mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging.

2.5 MB·English

by  Yadav, Rahul B

Yadavetal.BMCCellBiology2013,14:3
http://www.biomedcentral.com/1471-2121/14/3
RESEARCH ARTICLE Open Access
mTOR direct interactions with Rheb-GTPase and
raptor: sub-cellular localization using fluorescence
lifetime imaging
Rahul B Yadav1, Pierre Burgos1, Anthony W Parker1, Valentina Iadevaia2, Christopher G Proud2, Rodger A Allen3,
James P O'Connell3, Ananya Jeshtadi4, Christopher D Stubbs1* and Stanley W Botchway1*
Abstract
Background: The mammalian target of rapamycin (mTOR) signalling pathway has a key rolein cellular regulation
and several diseases. While it is thought that Rheb GTPase regulates mTOR,acting immediately upstream, while
raptor is immediately downstream of mTOR,direct interactions have yet to be verified inlivingcells, furthermore
thelocalisation of Rheb has been reported to have only a cytoplasmic cellular localization.
Results: Inthis study a cytoplasmic as well as a significant sub-cellularnuclear mTOR localizationwas shown ,
utilizing green and redfluorescentprotein (GFPand DsRed) fusionand highlysensitive single photon counting
fluorescence lifetime imaging microscopy(FLIM) of live cells. The interactionof themTORC1 components Rheb,
mTOR and raptor, tagged with EGFP/DsRedwas determined using fluorescence energy transfer-FLIM. The
excited-state lifetime ofEGFP-mTOR of~2400 pswas reduced by energy transfer to ~2200 ps in thecytoplasmand
to 2000 ps inthenucleus when co-expressed with DsRed-Rheb, similar results being obtained for co-expressed
EGFP-mTOR and DsRed-raptor. The localizationand distribution of mTOR was modified by amino acid withdrawal
and re-additionbut not by rapamycin.
Conclusions: The results illustrate thepower ofGFP-technology combinedwith FRET-FLIM imaging in thestudy of
theinteractionof signalling componentsin living cells, here providing evidence for a direct physical interaction
between mTOR and Rheb and between mTOR and raptorin living cells for the first time.
Keywords: FLIM, FRET, mTOR,GFP, Raptor, Rheb
Background whilst mTORC2 contains mTOR, rictor (rapamycin-
The mammalian target of rapamycin (mTOR) signalling insensitive companion of mTOR), mLST8, mSin1 and
pathway has a key role in cellular regulation and is in- protor [6-9], raptor and rictor being specific components
volved in multiple diseases. mTOR is a central regulator ofmTORC1andmTORC2respectively.
ofcellgrowth,aging,ribosomebiogenesis,proteinsynthe- Rheb (Ras homologue enriched in brain) is a small
sis, actin-cytoskeletal organization, autophagy and meta- GTP-bindingproteinthathasbeenshowntopromotecell
bolism.Italsoplaysavitalroleincouplingcellgrowthvia growth and control cell size in mammalian cells and also
signalling pathways, according to the availability of nu- inDrosophilamelangaster[10],isakeyproteinthatrelays
trientsandcellularenergysuppliesandoxygen[1].mTOR upstream signals to regulate mTORC1. The involvement
forms two distinct heteromeric complexes, mTORC1 and of Rheb in these important complexes is still unclear.
mTORC2. mTORC1 contains mTOR, raptor (regulatory However, Rheb is reported to bind directly to the amino
associated protein of mTOR), mLST8 and PRAS40 [2-5], terminal lobe of the mTOR catalytic domain and to acti-
vate mTOR kinase in a GTP/GDP-dependent manner
[11] in cell lysate studies, although a direct interaction is
*Correspondence:[email protected];[email protected] difficult to prove using this approach. Furthermore, evi-
1CentralLaserFacility,STFC,RutherfordAppletonLaboratory,Research
dence using the pull-down assay approach suggests Rheb
ComplexatHarwell,Didcot,OxonOX110QX,UK
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Yadavetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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associates with mLST8 and with raptor [11,12]. Both been made possible through imaging approaches. The
mTORC1 and mTORC2 complexes play key roles in sev- interactionofproteinsincellscanbefollowedbyutilizing
eral pathways that are involved in human cancers and in steady state FRET between protein pairs tagged with
otherimportantdiseases,makingthedevelopmentofinhi- appropriate GFP-fluorophores, such as enhanced GFP
bitors of these pathways a high priority for the pharma- (EGFP, enhanced green fluorescent protein) and mono-
ceutical/ biotechnologyindustries. meric red fluorescent protein (DsRed). With steady state
It has been reported that Rheb–TSC2 GAP activity may FRET the donor fluorophore, in this case GFP, is excited
stimulate mTOR phosphorylation and while Rheb is con- andwouldfluorescebutinsteadtheexcitedstateenergyis
sidereda“component”ofthemTORsignallingcomplex,as re-absorbed by an acceptor fluorophore, in this case
yet there is no convincing evidence of a direct interaction DsRed,whichitselfthenfluorescesatalongerwavelength,
reportedbetweenRhebandmTOR.Itisalsopossiblethat provided the two fluorophores, and by inference the pro-
RhebmaybindtoandactivatemTOR-interactingproteins teinstowhichtheyareattached,areclose(within~7nm).
