supporting information - pnas · 2014. 11. 29. · supporting information jain et al....

Supporting InformationJain et al. 10.1073/pnas.1419425111SI Materials and MethodsAntibodies and Other Reagents. All antibodies used were obtainedfrom commercial sources as follows: anti-Flag and biotinylatedanti-Flag M2 from Sigma-Aldrich; anti-Myc (9E10.2) and anti-HA (16B12) from Covance or Abcam (ab26228); anti-GFP fromRoche and Rockland Immunochemicals; anti-DEPTOR fromNovus Biologicals; and rictor, raptor, mLST8, and mTOR anti-bodies from Cell Signaling Technology. Raptor and rictor anti-bodies for immunoprecipitation were from Bethyl Laboratories,and mTOR antibody (N-19) for immunoprecipitation was fromSanta Cruz Biotechnology. All secondary antibodies for Westernblotting and SiMPull were from Jackson ImunoResearch Labs.Rapamycin was from Calbiochem, EZview Red anti-HA beadsand anti-Flag M2 beads from Sigma, NeutrAvidin from Thermo-Fisher, dithiobis(succcinimidyl propionate) (DSP) was fromPierce,and BSA from New England Biolabs.

Plasmids. Plasmids pRK5–HA–raptor (Addgene plasmid 8513)(1), pRK5–HA–mLST8 (Addgene plasmid 1865) (2) and pRK5–myc–rictor (Addgene plasmid 1860) (2) were obtained fromAddgene. pCDNA–Flag–mTOR has been described previously(3). YFP–raptor was constructed by inserting monomeric eYFP(A206K) cDNA into pRK5–HA–raptor between the HA tag andraptor sequence. YFP–rictor was constructed by replacing theraptor cDNA in YFP–raptor with rictor cDNA from pRK5–myc–rictor. pCDNA–mSin1–HA was constructed by cutting mSin1.1–HA from pMSCV–mSin1.1–HA (Addgene plasmid 12582) (4)using BglII and EcoRI, and inserted into pCDNA3 cut withBamHI and EcoRI. YFP–mTORwas created by inserting mTORcDNA into the peYFP–C1 plasmid (Clontech). YFP–PRAS40was constructed by replacing the raptor cDNA in YFP–raptorwith PRAS40 cDNA from pRK5–HA–PRAS40 (Addgene plas-mid 15481) (5). YFP–DEPTOR was constructed by replacing theraptor cDNA in YFP–raptor with DEPTOR cDNA from pRK5–Flag–DEPTOR (Addgene plasmid 21334) (6). mCherry–raptorwas constructed by replacing YFP cDNA in YFP–raptor withmCherry cDNA from pmCherry–C1 (Clontech).

Cell Culture and Transfection.HEK293 cells were grown in DMEMcontaining 10% (vol/vol) FBS at 37 °C with 5% (vol/vol) CO2. Alltransfection experiments were performed when cells were 60–70% confluent in 6-cm plates. Transfection of plasmids wascarried out using PolyFect (Qiagen) following the manufacturer’srecommendations. One day after transfection, cells (3.5 × 106)were lysed in 500 μL of ice-cold lysis buffer (40 mM Hepes, pH7.5, 120 mM NaCl, 10 mM pyrophosphate, 10 mM β-glycer-ophosphate, 2 mM EDTA, 1× protease inhibitor mixture, 0.3%CHAPS; designated “CHAPS buffer”) and analyzed by variousassays. To establish the YFP–mTOR stable cell line, HEK293cells were transfected with the YFP–mTOR plasmid, plated atlow density for single-clone colonies to form, and selected in 500μg/mL of G418. A cell clone expressing YFP–mTOR at a levelcomparable to that of endogenous mTOR was chosen. Endoge-nous mTOR was then knocked down by lentivirus-deliveredshRNA (Addgene plasmid 1856) (7) as previously described (8),and selected by 1 μg/mL of puromycin. For insulin stimulation,cells were cultured in serum-free medium for 24 h, followed bytreatment with 100 nM insulin for 30 min. For glucose/glutamine,amino acids, or leucine stimulation, cells were cultured in glucose/glutamine-free medium for 12 h, amino-acid-free medium for 2 h,or leucine-free medium for 1 h, followed by restimulation with

glucose/glutamine for 1 h, amino acids for 30 min, or leucine for15 min, respectively.

