Molecular Cell, Volume 64

Supplemental Information

An Autophagic Flux Probe

that Releases an Internal Control

Takeshi Kaizuka, Hideaki Morishita, Yutaro Hama, Satoshi Tsukamoto, TakahideMatsui, Yuichiro Toyota, Akihiko Kodama, Tomoaki Ishihara, TohruMizushima, and Noboru Mizushima

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Supplemental Figure 1. Validation of the GFP-LC3-RFP-LC3ΔG probe (related to Figure 1)(A) Overexpression of GFP-LC3-RFP-LC3ΔG does not affect autophagic flux and mTORC1 activity. MEFs with and without stable expression of GFP-LC3-RFP-LC3ΔG were starved (depletion of both serum and amino acids) for 4 h in the presence or absence of 100 nM bafilomycin A1 and analyzed by immunoblotting.(B) Uncleavable GFP-LC3ΔG-RFP-LC3ΔG does not serve as an autophagic flux probe. MEFs stably express-ing GFP-LC3-RFP-LC3ΔG or GFP-LC3ΔG-RFP-LC3ΔG were starved as in Figure 1D. The same histogram (gray) of probe (-) wild-type cells is shown in both GFP-LC3-RFP-LC3ΔG and GFP-LC3ΔG-RFP-LC3ΔG panels for comparison. Data represent mean ± SEM (n = 3).

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Supplemental Figure 2. Measurement of autophagic flux upon treatment with lysosomal inhibitors (related to Figure 2)The effects of lysosomal protease inhibitors on the level of GFP-LC3-RFP-LC3ΔG probe. HeLa cells stably expressing GFP-LC3-RFP-LC3ΔG were cultured in starvation medium for 6, 12, or 24 h with E64d (50 µM), pepstatin A (50 µg/ml), or a combination of both inhibitors. Data represent mean ± SEM (n = 3).

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Supplemental Figure 3. Induction of autophagy in fertilized zebrafish eggs (related to Figure 4)(A) A schematic diagram of a fertilized zebrafish egg and GFP-LC3 signals in a GFP-LC3-RFP-LC3ΔG transgenic zebrafish egg at the indicated time (min post fertilization (mpf)). GFP-LC3 puncta appeared throughout the egg except for the yolk from approximately 12 mpf. The indicated regions are magnified in the insets. Scale bar, 10 µm and 1 µm (insets).(B) Representative fluorescence and DIC images of fertilized eggs of wild-type and GFP-LC3-RFP-LC3ΔG transgenic zebrafish treated with or without 500 nM wortmannin at 1 and 2 h post fertilization. The graph shows the GFP/RFP fluorescence ratio in the blastomeres as a percentage relative to that of wortmannin-treated eggs at 1 hpf. The indicated regions are shown and magnified in the insets. Scale bar, 5 µm. Data are representative of two independent experiments.

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Supplemental Figure 4. Expression of GFP-LC3-RFP-LC3ΔG in various tissues in transgenic mice (related to Figure 6)Tissue homogenates from one wild-type and two GFP-LC3-RFP-LC3ΔG transgenic mice were subjected to immunoblotting using indicated antibodies. GFP-LC3, RFP-LC3ΔG, and endogenous LC3 were detected using anti-LC3 antibody. The asterisk indicates a potential degradation product, which is not authentic GFP-LC3.

PFG

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Supplemental Figure 5. Comparison between tfLC3 (RFP-GFP-LC3) and GFP-LC3-RFP-LC3ΔG (related to Figure 7)(A–C) Wild-type MEFs stably expressing tfLC3 (RFP-GFP-LC3) or GFP-LC3-RFP-LC3ΔG were cultured in regular, serum starvation, or amino acid and serum starvation medium in the presence or absence of 100 nM bafilomycin A1 for 24 h, and then subjected to ratiometric fluorescence microscopy (A), flow cytometry (B), and immunoblot analysis (C). The indicated regions are magnified in the insets (A). Scale bars, 40 µm and 10 µm (insets).

The same histogram (gray) of probe (-) wild-type cells is shown in both tfLC3 and GFP

-LC3-RFP-LC3ΔG panels for comparison (B).

1.0

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Legends to Supplemental Movies

Supplemental Movie 1. Visualization of fertilization-induced formation of LC3 puncta in a zebrafish embryo (related to Figure 4)Time-lapse images of GFP fluorescence signals taken every 3 min in a GFP-LC3-RFP-LC3ΔG trans-genic zebrafish embryo after fertilization. A number of GFP-LC3 puncta appeared throughout the egg except for in the yolk in the central region. Data are representative of four independent experiments. See Figure S3 for selected frames.

Supplemental Movie 2. Visualization of autophagy flux after fertilization in mouse embryos (related to Figure 6)Time-lapse images of GFP and RFP fluorescence ratios taken every 30 min in embryos, which were infected with GFP-LC3-RFP-LC3ΔG mRNA at the one-cell stage.