such as rictor, raptor or mLST8 rather than interacting Monitoring steady state FRET intensities is notoriously
withandactivatingmTORdirectly[1]. difficult and requires difficult corrections etc. A better
Raptor interacts with mTOR to form a nutrient- method is to monitor the decreased donor lifetime which
sensitivecomplexthatsignalstothecellgrowthmachinery is independent of the problems associated with steady
[2,3]. It has also been reported that the stability of the state intensity measurements and is evidence for a direct
mTOR-raptor complex increased when cells were starved physical interaction. We have previously shown that a re-
of amino acids or energy generating materials [3]. How- duction of as little as ~200 psin the excited state lifetime
ever,otherstudies[2]obtainednoevidenceforchangesin oftheGFPlabelledproteinrepresentsquenchingthrough
mTOR-raptor complex stability when cells were treated a protein-protein interaction [15-17]. In addition two-
with nutrient-rich and nutrient-poor conditions. The rea- photon-excitation fluorescence lifetime imaging micros-
sonforthediscrepancyintheobservationsbetweenthese copy (2P-FRET-FLIM) analysis as used here [15-17], pro-
two studies [2,3] is unclear since the former report [2] vides several advantages over the standard single photon
failed to demonstrate an impact of the nutrient status on method,includingreducedcellularcytotoxicityoftheexci-
the stability ofthe mTOR–raptor complex in mammalian tation light and reduced photobleaching of the fluoro-
cellsusingsimilarexperimentalconditions[3,13].Further- phore.Greatersensitivityoftheset-upisachievedthrough
more there is some evidence that raptor functions as a reduced sensitivityofthe excitation light (>900nm) by the
mTORscaffoldingprotein,thebindingtotheTORsignal- photomultiplier tube as a detector. Using the sensitive
ling(TOS)motifofmTORsubstratesbeingthoughttobe advancedimagingtechniqueoftime-correlatedsinglepho-
necessary for their effective mTOR-catalyzed phosphoryl- ton counting coupled with fluorescence lifetime imaging
ationinvivo[14].Hencethedynamicaspectsoftheinter- and molecular GFP fusion technology, it was possible for
action between mTOR, Rheb and raptor in living cells the first time to probe directly the nature of the inter-
requires new approaches for the detection of interaction, action between Rheb, mTOR and raptor as well as raptor
such as provided by the fluorescence resonance energy and mTOR whilst providing new insights into their sub-
transfer – fluorescence lifetime imaging (FRET-FLIM) cellularlocalization.
techniqueforobservationofappropriatelylabelledmateri- While the GFP-expression approach is excellent for
alsinanactiveenvironment[15-18]. providing evidence of potential interaction of proteins in
Several proteins involved in the mTOR signalling path- living cells, since it is an over-expression, one cannotex-
way, including phosphoinositide 3-kinase (PI3K) [19], clude the possibility that the labelled proteins may not
PDK1 [20], Akt [21], phosphatase and tensin homolog interact at their lower natural endogenous levels. Thus
(PTEN)[22],tuberin[23],andp70S6KanditssubstrateS6 there will always be some uncertainty in the approach.
[24]havebeenfoundtolocalizeinboththecytoplasmand However, alternative methods for assessing interactions
the nucleus. In addition, mTOR, but not raptor, has been are more limiting as they all involve pull-down assays
reported to shuttle between the cytoplasm and nucleus after solubilisation of the whole cell or cell fractionation
[25-27].Bycontrast,anuclearlocalizationofRhebhasnot which destroys localization information and itself intro-
beenreported. ducesinteractionartefactsasitmixesorganellecontents.
Fluorescence lifetime imaging utilizing confocal single For these reasons the GFP approach is widely used and
and multiphoton excited state emission microscopy is the best method available for assessing potential inter-
together with time-correlated single photon counting actions of cell components as long as one is aware of its
(TCSPC) provides an unambiguous determination of the limitations.
location of fluorophores. With the discovery of green In this study, we report on the significant nuclear
fluorescent protein (GFP) technology, significant new in- localizationofRhebtogetherwithhighlevelsofaconstant
formation on the inner workings of the living cell has pool of mTOR (but not raptor) in HEK293, CHO and
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HeLacellsusingadvancedreal-timemicroscopy.Interest-
ingly, raptor was absent from the cell nucleus under all
conditions investigated. A constant and direct interaction
was observed between Rheb-mTOR and raptor-mTOR,
butnotbetweenRhebandraptorinthecellcytoplasm.
