the

extracellularmedium(e.g.blood,ECM).(3)Thereceiver

isthereceptorsonthetargetcells,where(4)thedecoder,

the intracellular signalling machinery transmits and

decodesthesignalsto(5)thedestination,thatis,thecell nucleustodeterminecellfate.Althoughintracellularsignal

transmissionanddecodingoccursimultaneously,theyare

distinctprocessthatcanbeuncoupled.A

well-character-izedexampleoftheseprocessesisthefamilyofreceptor

tyrosinekinases(RTKs).Inthiscase,signallingisinitiated byaligandbindingtoitsreceptor,whichleadstoreceptor activationandrecruitmentofintracellularscaffoldproteins [3]. Next, intracellular signalling scaffolds and kinases

amplify thesignal and finally, lead to changes in target

proteinsthatmodifythecellularstate[4].Forthispathway,

informationistransmittedanddecoded/processedthrough

phosphorylation[5],andotherpost-translational modifica-tionssuchasubiquitination[6,7]orSUMOylation[8].Key propertiesofsuchabiologicaldecoderare:(1)specificity,

which is the capacity to translate a given input into a

specific output; (2) multiplexing, which is the abilityto

processdifferentsignalsthrougharelativelylimited num-berofcorecomponentsinordertotransferinformation;(3)

robustness, which is the capacity to maintain signalling

functionsdespiteinternal(e.g.levelsofsignaltransducers) orexternal(e.g.levelsofsignallingmolecules)fluctuations;

(4) adaptation, which is the ability to adjust the signal

processingnetworkaccordingtothecellularcontext(cells withintheirtissue)andsignalsthatactconcomitantly.How

these properties arise from the cellular machinery and

contributeto cellhomeostasisandcell fateremainopen

questions.

Recent results suggest that both thenetwork topology,

thatis,networkmotifssuchasfeedbackloops[9,10],and

the spatial organization of signalling components

(reviewed in [11]) regulate signalling specificity and

robustness.Animportantelementofthecellulardecoder

is endocytosis. Concomitant with receptor activation,

ligand binding also triggers the internalization of the

signallingreceptorsfromtheplasmamembraneintoearly

endosomes,wherereceptorsaresortedfordegradationto

late endosomes and lysosomes or recycled back to the

plasma membrane tobe reutilized(Figure 1). The

bal-ance among internalization, degradation, and recycling,

regulates the ratio of surface receptor and intracellular

pools, and, as such, the sensitivity of the cell to the

concentrationofligand.Thisparticularroleof

endocyto-sis on thelevels of surface receptors and its impact on

responsiveness to external signals has been extensively

reviewed (e.g. see [12,13]). Here, we focus on novel

insights into the role of the endocytic system as an

essential part of the cellular machinery that decodes

signals foraspecificcellfatedecision.

Compartmental

organization

of

the

endocytic

network

Tounderstandtheroleoftheendocyticsysteminsignal

entry routes and endosomal compartments that could

bestowregulationforthedecoder.Receptorsatthecell

surfacecanbeinternalizedthroughseveraldistinct

endo-cyticroutesandmechanisms([14]andDiFioreetal.,this

issue). The best-characterized pathway in molecular

terms is Clathrin-mediated endocytosis (CME). CME

requires theGTPaseDynamin.Reagentsthat blockits

GTPaseactivityinhibitCME.Note,however,that

Dyna-min is dispensable for other forms of endocytosis. In

additiontothewell-knownmacropinocytosisand

phago-cytosis,severalClathrin-independent endocytosis(CIE)

mechanismshavebeendiscoveredthatdifferonthebasis

ofmorphologicalfeatures,cargospecificity,andmolecular

requirements[14].Theseinclude Dynamin-dependent

mechanismssuchascaveolaeandRhoA-dependent

vesi-clesbutalsoavarietyofDynamin-independentvesicles,

such as Clathrin-independent tubulovesicular carriers

CLIC/GEECorvesiclesthataredifferentiallyregulated

either byCdc42, Arf6 and Flotillin[14]. Recently,the

protein endophilin, a component of CME, has been

implicated in a form of rapid CIE (Fast

endophilin-mediated endocytosis (FEME); [15]). CIE pathways

can contribute to RTK internalization. For example,

RTKscanbe internalizedbycaveolae [16] andFEME

[15].Thedifferentinternalizationpathwayscouldleadto

changesinsignallingoutput.Foradetaileddiscussionon

themechanismsofendocytosisandtheirroleinsignalling

seeDiFiore’sreview inthesameissue.