Immunoprecipitation and in Vitro Kinase Assay. Cells were rinsedonce with ice-cold PBS and lysed in ice-cold CHAPS buffer. Thelysates were cleared by centrifugation at 10,000 × g for 10 min,and then subjected to immunoprecipitation at 4 °C with anti-HAbeads or anti-Flag M2 beads for 2 h. For GFP or mTOR im-munoprecipitation, anti-GFP or anti-mTOR (N-19) antibodywas incubated with the cell lysates for 2 h, followed by incubationwith protein G beads for 1 h. The beads were washed three timeswith lysis buffer and then boiled in 2× SDS sample buffer for 5min. Samples were analyzed by Western blotting. mTOR kinaseassays were performed as previously described (8). mTORC1kinase assays were carried out at 30 °C for 30 min in 25 mMHepes (pH 7.4), 50 mM KCl, 10 mM MgCl2 and 250 μM ATP,with 100 ng GST–S6K1 as the substrate. mTORC2 kinase assayswere carried out at 37 °C for 30 min in 25 mM Hepes (pH 7.4),100 mM potassium acetate, 1 mMMgCl2, and 500 μMATP, with250 ng His–Akt as the substrate.

Western Blot Analysis. Cells were lysed in either CHAPS buffer ora buffer containing 20 mM Tris·HCl, pH 7.5, 0.1 mM Na3VO4,25 mM NaF, 25 mM glycerophosphate, 2 mM EGTA, 2 mMEDTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 1×protease inhibitor mixture (Sigma-Aldrich), 0.3% Triton X-100.The lysates were cleared by centrifugation at 10,000 × g, thenmixed with 2× SDS sample buffer and boiled for 5 min. Proteinswere resolved on SDS/PAGE and transferred onto PVDFmembrane (Millipore), followed by incubation with various an-tibodies according to the manufacturer’s recommendations. De-tection of horseradish peroxidase-conjugated secondary antibodieswas performed with Western Lightning Chemiluminescence Re-agent Plus (Perkin-Elmer). Results were developed on X-ray filmsand scanned with an Epson scanner (Perfection 2400).

In-Cell Cross-Linking. Chemical cross-linking was performed asdescribed by Sancak et al. (9) Briefly, cells were grown in 6-cmplates and incubated with 3 mL of fresh culture media containing1 mg/mL DSP (2.5 mM) for 10 min at 37 °C, and 5% CO2. DSPwas prepared fresh as a stock solution of 250 mg/mL in DMSO.After the incubation time, DSP was quenched by adding Tris·HCl(pH 8.0) to a final concentration of 100 mM in fresh cell culturemedium. After an additional 10-min incubation at 37 °C, 5% CO2cells were washed on ice twice with cold PBS and lysed in 500 μLof lysis buffer (with CHAPS or Triton; see below). Cell lysateswere diluted 50-fold and immediately infused into chambers forSiMPull analysis.

Single-Molecule Imaging and Spot Counting. Cells (3.5 × 106)growing in 6-cm plates were lysed in ∼500 μL CHAPS buffer orTriton buffer (40 mM Hepes, pH 7.5, 120 mM NaCl, 10 mMpyrophosphate, 10 mM β-glycerophosphate, 2 mM EDTA, 1×protease inhibitor mixture, 0.3% Triton) to yield a final lysateconcentration of 3.5 mg/mL (Bradford assay). For cell mixingexperiments, cells were scraped in CHAPS buffer, mixed at 1:1ratio, and then incubated on ice for 15 min. Alternatively, cellswere trypsinized and resuspended in PBS, mixed at 1:1 ratio, andthen pelleted, followed by lysis in CHAPS buffer. Lysates werecleared by centrifugation at 10,000 × g for 10 min and diluted 20-to 100-fold (in most cases 50-fold) to obtain a surface densityoptimal for single-molecule analysis (∼300 molecules in 2,500μm2 imaging area). Dilutions were made either in detergent-free