Results
Rheblocalizestobothcytoplasmicandnuclearregionsin
mammaliancells
The functionality of EGFP-Rheb was first examined by
Western blots using an antibody to the phosphorylated
form of S6-K1 as induced by mTORC1 signalling (see
Figure 1). S6-K1-phosphorylation (Row D, lane 2) was
increased due to EGFP-Rheb expression, which was
therefore functional. The data indicate that it is Rheb
that largely controls thelevelof S6-K phosphorylation. If
either of the mTOR or raptor had been non-functional
they would have reduced the level of S6-K phosphoryl-
Figure2EGFP-RhebandDsRed-Rhebexpressioninmammalian
ation, in fact it was increased by a small but significant
cells.A)HEK293cellsandB)HeLacellsweretransientlytransfected
amount by EGFP-mTOR and DsRed-raptor expression.
withEGFP-Rheb(leftpanel)andDsRed-Rhebvector(rightpanel).
Therefore EGFP-mTOR and DsRed-raptor were likely 24hfollowingtransfection,andthelivecellsanalyzedunderaNikon
alsofunctional. TE2000Uconfocalmicroscope.Bar8μm.
EGFP and DsRed tagged proteins were observed to be
efficientlyexpressedinseveralmammaliancelllinesunder
investigation. Figure 2 shows confocal images of the tran- three cell lines. Fluorescently tagged Rheb, raptor and
siently expressed fluorescent proteins fused with Rheb mTOR were confirmed by SDS-PAGE and gel electro-
protein in HEK293 and HeLa cells. The transfection ex- phoresisinthatthecorrectfluorescentproteinwastagged
pressionlevelsforthe celllinesusedinthisstudydiffered to mTOR, Rheb and raptor and that they were con-
somewhat, in that HEK293 cells showed more than 80% structed and expressed correctly (data not shown). The
transfection efficiency, compared to CHO and HeLa cells main localization of Rheb and mTOR appeared to be the
at~60%(CHOimagedatanotshown),likelyduetodiffer- perinuclearregionsintheGolgiandER.
ences in the optimal conditions for transfection for the To determine if EGFP-Rheb was present within the
cell nucleus, we obtained a 3D-multiphoton TCSPC
image Z-stack of HEK293 cells expressing the fluores-
cent protein (Figure 3A-K). Using this technique, it was
possible to observe the presence of EGFP-Rheb protein
throughout the cell with significant expression levels
withinbothcytoplasmicandnuclearregions.Takinginto
account the average fluorescence intensity of EGFP-
Rheb within the cytoplasmic/Golgi regions (52000 pho-
ton counts per second, Figure 3F red box), compared to
that within the nuclear region (26000 photon counts per
second, Figure 3F yellow box), we can estimate Rheb ex-
pression levels of ~40% within the cell nucleus and
Figure1FunctionalityofEGFP-Rheb,EGFP-mTORandDsRed- ~60% within the cytoplasm, the Golgi apparatus and ER
raptordeterminedbyS6-kinaseactivity.EGFP-Rheb,EGFP-mTOR together. In HEK293 cells expressing EGFP-Rheb a cyto-
andDsRed-raptorwereexpressedinHEKcellsandgelswererunand plasmicand nuclear localization was found (Figure 4). In
blottedwiththerespectiveantibodiestoRheb,mTOR,raptorandS6-
addition we performed immunohistofluorescent staining
kinase(thr389).TheexpressedlevelofEGFP-RhebisshowninRowA,
using an anti-Rheb antibody (Figure 4), although, as is
lanes2,5and7buttheantibodywasunabletopickupendogenous
levels.TheendogenouslevelsofmTORcanbeseeninRowB,lanes1, well known, Rheb antibodies are of somewhat low
2,4and7(withincreaseddensitywhenEGFP-mTORisexpressed quality, perinuclear and nuclear staining is apparent.