Allinternalizationpathwaysconverge attheearly

endo-some, where cargo is sorted and transported to

subse-quentcompartments(Figure1).Itisimportanttobearin

mind that these functions result from the collective

propertiesof several hundredsof individual endosomes

that form a dynamic network. One can view the

earlyendosomenetwork asafunnel-likesystem[17,18]

(Figure2).Endocyticcargosuchas RTKsprogressively

flowfromsmallendosomesatthecellperipherytolarge

endosomes in the centre,where eventuallyearly

endo-somes that are positive for the small GTPase Rab5

convertinto Rab7-positivelate endosomes [19,20].

Pro-gressively, the internalized receptors are incorporated

intointra-luminalvesicles(ILV),leadingtodegradation.

The endosomes undergo fusion and fission reactions,

whichshapethedistribution(inspace,numberandsize)

oftheorganellesand,thus,thekineticsandfateofcargo

transportin thenetwork.

This organization already provides a simple means to

regulate cargo flowthrough thenetworkby modulating

thekineticsoftransportand,consequently,theduration

of signalling beforedegradation [21].However, recent

Figure1 Recycling endosomes Rac1 APPL1 + EEA1 CME CIE Lysosome EEA1 Grb2 Shc1 Pyk2 Stat3 Rac1 endosomes Vav2 PTP1B Perinuclear APPL1 Akt GSK-3β Macropinosome LAMTOR2 K-Ras Late endosome

Current Opinion in Cell Biology

Modelofbasicorganizationoftheendocyticpathway.MoleculescanbeinternalizedfromtheplasmamembraneeitherbyCMEorvariousforms ofCIE,includingmacropinocytosis.TheycanbetransportedtotwodistincttypesofRab5-positiveearlyendosomes,distinguishedbythe presenceoftheRab5effectorsAPPL1orEEA1andPI(3)P.Thearrowsindicatethedirectionofcargoflowbetweendifferentendocytic compartments.ThetwoendosomescommunicatethroughadoubleAPPL1+EEA1-positivecompartment.Fromtheseendosomes,cargocan eitherberecycledtotheplasmamembraneviatubularcarriers,directlyorpassingthroughperinuclearrecyclingendosomes,ortransportedto lateendosomesandlysosomes.Inthecourseofthisprocess,cargoisincorporatedinintra-luminalvesicles(ILV)anddegraded.Themodel, whichishighlysimplified,emphasizesthediversityofendosomalcompartmentswheresignallingreceptorscantraffic,withdifferentkineticswhile encounteringdifferentadaptorsandeffectors.

evidence further suggests that early endosomes are a

heterogeneous populationof distinctorganellesin

com-munication with each other.The canonical early

endo-someisdefinedbythepresenceofRab5,anditseffectors,

such as EEA1, that are recruited to the membrane in

a phosphatidylinositol 3-phosphate PI(3)P-dependent

fashion [20,22]. Some Rab5-positive early endosomes

contain theeffectors APPL1 and APPL2. These

endo-somesareenrichedinthesub-corticalregionofthecell,

and are devoid of EEA1 and PI(3)P[23,24]. Afraction

(15–30%)ofendosomescontainbothAPPL1andEEA1

(APPL1+EEA1 endosomes [23,24]). It has been

pro-posed that, similar to Rab5-to-Rab7 conversion [19],

APPL1vesiclesaretransportintermediatesinthe

gener-ationofEEA1-positiveearlyendosomes[25,26].Recent

work, however, hasprovided evidence for APPL1- and

APPL1+EEA1andEEA1endosomesrepresenting

dis-tinctpopulationsofearlyendosomeswithdistinctspatial

distributionsandcargosortingactivity.Thepresenceof

distinct classes of early endosomes offers additional

modes of signalling regulation by RTKs, becauseeach

endosomeclasscansortcargospecificallyandprovidesa

distinct membrane platform for signalling adaptors and

effectors, before transport to late endosomes and

lyso-somes.