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lysis buffer or 20 mM Tris, pH 8.0, 50 mM NaCl; similar resultswere obtained in both cases. A prism-type total internal reflectionfluorescence microscope was used to acquire single-moleculedata. Quartz slides were passivated with 200 mg/mL methoxypolyethylene glycol (PEG) containing 2.5% (wt/wt) biotinylatedPEG. YFP- and mCherry-tagged proteins were excited at 488 nmand 568 nm, respectively. Band pass filters (HQ 535/30, ChromaTechnology for YFP and BL 607/36, Semrock for mCherry) wereused to collect the emitted fluorescence signal. All experimentswere performed at room temperature (22–25 °C) unless speci-fied. Mean spot count per image (imaging area 2,500 μm2) andSD were calculated from images taken from 20 or more dif-ferent regions.

Photobleaching Analysis and Single-Molecule Colocalization. Single-molecule fluorescence time traces of surface immobilized YFP-tagged proteins weremanually scored for the number of bleachingsteps. To avoid false colocalization, samples were immobilized atan optimal surface density (∼300 molecules in 2,500 μm2 imagingarea) by adjusting the dilution factor for each lysate. The fluo-rescence trace of each molecule was classified as having one tofour bleaching steps or was discarded if no clean bleaching stepscould be identified. At least 400 molecules were evaluated for

each experiment; the total number of molecules successfullyscored as bleaching in one to four steps (N) is depicted in thefigures. The intensity of molecules scored as bleaching in oneand two steps was plotted to verify scoring: on average we expectthe fraction of molecules bleaching in two steps to be twice asbright as the molecules bleaching in one step. The intensity ofdiscarded molecules was also plotted to ensure unbiased scoringas observed via lack of enrichment of any specific intensities. Toconvert the photobleaching step distribution to monomer/dimerfraction, the percentage of molecules bleaching in two steps wascompared with the calibration experiment in Fig. 1I. This con-version was performed only when >90% of the moleculesbleached in one or two photobleaching steps. For single-moleculecolocalization, two separate images were acquired imaging YFPor mCherry in the same region of interest. Positions of YFP andmCherry molecules were determined to half-pixel accuracy byfitting a Gaussian point spread function. Molecules lying within1-pixel distance (pixel size, ∼150 nm) were said to be colocalized.For determining false colocalization by chance, two different re-gions were imaged and similar analysis was performed; colocali-zation by chance was observed to be ∼1%.

1. Kim DH, et al. (2002) mTOR interacts with raptor to form a nutrient-sensitive complexthat signals to the cell growth machinery. Cell 110(2):163–175.

2. Sarbassov DD, et al. (2004) Rictor, a novel binding partner of mTOR, defines a rapa-mycin-insensitive and raptor-independent pathway that regulates the cytoskeleton.Curr Biol 14(14):1296–1302.

3. Vilella-Bach M, Nuzzi P, Fang Y, Chen J (1999) The FKBP12-rapamycin-binding domainis required for FKBP12-rapamycin-associated protein kinase activity and G1 pro-gression. J Biol Chem 274(7):4266–4272.

4. Frias MA, et al. (2006) mSin1 is necessary for Akt/PKB phosphorylation, and its isoformsdefine three distinct mTORC2s. Curr Biol 16(18):1865–1870.

5. Vander Haar E, Lee SI, Bandhakavi S, Griffin TJ, Kim D-H (2007) Insulin signalling tomTOR mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol 9(3):316–323.

6. Peterson TR, et al. (2009) DEPTOR is an mTOR inhibitor frequently overexpressed inmultiple myeloma cells and required for their survival. Cell 137(5):873–886.

7. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM (2005) Phosphorylation and regulationof Akt/PKB by the rictor-mTOR complex. Science 307(5712):1098–1101.

8. Yoon MS, Du G, Backer JM, Frohman MA, Chen J (2011) Class III PI-3-kinase activatesphospholipase D in an amino acid-sensing mTORC1 pathway. J Cell Biol 195(3):435–447.

9. Sancak Y, et al. (2008) The Rag GTPases bind raptor and mediate amino acid signalingto mTORC1. Science 320(5882):1496–1501.



HA-RaptorYFP-mTOR

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Fig. S1. Characterization of fluorescent protein-tagged mTORC1 subunits. (A) YFP–mTOR stable cells were deprived of glucose/glutamine for 12 h, aminoacids for 2 h, or leucine for 1 h, and then restimulated by complete growth medium for 1 h (glucose/glutamine), 30 min (amino acids), or 15 min (leucine). (B)HA–raptor and YFP–mTOR were coexpressed in HEK293 cells and YFP immunoprecipitates from cell lysates were subjected to mTORC1 kinase assay using GST–S6K1 as substrate. (C) Empty vector, YFP–raptor, or mCherry–raptor was transfected into HEK293 cells. Cell lysates were subjected to HA immunoprecipitationfollowed by Western blotting. (D) HA–YFP–raptor and Flag–mTOR were coexpressed in HEK293 cells and HA immunoprecipitates from cell lysates weresubjected to mTORC1 kinase assay. (E) HA–PRAS40 or HA–YFP–PRAS40 were transiently expressed in HEK293 cells and cell lysates were subjected to HA im-munoprecipitation. Endogenous mTOR and raptor were detected by Western blotting. (F) Empty vector, HA–PRAS40, or HA–YFP–PRAS40 were coexpressedwith myc–S6K1 in HEK293 cells. pT389–S6K1, myc–S6K1, and PRAS40 were detected by Western blotting. (G) HA–DEPTOR or HA–YFP–DEPTOR were transientlyexpressed in HEK293 cells and cell lysates were subjected to HA immunoprecipitation. Endogenous rictor and raptor were detected by Western blotting.



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Fig. S2. Comparison of sensitivity of SiMPull and Western blotting (WB). (A and B) Comparison of SiMPull with WB. Lysate from YFP–mTOR cells was diluted asindicated. In SiMPull, YFP–mTOR was captured using an antibody against GFP. WB was performed using the same anti-GFP antibody or an antibody againstmTOR. (C–E) Comparison of SiMPull with coimmunoprecipitation. (C and D) Lysate from YFP–mTOR cell line was diluted as indicated and applied to SiMPullchambers coated with an antibody against raptor. (E) Conventional immunoprecipitation for raptor using various dilutions of YFP–mTOR expressing cells,followed by WB for YFP–mTOR and raptor.



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Fig. S3. mTORC1 is dimeric. Epitope-tagged mTORC1 components were transiently expressed and captured on single-molecule imaging chambers as depictedin schematics (A, C, and E, Left). (A and B) YFP–raptor and mTOR formed dimers when coexpressed. (C and D) PRAS40 when pulled down via mTOR was dimeric.(E and F) DEPTOR associated with mTORC1 was dimeric. (G) YFP–raptor and Flag–mTOR were cotransfected (CT), or transfected separately (raptor, mTOR). Formixed, lysates expressing mTOR and raptor separately were mixed at a 1:1 ratio. Expression levels in these lysates were analyzed by Western blotting. (H)SiMPull analysis of mTOR–raptor interaction in lysates in G. (I) Cells were transfected as in G and H. For mixed, cells were trypsinized and mixed at 1:1 ratio,followed by lysis. SiMPull analysis was performed as in H. Error bars depict SD of the mean across 20 or more imaging areas. (Scale bars, 5 μm.)