(lanes3,5and6,highMWtoocloseforlargemigrationdifference). Co-localization studies using a Golgi fluorescent
TheendogenouslevelsofraptorcanbeseeninRowC,lanes1,2,3,
probe (BODIPY Texas Red ceramide, Invitrogen) and a
and5,withlanes4,6and7showingDsRed-raptorexpression.
probe for the endoplasmic reticulum (ER-Tracker Red;
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Figure33DStackimagesofmultiphoton-inducedfluorescenceofHEK293expressingEGFP-Rheb.FluorescencefromTCSPCimagesof
liveHEK293cellswasacquiredusingmultiphotonexcitation(920nmlaserexcitation,520nmemission)following24htransfection.Rawdata
presentedwithoutfurtherimageprocessing.ImagesshowclearRhebnuclearlocalization.Yellowandredboxes(F)refertonuclearandER/Golgi
regionstakenforcomparativephotoncount.Imagesize120x100μm.
glibenclamide BODIPY TR, Invitrogen) revealed that
EGFP-RheblocalizedtoGolgiandER(see Figures 5,6).
mTORshowssomenuclearbutmainlyERandGolgi
localizationinmammaliancells
The localization of mTOR in HEK293, HeLa and CHO
cells was studied by transient transfection with EGFP-
tagged at either end of mTOR. Cells were examined for
expressionfollowing24hand48hoftransfection.HEK293
and HeLa cells showed mTOR expression mainly in the
cytoplasmwithsomedetectablebutweakpresencewithin
thecellnucleusasshowninFigure7.
Using the TCSPC technique, we observed the average
Figure4ImmunohistochemicalstainingofRheb.HEK293cells percentage count in three independent experiments to be
expressingEGFP-Rheb(LEFTPANEL)orcellsfixedin4% ~20%EGFP-mTORinthenucleusand~80%withincyto-
formaldehydeandtreatedwithanti-Rhebantibodyinconjugation plasmic regions of HEK293 cells (error of ± 6%, SD in
withTexasred-labelledsecondaryantibody(RIGHTPANEL)were
both regions) (data not shown) (note that this quantifica-
imagedwithconfocalmicroscopy.Bar10μm.
tion information is lost within standard confocal laser
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Figure5Sub-cellularlocalizationofRhebonER.A)HEK293cells;B)HeLacellsweretransientlytransfectedwithEGFP-Rheb.Twenty-four
hoursfollowingtransfectioncellswerestainedwith1μMERTrackerredandlivecellsanalyzedbyconfocalmicroscopy.Theimagesrevealthat
RheblocalizestotheERBar8μm.
scanning microscopy images). The maximum nuclear ex- HeLa (Figure 8C) and CHO cells (Figure 8D) showed a
pressionofEGFP-mTORobservedinpopulationsofHeLa similar pattern ofexpression.
cellwas~32%(±8%)whileinCHOcellsthenumberwas
~26%(±6%inbothregions)(datanotshown). EGFP-mTORshowsadirectinteractionwithDsRed-Rheb
thatisunaffectedbyrapamycin
ToinvestigatemTORbinding-partnerinteractions,transi-
Raptorexpressionisobservedonlyinthecytoplasm ently expressing EGFP-mTOR in HEK293 and HeLa cells
The localization of raptor was studied by transiently were first obtained and analyzed using FLIM, to obtain
transfecting HEK293, HeLa and CHO cells with a vector the control excited state lifetime (donor chromophore)
for DsRed-raptor. Figures 8A, B show that in HEK293 48h after transfection (Figure 9). A lifetime image of
cells DsRed-raptor was predominantly within the cyto- HEK293 cells transfected with EGFP-mTOR for 48h is
plasm, at 24h and 48h after transfection in punctate shown in Figure 9, with the corresponding FLIM image.
structures. Transient transfection of DsRed-raptor in In three independent experiments the lifetime of EGFP-
Figure6Sub-cellularlocalizationofRhebonGolgi.A)HEK293cells;B)HeLacellsweretransientlytransfectedwithEGFP-Rheb.Twenty-four
hoursaftertransfectioncellswerestainedwith5μMBODIPYTRC5ceramideandlivecellsanalyzedbyconfocalmicroscopy.Theimagesreveal
thatRheblocalizestotheGolgi.Bar8μm.
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mTOR averaged for all of the cells was found to be 2400
±100ps.EGFP-mTORlocatedwithinthenucleusshowed
asimilarlifetimetothatinthecytoplasm.
HEK293 cells co-expressing EGFP-mTOR with DsRed-
Rheb over a 48h of transfection is shown in Figure 10A.
The average excited state lifetime of the EGFP-mTOR in
thepresenceofDsRed-Rhebwasdeterminedtobe2200±
50psinthreeindependentexperiments,thereducedlevel
duetoadirectinteractionbetweenRhebandmTOR,due
to energy transfer from EGFP to DsRed. In HeLa cells, a
similarquenchinginthelifetimeofEGFP-mTOR(2100±
100ps)wasobservedinco-transfectedcellsagainshowing
adirectinteractionandevidenceforadirectmTOR/Rheb
interaction in HEK293 cells was observable whether the
EGFP was at the N- or C- termini of mTOR (data not
shown).