Signalling

from

endosomes

There is an ongoing debate whether signalling from

endosomes is actually required for the signalling

re-sponse. Cansignals propagate directly from the plasma

Figure2 endosome fusion endosome fission conversion RAB5 endosome RAB7 endosome CCVS RAB5 endosome recycling

Current Opinion in Cell Biology

Funnel-likeorganizationoftheearlyendosomalnetworkandsignallingquanta.EndocyticcargosuchasEGF-EGFRcomplexesinternalizedfrom thesurface,forexample,viaclathrin-coatedvesiclesCCVs,aretransportedtoRab5-positiveearlyendosomes(seeFigure1).Bytracking individualendosomesaswellasendosomalpopulations,itwasshownthatcargoprogressivelyflowsfrommanysmallearlyendosomesatthecell peripherytofewlargeendosomesatthecentre,resemblingafunnel-likeshapedistributionofendosomes.Thisisbecauseearlyendosomes progressivelyundergofusionandfissionreactions,propagatingwithtimecargotolargerendosomes.Thekineticsofthesetwoprocessesaswell asofmembranerecyclingtothesurfaceshapethedistributionofendosomesintermsofnumber,sizeandlocation.ThefewlargeRab5-positive earlyendosomesthataccumulateintheperinuclearregioneventuallyconvertintoRab7-positivelateendosomesleadingtocargodegradation. Duetothesefeatures,theearlyendosomalnetworkfunctionsasadigital-analoguecomputerthatregulatesspecificityandrobustnessofthe signallingresponse.Uponinternalization,activep-EGFR(singleEGF-EGFRred)isdistributedandpackagedinearlyendosomesassmallclusters orquantaofp-EGFR(tripleEGF-EGFRred),whereasunphosphorylatedEGFRisdistributedrandomly.Withtime,thenumberofendosomeswith p-EGFRquantadecreasesduetop-EGFRdephosphorylation(EGF-EGFRblack)andsequestrationintointra-luminalvesicle(ILV),leadingto degradationinlateendosomes/lysosomes(EGF-EGFRgrey).

membrane to thenucleus?Are endosomes essential for

signaldecoding?StudiesusingDynaminknock-out(KO)

cells showed that reduced endocytosis did not prevent

MAPKsignalling[27],suggestingthatendocytosisisnot

an absolute requirement for signal transmission to the

nucleus. However, stimulation with low (physiological)

concentrationsof EGF leadsto differentErkactivation

kineticsinDynaminKOcompared tocontrolcells[27].

InhibitionofreceptorCMEorubiquitinationchangesthe

EGF-inducedtranscriptionalresponse,similartoEGFR

overexpression[28].Interestingly,stimulatingcellswith

PMA,acompoundknowntorewirethesignaldecoding

network[10],causessubstantialchangesinthetemporal

pattern of the transcriptional response [28], similar to

thoseproducedbyinhibitionof receptorinternalization.

TheeffectofClathrinandDynamin-2knockdownisless

pronounced than thatof PMA, mostlikelybecausethe

block of EGFR CME can be compensated by CIE

mechanisms [14,29]. By contrast, reduction of EGFR

degradation by silencing components of the ESCRT

complex(TGS101,Alix)doesnotaffectthe

EGF-depen-denttranscriptionalresponse[28],arguingthatregulation

of signallingtransmission isnot dueto degradation but

takesplaceatapreviousstage(seebelow).Byblocking

(dynamin-dependent) endocytosis, the levels of active

receptorsaberrantlyincreaseatthecellsurface.Insuchan

artificial situation, both the precise temporal activation

patternofdownstreamsignallingmolecules[27]andthe

magnitudeofthetranscriptionalresponse[28]arealtered.