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Fig. S4. mTORC1 cross-linking assay. (A and B) YFP–mTOR stable cells were lysed in buffer containing 1% Triton X-100 or 0.3% CHAPS. For cross-linking, cellswere treated with DSP cross-linker before cell lysis. mTORC1 was pulled down using anti-raptor antibody. (C) Fluorescence photobleaching step distribution forYFP–mTOR in mTORC1, for cells lysed in 0.3% CHAPS without or with DSP. Error bars depict SD of the mean for more than 20 images. (Scale bar, 5 μm.)



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Fig. S5. mTORC2 oligomerization and incorporation of fluorescently labeled subunits. (A) Recombinant proteins were transiently expressed in HEK293 cells asindicated, followed by Flag or HA immunoprecipitation and Western blotting. (B) Recombinant proteins were transiently expressed in HEK293 cells as in-dicated, followed by Flag immunoprecipitation and Western blotting. (C) Recombinant proteins were transiently expressed in HEK293 cells as indicated,followed by GFP immunoprecipitation and in vitro mTORC2 kinase assays using Akt as a substrate. (D) Recombinant proteins were transiently expressed inHEK293 cells as indicated, followed by Flag immunoprecipitation and Western blotting. (E) Recombinant proteins were transiently expressed in HEK293 cells asindicated, followed by HA immunoprecipitation and in vitro mTORC2 kinase assay using Akt as a substrate.



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Fig. S6. mTORC2 is dimeric. (A) YFP–mTOR was coexpressed with mSin–HA, HA–mLST8, and Flag–rictor. mTORC2 complexes were captured on SiMPull usingFlag antibody. (B) Fluorescence photobleaching step distribution (Left) and intensity of scored molecules (Right) for YFP–mTOR in mTORC2. (C) YFP–DEPTORwas coexpressed with core mTORC2 components in HEK293 cells and was pulled down via Flag–rictor. (D) Fluorescence photobleaching step distribution (Left)and intensity of scored molecules (Right) for YFP–DEPTOR in mTORC2. (E) mTORC2 subunits were cotransfected (CT) or transfected separately (rictor/mSin andmTOR/mLST8) followed by lysate mixing at 1:1 ratio (mixed). Expression levels were analyzed by Western blotting. (F) mTORC2 from CT, mixed, and rictor/mSinlysates was capture using Flag antibody. (Upper) For mixed, lysates were mixed at a 1:1 ratio. (Lower) For mixed, cells were trypsinized and mixed at 1:1 ratio,followed by lysis. (G) YFP–mTOR stable cells were lysed in CHAPS buffer. Some cells were incubated with DSP cross-linker before cell lysis. mTORC2 as pulleddown using anti-rictor antibody. (H) Fluorescence photobleaching step distribution was analyzed for samples in G. (Scale bar, 5 μm.)



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Fig. S7. Two-color SiMPull for mTORC1 and mTORC2. (A) mCherry–raptor and YFP–rictor were coexpressed with Flag–mTOR, mSin–HA, and HA–mLST8. Flag–mTOR is captured using a surface-immobilized Flag antibody. Two different imaging areas were visualized for YFP and mCherry to estimate the overlap bychance. The observed overlap was ∼1% across 23 imaging areas over four independent experiments. (B) mCherry–raptor, YFP, Flag–mTOR, mSin–HA, and HA–mLST8 were coexpressed. Flag–mTOR is captured using a surface-immobilized Flag antibody. A background level of fluorescence was observed in the YFPchannel and these fluorescent spots did not colocalize with mCherry (0% overlap). (C) Fluorescence photobleaching step distribution for YFP–rictor from A. (D)Expression levels of mTORC components when mTORC1 and mTORC2 were expressed together, separately, or when lysates mixed at 1:1 ratio. (E, Top) Co-localization of mTORC1 and mTORC2 when cotransfected in the same cells. (Bottom) Colocalization of mTORC1 and mTORC2 when cell lysates are mixed. Oncapturing Flag–mTOR, YFP–rictor (Left), mCherry–raptor (Center), and overlapping spots (Right) were observed. (F) The box plot shows distribution of overlaypercentage from three independent experiments (n = 15 images) calculated after subtracting the false colocalization. Mean is shown as square marker andmedian as center line. (Scale bar, 5 μm.)