Analysis of the average lifetime of the EGFP-mTOR
fromthenuclearregionsinHEK293cellswasdetermined
to be ~2000 ± 100 ps in three independent experiments.
HeLa cells showed similar expression in the nuclear
Figure7EGFP-mTORexpressespredominantlyinthe regions of EGFP-mTOR and DsRed-Rheb co-transfected
cytoplasmofmammaliancells.EGFPtaggedmTORexpression cells (data not shown). Quenching in the lifetime of the
wasstudiedinHEK293,HeLaandCHOcells.Thecellswere
donor (EGFP-mTOR) from 2400 ± 100 ps to 2000 ± 100
transientlytransfectedwithEGFPtaggedmTORandexpressionwas
ps in the nuclear regions indicates that EGFP-mTOR and
analyzedinlivecellsfollowing48hoftransfectionusingaNikon
TE2000UinvertedmicroscopeandEC1confocalsystem.(EGFP- DsRed-Rhebinteract(duetoexcitedstateenergytransfer)
mTOR;EGFPtaggedattheN-terminalofmTORandmTOR-EGFP; differently (i.e. they are closer) in the cell nucleus than in
EGFPtaggedattheC-terminalofmTOR).Bar8μm. the cytoplasm, likely brought about by a different con-
formation of the mTOR/Rheb complex. Importantly, in
this study EGFP-mTOR and DsRed-Rheb were consist-
entlyobservedtobeexpressedatsimilarlevelsinHEK293
cells (~40%), whilst these levels were different when
expressed alone (~30% for mTOR alone but ~40% for
cellsalsoexpressingRheb).
The effect of rapamycin on the direct interaction of
EGFP-mTORandDsRed-RhebexpressedinHEK293cells
wasdeterminedbya24htreatment(100nM).TheEGFP
lifetime(2200±100ps, dataofthreeindependentexperi-
ments) wasunaffected bythe treatment showingrapamy-
cinhadnoimpactontheinteraction(seeFigure10B).
Although the interaction of Rheb with mTOR has been
widely inferred by indirect approaches this is the first live
cell imaging work showing the interaction occurs and
does so in specific regions of the cell. Although it suffers
from the disadvantage that we are looking at over-
expression it enables us to examine the mechanisms and
consequencesoftheinteractioninalivingcell.
RaptorinteractsdirectlywithmTORbutnotwithRheb
andthemTOR-raptorinteractionisunaffectedby
Figure8Sub-cellularlocalizationoftransientlytransfected
DsRed-raptorindifferentmammaliancelltypes.DsRedtagged rapamycin
raptorwasexpressedinHEK293cellsforA)24handB)48h,HeLa EGFP was tagged to either end of mTOR and DsRed was
cellsfor48h,C)andCHOcellsfor48h,D).Livecellswereanalyzed taggedtotheN-terminalofraptorandlifetimesmeasure-
forvarioustimesfollowingtransfectionusingaNikonTE2000U
ments of GFP observed. The excited state lifetime of the
confocalmicroscope.Bar8μm.
donor EGFP-mTOR was determined in HEK293 cells co-
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Figure9FluorescencelifetimeimagingofEGFP-mTORexpressedinHEK293cells.A)ConfocalimageofEGFP-mTORexpressedinHEK293
cellsfollowing48hoftransfection;B)Lifetimeimageofthesamecells(colourcodingforthelifetimesisshownin(C)),imagesize120x110μm;
C)Lifetimedistributiontakenfortheentirearea(whiteborder)shownin(B).Thedatacollectiontimeforlifetimeimageswereoptimally3
accumulationsof30sec.withanimagedimensionof128×128pixels(thebluecrosslinesareforsinglepixellifetimevalueswhichwerenot
used).Afluorescencelifetimevaluewasdeterminedfromthedistributioncentrein(C),representingavaluefortheentireareashownin(B)and
was~2450±100ps,takenfromthreeindependentexperiments.
expressing DsRed-raptor. The average lifetime of the EGFP-mTOR and DsRed-raptor directly interact under
whole area of the image was 2300 ± 100 ps (Figure 11A). normal growth conditions within the perinuclear regions,
The corresponding confocal images of the co-expressed asindicatedbyGolgiandERconfocalco-localizationdata.