These changes can result in incorrect signal decoding

sinceitisknownthatalteringsignallingactivation

kinet-icsleadstosignallingnetworkrewiringandsignal

misin-terpretation[10]. Weconclude from these studiesthat,

although endocytosis may not be strictly necessary for

signal transmission per se, it appears to be required for

correctsignaldecoding.

Numerous studies have provided evidence that, under

normalconditions, signallingcontinues following

recep-torendocytosisinendosomes.Earlybiochemicalworkon

EGFR and insulin signalling revealed receptor

recruit-ment of adaptor proteins (SHC, GRB2 and mSOS) in

endosomes[30,31].Recently,differentquantitative

tech-niques have confirmed these initial observations.

Live-cellimagingrevealed sustainedlocalization of the

fluo-rescently-tagged signalling adaptor Grb2 to endosomes

[32].Phosphorylation of c-Met in endosomes leads to

recruitment of the guanine nucleotide exchange factor

Vav2andsustainedactivationofRac1[33].Workusinga

conformationspecificintracellularnanobodyshowedthat

activationoftheb2-adrenoceptorGPCR issustainedin

endosomes, contributing to the overall cyclic AMP

re-sponse [34].A recentstudy usingquantitative

immu-nofluorescenceandFRETshowedthatasub-population

ofEGFRremainsactivatedinearlyendosomeswhereit

recruits the signalling adaptor Shc [35]. Importantly,

thisstudyalsoshowedthatEGFRwasdephosphorylated

before sequestration into ILVs, arguing that signal

quenching(receptordephosphorylation)anddegradation

canbeuncoupled.By contrast todegradation,

de-phos-phorylationisareversibleprocessandcanbeexploitedto

fine-tunesignallingthroughtheendocyticsystem.

Alto-gether,thesefindingssupporttheconceptthatsignalling

isnotlimitedtotheplasmamembranebutcontinues in

endosomes,whichactas signallingplatforms.

Qualitative

regulation

of

signalling

specificity

by

endocytosis

Alargefractionofsignallingadaptorsidentifiedby

mass-spectrometry [36,37] is localized to endosomes (see

Table1).Arecentreviewofproteindatabasesidentified

nearly50endosomalscaffoldsthatcouldregulate

multi-plesignallingpathwaysincludingWnt,NotchandMAPK

[38].Furthermore,thesescaffoldsarelocalizedtospecific

endosomalcompartments.Forexample,thekinasePYK2

is recruited specifically to early endosomes, where it

sustains EGFR-mediated STAT3 activation [39]. The

LAMTOR complex is restricted to late endosomes,

where it regulates mTOR and MAPK signalling [40].

This organization allows for different mechanisms of

signalling regulation.For example, sorting of the same

receptortodifferentcompartmentscangiveriseto

sub-stantiallydifferentsignallingresponsesand cellular

out-comes. This is the case for the c-Met receptor, which

triggersacuteRac1activationwhentransported toearly

endosomes but leads to PI3K-dependent and

Vav2-de-pendentsustainedRac1activationonlyfromperinuclear

endosomes[33].Table1 summarizesthelocalizationof

signallingmolecules recentlyfoundin endosomes.