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es

IP Flag IgGIP Flag IgGY

FPFlag-Raptor

Anti-Flag

YFP-PRAS40

1 2 3 40

50

100

%of

mol

ecul

es

Number of steps0 400 800

0

50

100

Num

bero

fmol

ecul

es

Intensity (a.u.)

E F

Rap0

500

1000

-+

Anti-GST

YFP-mTOR

GST-FKBP12Rapamycin

Rap + -

YFP

0 400 8000

75

150

Num

bero

fmol

ecul

es

Intensity (a.u.)1 2 3 4

0

50

100

%of

mol

ecul

es

Number of steps

α-RaptorLeu

0

250

500

Num

bero

fmol

ecul

es

- ++ +

YFP

-mTO

R

Leu - +

IP: Raptor

1 2 3 40

20

40

60

%of

mol

ecul

es

Number of steps

N = 1154- Leu

1 2 3 40

20

40

60 N = 1034+ Leu

RaptorYFP-mTOR

Anti-Rabbit

Anti-Raptor

C D

Fig. S8. mTOR subcomplexes and effect of leucine stimulation. (A) YFP–PRAS40 and Flag–raptor were coexpressed and SiMPull was performed with Flagantibody. (B) YFP–PRAS40 bound to raptor was monomeric as indicated by photobleaching step distribution. (C) YFP–mTOR stable cells were starved (−) ofleucine (Leu) for 1 h followed by restimulation (+) with leucine for 15 min. mTORC1 was pulled down via endogenous raptor followed by SiMPull analysis. (D)mTORC1 dimeric stoichiometry did not change upon leucine stimulation. (E) Lysate from YFP–mTOR stable cells was applied to SiMPull chambers coated withpurified FKBP12 with or without rapamycin. YFP–mTOR was captured via FKBP12 in the presence of rapamycin. (F) YFP–mTOR bound to FKBP12–rapamycinwas monomeric as indicated by photobleaching step distribution. (Scale bars, 5 μm.)



IP: m

TOR

ControlIP: Goat IgG

0

175

350

Num

bero

fmol

ecul

es

Rap (nM) 0 2 10 100 Con

Total mTOR (IP: mTOR)Rapamycin (nM) 0 2 10 100

ApT389 S6K1

S6K1

4EBP1

Rapa (nM)2 10 100DMSO

pT37/46 4EBP1

pS65 4EBP1

BmTOR

mTOR

Raptor

Raptor

Rictor

RictorLysate

IP: mTOR

(hours) 0 2 24 Rapa 100nM

C

0

250

500

Num

bero

fmol

ecul

es

ConRap (h) 0 6

0

250

500

Num

bero

fmol

ecul

es

ConRap (h) 0 6 12 24

IP: m

TOR

Rap (h) Con0 6 12 24

ConRap (h) 0 6

IP: R

apto

r

mTORC1 (IP: Raptor)

Total mTOR (IP: mTOR)D

E

Fig. S9. Effect of rapamycin on mTOR complexes. (A) HEK293 cells were treated with 2, 10, or 100 nM rapamycin for 30 min. Cell lysates were analyzed byWestern blotting with the indicated antibodies. (B) Cells were treated with 100 nM rapamycin for 0, 2, and 24 h. Cell lysates were subjected to mTOR im-munoprecipitation and analyzed by Western blotting. (C) YFP–mTOR stable cells were treated with various doses of rapamycin for 30 min, and SiMPull wasperformed with an antibody against endogenous mTOR. (D) YFP–mTOR stable cells were treated with 100 nM rapamycin for various lengths of time, andSiMPull was performed with an antibody against endogenous mTOR. (E) Cells were treated as in D, and SiMPull was performed with an antibody againstendogenous raptor. Error bars in C–E represent SD of the mean across 20 or more imaging areas. (Scale bar, 5 μm.)


supporting information - pnas · 2014. 11. 29. · supporting information jain et al....

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