cells excited separately at 488 nm for EGFP-mTOR and SimilarresultswerealsoobservedinHeLaandCHOcells
543 nm for DsRed-raptor, respectively, as shown in (data not shown). Analysis of cells co-expressing EGFP-
Figure 11A, shows high expression levels (>70% of cells) Rheb (donor) and DsRed-Raptor (acceptor) in HEK293
of both proteins. The quenched lifetime of the donor and HeLa cells gave an unquenched excited state average
fluorophore in co-transfected cells suggests that the lifetimeofthedonorof2400±100psindicatingalackof
Figure10DsRed-RhebdirectinteractionwithEGFP-mTORinHEK293cellsandlackofeffectofrapamycin.(A,B)LEFTPANELS:Confocal
488nmexcitationimagesofEGFP-mTOR(co-expressedwithDsRed-Rheb,after48hoftransfection)and543nmexcitationimagesofDsRed-
Rheb,without(A)andwith(B)rapamycin(100nM)treatmentfor24h.(A,B)MIDDLEPANELS:Lifetimeimagesoftheco-transfectedcells(colour
codingforthelifetimesisshownintheRIGHTPANELS).(A,B)RIGHTPANELSThelifetimedistributionstakenfortheentirearea(whiteborder)as
shownintheMIDDLEPANELS.Thelifetimevaluefromthedatain(A)RIGHTPANELfortheEGFP(attachedtomTOR)isreduceddueby
quenchingbyDsRed(attachedtoRheb)from~2450±100psforEGFPalone(seeFigure9)to2200±100ps(n=3)here,thusshowingadirect
interaction.Thedatain(B)RIGHTPANELforthelifetimeoftheEGFP(attachedtomTOR),aftertreatmentwithrapamycintherewasnoreliefof
thequenchingindicatingitdoesnotimpactonthedirectinteraction.Thedatacollectiontimeforlifetimeimageswasoptimally3accumulations
of30sec.withanimagedimensionof128×128pixels,bar8μm.
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Figure11EGFP-mTORdirectinteractionwithDsRed-raptorinHEK293cellsandlackofeffectofrapamycin.(A,B)LEFTPANELS:Confocal
488nmexcitationimagesofEGFP-mTOR(co-expressedwithDsRed-raptor,after48hoftransfection)and543nmexcitationimagesofDsRed-
raptor,without(A)andwith(B)rapamycin(100nM)treatmentfor24h.(A,B)MIDDLEPANELS:Lifetimeimagesoftheco-transfectedcells
(colourcodingforthelifetimesisshownintheRIGHTPANELS).(A,B)RIGHTPANELSThelifetimedistributionstakenforthetheregiontaken
hereiswithintheareaofthethinredline(nottheentireareaaspreviousFigures)asshownintheMIDDLEPANELS.Thelifetimevaluefromthe
datain(A)RIGHTPANELfortheEGFP(attachedtomTOR)isreducedduetoquenchingbyDsRed(attachedtoraptor)from~2450±100psfor
EGFPalone(seeFigure9)to2300±100ps(n=3)here,thusshowingadirectinteraction.Thedatain(B)RIGHTPANELforthelifetimeofthe
EGFP(attachedtomTOR)forthecelldemarkedbythethinredline(thecellbelowbeingomittedasitisnotexpressingDsRed-raptor,seeLEFT
PANEL),aftertreatmentwithrapamycintherewasnoeffectonthelifetimecentre(2300ps±100ps).Thedatacollectiontimeforlifetimeimages
wasoptimally3accumulationsof30sec.withanimagedimensionof128×128pixels,bar8μm.
Figure12Aminoacidstarvationandre-stimulationdoesnotaffectthedirectinteractionbetweenEGFP-mTORandDsRed-Rhebin
perinuclearregions.(A-C)LEFTPANELS:Confocal488nmexcitationimagesofEGFP-mTOR(co-expressedwithDsRed-Rheb,after48hof
transfection)and543nmexcitationimagesofDsRed-Rheb,forHEK293cellsserum-starvedovernightandthenaminoacidstarvedinD-PBSfor1
h(A)followingwhichtheaminoacidswereaddedback(B,C).Punctatestructuresappearafteraminoacidremovalanddisappearafteradding
backtheaminoacidandappearnottocontainRheb.A-C)MIDDLEPANELS,Lifetimeimagesoftheco-transfectedcells(colourcodingforthe
lifetimesisshownintheRIGHTPANELS).(A-C)RIGHTPANELSThelifetimedistributionstakenforthecellarea(withinredline)asshowninthe
MIDDLEPANELS.A,revealingbothquenchedandunquenchedEGFPreflectingthemixtureofperinuclearmTOR/RhebandpunctatemTOR),as
reflectedinthelifetimeimage;B)thesamecellsafter10minofaminoacidre-stimulation;C)after60minutesofaminoacidre-stimulationwhen
thepunctatestructureshaddisappeared.Atalltime-pointstheareamarkedbythewhitecircle,mainlyER/Golgi,showsaquenchedlifetimei.e.