Several studies have reported interactions of APPL1

with specific receptors and components of signalling

pathwaysincludingadiponectinreceptors[41],TRAF2

Table1

Summaryofrecentlydiscoveredsignallingproteinslocalizedto distinctendosomecompartments

Protein Signalling pathway

Endosome compartment

Reference

Akt PI3K-Akt APPL+endosomes [44] GSK-3b PI3K-Akt,Wnt APPL+endosomes [44] GRB2 MAPK(EGFR) Earlyendosomes [32_]

SHC1 MAPK(EGFR) Earlyendosomes [35] PYK2 JAK/STAT Earlyendosomes [39] LAMTOR2 MAPK,mTOR Lateendosomes [40] PTP1B Broadspectrum

phosphatase

ER-lateorrecycling endosomecontacts

[46,47,49_]

VAV2 PI3K-Rac(c-Met) Uncharacterized perinuclearendosomes

[33]

KRas MAPK Lateendosomes [64]

STAT3 JAK/STAT Earlyendosomes [65]

Rac1 RTK Earlyendosomes [66]

Adenylyl Cyclase

cAMP(TSHR) Uncharacterized endosomes

in theNF-kbpathway [42], Akt[43,44],andTAK1 in

thep38MAPKpathway[45].Theseresultssuggestthat

APPL1endosomesplayaspecializedfunctionaspartof

thesignal decoder.Inaddition,RTKsentering APPL1

endosomescanbetemporarilyprotectedfrom

degrada-tion, asshownbythe enhancedERK activation

down-streamofEGFR[26].Changingtheproportionofcargo

flowbetweenAPPL1-earlyandEEA1-earlyendosomes

could thus result in changesin signalling.

The

endosomal

system

as

an

analogue-digital

computer

The cytoplasmisnothomogeneous,andthe

concentra-tionof moleculescanchangeinspace.Thismeansthat

thespatialdistributionofreceptorsinthecytoplasmcan

modulatesignalling.Forexample,deactivationof

signal-ling receptors is regulated by compartmentalization.

PTP1B,awell-characterizedphosphataseofEGFR,

loca-lizestotheER[46].ThisresultsinagradientofPTP1B

from the periphery to the perinuclear area of the cell

wheretheterminationofsignallingishigherinlate[47]or

recyclingendosomes[48].RecentstudiesonbothEphA2

[48] and EGFR [49] showed thatligand-independent

autonomousactivationwassuppressedviatransporttothe

phosphatase-richperinucleararea,whereasligand-bound

receptors were transported to early endosomes where

signalling was prolonged. This mechanism could have

a key role in signalling robustness as it allows high

sensitivitywhilesuppressing autonomousactivation.

Nano-clusters (less than ten molecules) of activated

receptors or of Ras at the plasma membrane regulate

intracellular signal transduction [50,51,52,53]. Recent

developments in quantitative microscopy revealed that

active (phosphorylated) EGFR forms small clusters of

80moleculesonearlyendosomes[35].Increasingthe

EGFconcentrationdoesnotproducelargerclustersbuta

higher number of clusters. Remarkably, increasing the

numberofclustersthroughamildreductioninendosome

fusion is sufficient to change the signalling outcome.

Theseclusterscanbeconsideredasquantaofsignalling

informationthatconferregulationandrobustnesstothe

cellularresponseagainstfluctuationsinligandorreceptor expression [35].

Experimental data suggest that quanta formation is an

emergent property of positive and negative feedback

loops between receptor activation and

de-phosphoryla-tion. Thepositive feedbackloop is dueto thecatalytic

auto-activation of receptors in a cluster [54,55]. The

activationofEGFRalsoleadsto

phosphotyrosine-medi-atedrecruitmentandlocalactivationofphosphatases(e.g.

PTPN11/SHP2)in theclusters,thusforminganegative

feed-back loop [35,56]. Mathematical simulations

showed thatsuchanauto-inhibitoryloop,together with

fast phosphatase diffusioninto thecytosolcangiverise

to self-organized clusters of activated receptors of a

characteristic size [55], a mechanism first described

byTuring[57].Sinceitisknownthatmultiplereceptors canrecruitphosphatasestotheircytoplasmictails,quanta formationisinalllikelihoodageneralpropertyof

signal-lingreceptorsinendosomes.The formationofp-EGFR

quanta on endocyticmembranes by Turingmechanism

has interesting consequences. The endosomal quanta

behave as a digital-analogue computer. Clustering of

activated EGFRonendosomes isequivalent to an

ana-logue-to-digitalconversionofsignalling.Thebasic

prop-erties of the endosomal network, such as endosome

fusion, fission and sequestration of receptors into ILV

(Figure2)allowperformingcalculusoperations(addition

and subtraction) on quanta. For example, the Turing

mechanism ensures that the fusion of two endosomes

eachcontainingap-EGFRclusterdoesnotresultintwo

quanta or a cluster of double size, but keeps only one

quantumperendosome.Thisissimilartothebitsaddition

insingle-bitprocessors(digitalsummationbymodule2).