RhebinteractingwithmTOR.Bar8μm.
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a direct interaction between Rheb and raptor (data not rapidly after addition of serum (FCS) alone (data not
shown). shown). Also amino acid starvation and re-stimulation in
HEK293 cells were co-transfected with EGFP-mTOR HEK293 cells led to a distinct region of interaction, indi-
and DsRed-raptor, and 24h later were treated with rapa- cated by different levels of EGFP-lifetime quenching, to
mycin at 2h or 24h. There was no effect on the EGFP- ~2100psintheperinuclearregionandto~2400psinthe
mTOR(quenched)excitedstatelifetime(~2300±100ps) punctuate structures. Again the results indicate that Rheb
indicatingthattheinteractionwasnotaffectedbyrapamy- andmTORmayinteractdifferentlyinthetworegions.Itis
cin treatment (Figure 11B). Although there may be mar- important to note that the EGFP-Rheb subcellular
ginal unquenched EGFP (shoulder at 2400–2500 ps, localizationanddistributionisunaffectedbythistreatment
Figure 11B RIGHT PANEL), however, because the
mTOR-raptor interaction is not as close as Rheb-mTOR
(quenchedEGFPlifetime2300psvs2200ps,respectively) Aminoacidstarvationdoesnotaffectthelocalizationor
itis difficulttobe certain if thisindicatesmarginal effects thedirectinteractionofmTORwithraptor
ofRapamycin. The mTOR-raptor interaction was also observed in
small punctate-like structures in the cytoplasm as for
Aminoacidstarvationmodifiesthelocalizationandthe Rheb in the perinuclearregion, under amino acid starva-
natureofthedirectinteractionofmTORwithRheb tion conditions (Figure 13). Under conditions of amino
Under conditions of amino acid starvation, punctate acid starvation and subsequent re-addition showed no
structures or granules were observed for EGFP-mTOR significant change in the EGFP-mTOR lifetime (2200 ±
(Figure 12A). When amino acids were added back the 100 ps) (Figure 13) and therefore we conclude the inter-
punctuate structures gradually diminished and had dis- action between EGFP-mTOR and DsRed-raptor remains
appeared by ~60 mins (see Figure 12C), or much more unchanged. Also the localization and distribution of
Figure13Aminoacidstarvationandre-stimulationeffectonthedirectinteractionbetweenEGFP-mTORandDsRed-raptor.(A-C)LEFT
PANELS:Confocal488nmexcitationimagesofEGFP-mTOR(co-expressedwithDsRed-raptor,after48hoftransfection)and543nmexcitation
imagesofDsRed-raptor,forHEK293cellsserum-starvedovernightandthenaminoacidstarvedinD-PBSfor1h(A)followingwhichtheamino
acidswereaddedback(B,C).Punctatestructuresappearafteraminoacidremovalanddisappearafteraddingbacktheaminoacid.(A-C)
MIDDLEPANELS,Lifetimeimagesoftheco-transfectedcells.Thecolourcodingforthelifetimeisshownintherightpanelwhichisalifetime
distributionoftheareaofEGFP-mTORandDsRed-raptorco-expressingcells(areamarkedbyredlineinthemiddlepanel)(A-C)RIGHTPANELS
Thelifetimedistributionstakenforthecellarea(withinredline)asshownintheMIDDLEPANELS.A,revealingquenchedEGFPreflectingadirect
interactionofmTOR/raptorbothinthepunctatestructuresandotherregions.NotethatthelifetimeofEGFPmTORisslightlyreduced,toabout
2350ps,possiblyindicatingthatinthepunctatestructurestheremaystillbesomemTOR-raptorinteractionbut'looser'underaminoacid
starvationconditions(compareFigure11).(AcellswithonlyEGFP-mTORexpressionisshownforcomparison,whitecircle).Afteraminoacidre-
stimulation(B,C)thepunctatestructuresdisappearandthequenchedlifetimefortheEGFPreturnsto<2300PS.
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DsRed-raptor is unaffected by the amino acid starvation Ithasalsobeenreportedbyusingtimedimagingoflive
treatment. cellsthat,followingbriefassociation withER, EGFP-Rheb
localizes to highly-ordered distinct structures within the
cytoplasm that display the characteristics of Golgi mem-
Discussion branes [28]. Rheb was also reported to be localized with
Thisstudyisthefirsttoshowadirectinteractionbetween mitochondria[31],alongwithFKBP38andmTOR[32].It
Rheb and mTOR in specific regions of living cells. Al- has also been shown that GFP-tagged Rheb co-localizes
though previous studies have suggested an interaction with Rab7, a marker for endosomal and lysosomal struc-
using pull-down approaches [11], the ability to be able to tures [33]. Our studies did not reveal a co-localization of
study localization and interaction on a (live) cell-by-cell EGFP-RhebwithMitotracker(datanotshown)inkeeping
basis is important when studying a signalling complex with observations that ectopically expressed Rheb does
which may play differing roles simultaneously in different not localize to mitochondria [30]. Previously it has been
parts of the cell, as appears to be the case with the reported that EGFP-tagged Rheb showed a granule-like
mTORC1 complex. The FRET-FLIM approach offered fluorescence pattern in the cytoplasm, while not being
two other advantages. It was able to show a direct inter- foundattheplasmamembraneorinthenucleus,andthat
actionof bothRhebandraptorwithmTOR,alsodifferent it has mainly an endomembrane localization [34]. How-
conformations of the interacting components could be ever, several upstream regulators of mTOR have been
distinguished by this technique, such information being reported to be expressed in the nucleus as well as in the
unavailableusingapull-downassayapproach. cytoplasmandmTORplaysanimportantroleintheregu-
BothEGFP-and DsRed-taggedRhebappearedtolocate lationoftranslation.Evidenceforanuclearlocalizationof
inperinuclearregionsintheGolgiandER,withnodiffer- mTOR has also been provided previously based on data
ence in the expression levels in transfected cells. Previous from immunofluorescence in conjunction with cell frac-
studies of GFP-tagged Rheb have also shown such peri- tionationandWesternblotanalysis[25-27].
nuclear accumulation [28-30]. The insensitive nature of Other components of the pathway such as tuberin,
confocalimageacquisition(i.e.,becauseofanaloguesignal Rheb and p70S6K also localize to the cytoplasm (see
thresholding) can make it difficult to set the threshold of Introduction). Indeed, tuberin, an upstream regulator of
standard confocal photomultiplier tubes without losing Rheb has been reported to localize in the cytoplasm and
weaker signals. Consequently, gain and background levels nucleus [23], similar to p70S6K, one of the major sub-
can lead to either signal loss or oversampling. The use of strates of mTORC1 [24]. In addition, mTOR was also
highly sensitive time-correlated single photon counting, found to shuttle between the cytoplasm and the nucleus,
such as TCSPC, allows fluorescence photons to be this being required to regulate the mitogenic stimulation
detected above any background level as well as the ability of p70S6K activation and 4E-BP1 phosphorylation [25].
toquantifythe numberofphotonsdetected.Thus, inthis This study provides supporting evidence for the nuclear
current study using TCSPC, we report persistent and ob- localization of Rheb for the first time as well as mTOR
servable EGFP-Rheb presence within both cytoplasmic in live cells. The nuclear localization of mTOR is likely
andalsothesub-cellularnuclearregions,althoughthesig- an evolutionarily conserved phenomenon and may play
nal from the nucleus is somewhat weak and it may pos- an essential role in mTOR signalling and functioning as
siblybeduetothefactthatitisover-expressed.However, previously suggested [35]. With regard to raptor, previ-
regarding a nuclear Rheb presence it is important to note ous studies using immunofluorescence have shown that
(i) it hasbeensuggested that mTOR shuttlesbetween the raptor is localized within cytoplasm [33]. The live cell
nuclear region and cytoplasm [25-27], and (ii) that confocal imaging here also showed a cytoplasmic
mTORC1 plays a key role in ribosome biogenesis, a localization and absence from the nucleus. Furthermore
process which occurs in the nucleolus [1]. The presence lifetime data revealed that raptor interacts with mTOR
ofRhebwithinthecellularnucleussuggestsitmightinter- both in the cytoplasm and also within the amino-acid
act with mTOR in that compartment. Although the role starvation-induced punctate structures within the cyto-
for Rhebwithin the cell nucleus is unclear and whether it plasm(seebelow).
simply resides there or can indeed shuttle in and out is The involvement of Rheb in the mTORC1 complex
not known, however, it islikely tohave a rolein directing has remained an important point of interest since it was
mTOR localization and therefore downstream signalling shown to interact with mTOR due to the potential of its
within the nucleus. The interaction between Rheb and ability to control activity of the complex through its
mTOR observed is unlikely to be due to mis-localization GDP/GTP bound state [11,12,36]. Rheb is reported to
sincerandomlydistributedproteinmoleculeswillbesuffi- bind directly to the amino terminal lobe of the mTOR
ciently far apart (>>10 nm) to limit direct stable catalytic domain and to activate mTOR kinase in a
interaction. GTP/GDP-dependent manner in cell lysate studies.