Therefore, the kineticsof endosome fusion and fission

(theanaloguecomponent)regulatesthenumberand

life-timeofthequanta,whichinturnmodulatessignal

decod-ing[35].

Feed-back

regulation

of

endocytosis

The previous examples underscore the importance of

endosomal sortingforsignaltransduction.Anadditional

levelofregulationcomesfromthefeedbackthat

signal-ling pathways exert on the endosomal system itself

(reviewedin[58]).Phospho-proteomicanalysisofEGFR

downstream targets identified multiple endosomal

pro-teins [37]. Phosphorylation of endosomal proteins can

leadto substantialchangesin cargo uptakeand sorting.

For example, Akt-mediated phosphorylation of

PIK-FYVE regulates EGFR degradation [59], whereas

p38-mediatedphosphorylationofEEA1promotesearly

endo-somefusion[60].Butevenmoreimpressiveistheextent

towhichsignallingpathwaysregulatetheendocytic

sys-tem. Genomicsurveys by RNAinterference revealed a

profound influence on endocytosis and the endosomal

network by avariety of signalling pathways (e.g. MAP

Kinase,Notch,Wnt,TGFb,amongothers.)[18,61].

Sig-nallingmoleculescanthusmodifytheendosomalsystem

by causing specific changes in endosome fusion and

fission, which implychangesin number, size and

intra-cellularpositionoftheendosomes,andconsequently,in

the trafficking fate of signalling molecules themselves

[35,62]. The combined action of simultaneous

signal-ling pathways can lead to a specific spatio-temporal

organization of endosomes that, in turn, can regulate

signallingspecificityandresponsiveness.Thismeansthat

re-adjustmentoftheendocyticsystemdependingonthe

cell state canmodify the decoding of signals. Through

thismechanism,endosomes allowformultiplexing, that

is, a weighted integration of different types of signals

whichshareacommondown-streamsignallingmolecular

Concluding

remarks

Our understanding of signalling endosomes, first

de-scribedusing biochemical techniques, hasbeen greatly

expanded due to the advancement in high-resolution

quantitative microscopy applied to single cells or

sub-cellularstructures.Abetterunderstandingofthese

mech-anismswillrequirefastlive-cellimagingtostudy

spatio-temporal dynamics with sufficient precision but also

resolving molecular nano-clusters below the diffraction

limit. A current limitation, however, is that signalling

moleculesonendosomeshavebeen exploredmainly in

cellculturesystems. Inthecontext ofmulticellular

sys-tems, the bidirectional regulation of endocytosis and

signallingmay leadto a more complex signaldecoding

thanappreciatedsofar.Forexample,thespatial

regula-tionofVEGFRendocytosisbyPKCinretinalvasculature

leadstoanendocytosisgradientthatisrequiredforvessel patterning[63].Therefore,amajorchallengewillbeto

studytheroleplayedbyendosomalsignallingquantaand

otherregulatorymechanismsinphysiologicalprocessesin

vivo.

Acknowledgements

WethankDrsKonstantinaDiamantaraandFoShengHsuforcritical readingofthemanuscript.

Theauthorscommentedonpublishedworkfinanciallysupportedbythe VirtualLiverinitiative(www.virtual-liver.de)fundedbytheMaxPlanck Society(MPG),theGermanFederalMinistryofResearchandEducation (BMBF)andtheGermanResearchFoundation(DFG)andtheGottlieb DaimlerundKarlBenzStiftung.

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