1

EXTENDED EXPERIMENTAL PROCEDURES

Cell lines and transfection

Atg5-/- MEFs have been described previously (Kuma et al., 2004). Stable cell lines were generated using a

retroviral expression system as previously described (Itakura et al., 2012). Single clones of the cells

retrovirally transfected with GFP-LC3-RFP-LC3ΔG were isolated and proper expression of the probe was

confirmed by genomic PCR using primer I (5′- CGCCGCCGGGATCACTCTCG -3′) and Primer II (5′-

CCACCACACTGGGATCCTTA -3′), which amplified the 1,533 bp GFP-LC3-RFP-LC3ΔG fragment,

and immunoblotting or flow cytometry. Note that GFP-LC3ΔG caused by homologous recombination

results in amplification of a 480-bp fragment. Wild-type and Atg5-/- MEF clones expressing GFP-LC3-

RFP-LC3ΔG (#3–7 and #4–5, respectively), wild-type MEF clone expressing tfLC3 (#13), and HeLa cell

clone expressing GFP-LC3-RFP-LC3ΔG (#6) were used. For th e evaluation of GFP-LC3-RFP, bulk

transformants without clone isolation were used directly.

Antibodies and reagents

Rabbit polyclonal antibody against rat LC3B (NM1) has been described previously (Quy et al., 2013).

Antibodies against GFP (A-6455, Molecular Probes), RFP (R10367, Molecular Probes), p62 (PM045,

MBL), phospho-S6K1 (Thr389) (9206, Cell Signaling), cleaved caspase-3 (9664, Cell Signaling),

myosin

heavy chain type I (M8421, Sigma-Aldrich), myosin heavy chain type IIa (SC-71, Developmental Studies

Hybridoma Bank), HSP90 (610419, BD Biosciences), and β-actin (A2228, Sigma-Aldrich) were used.

Pepstatin A and E64d were purchased from Peptide Institute. Information about other drugs and reagents

are summarized in Table S1.

Immunoblotting

Cells were collected in ice-cold phosphate-buffered saline (PBS) by scraping, and precipitated by

centrifugation at 3,800 × g

for 1 min. Cells were then suspended in lysis buffer (50 mM Tris-HCl, pH 7.5,

150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and Complete EDTA-free protease inhibitor cocktail

(Roche)). Zebrafish embryos were homogenized with the same lysis buffer. The lysates were clarified by

centrifugation at 17,400 × g

for 15 min and boiled in sample buffer (46.7 mM Tris- HCl, pH 6.8, 5%

glycerol, 1.67% sodium dodecyl sulfate, 1.55% dithiothreitol, and 0.02% bromophenol blue). Samples

were separated by SDS-PAGE and transferred to Immobilon-P polyvinylidene difluoride membranes (

Millipore). Immunoblotting was performed with the indicated antibodies and the signals were visualized

with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) or Immobilon

Western Chemiluminescent HRP Substrate (Millipore). Signal intensities were analyzed with an LAS-

3000 mini imaging analyzer (FUJIFILM). Contrast and brightness adjustment was applied equally over

the whole image with Photoshop Elements 5.0 software (Adobe).

2

Flow cytometry

Cells were washed with PBS and harvested with 0.05% trypsin-EDTA at 37°C for 5 min (note that

prolonged trypsinization may cause loss of fluorescence). The dishes were placed on ice immediately

after trypsinization and the cells were transferred to siliconized 1.5 ml tubes (131-615CH WATSON) with

ice-cold PBS. The cells were centrifuged at 2,300 × g for 2 min, suspended in ice-cold PBS, and analyzed

with an EC800 cell analyzer (Sony) equipped with a 488-nm laser and 561-nm laser. At least 10,000

events for each sample were acquired. Data were processed with Kaluza software (Beckman Coulter).

Fluorescence microscopy

MEFs or HeLa cells were placed on a glass-bottomed dish (627870, Greiner) or chamber slide (80826,

Idibi). Cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) in PBS for 10 min.

Tissue samples were prepared as follows. Mice were anesthetized with avertin (i.p.) or isoflurane gas

(inhaled) and immediately perfused with 4% PFA in PBS. Frozen sections were prepared as previously

described (Mizushima et al., 2004). Cell samples, tissue samples, and zebrafish eggs were observed with

a fluorescence microscope (IX81; Olympus) equipped with a CCD camera (CoolSNAP HQ2,

Photometrix).

For live-imaging of zebrafish, embryos were anesthetized with 0.03% tricaine (Sigma-

Aldrich), placed in water on a glass-bottomed dish and imaged using a confocal microscope (FV1000,

Olympus) equipped with an objective lens (UPLSAPO30XS, Olympus). To visualize fluorescence in

mouse embryos, embryos microinjected with mRNA were placed in 1–2 µl Hepes-buffered potassium

simplex optimization medium (KSOM) under mineral oil in a glass-bottomed dish (Matsunami Glass)

and observed under an inverted microscope (Olympus IX70) equipped with a DP72 CCD camera

(Olympus) and 10x objective lens (UPlanFL NA 0.3, Olympus). Images were captured using

LuminaVision software (Mitani Corporation).

For live-cell imaging of mouse embryos, embryos were cultured in 5 µl KSOM supplemented

with amino acids under mineral oil (Fuso Pharmaceutical Industries) in a glass-bottomed dish, and

fluorescence images were captured at 30-min intervals using a real-time cultured cell monitoring system

(ASTEC Co., Ltd) with a 20x objective lens (Plan Fluor ELWD, NA0.45, Nikon). Internal temperature

was maintained at 37°C in 5% CO2 and 90% N2.

Immunohistochemistry of mouse muscles were performed as previously described (Mizushima

et al., 2004). After blocking with 5% normal goat serum and 0.5% Triton X-100 in PBS for 30 min,

sections were incubated with antibodies against myosin heavy chain type I or IIa at room temperature for

30 min, and then with Alexa Fluor 660-conjugated goat anti-mouse IgG or Alexa Fluor 647-conjugated

goat anti-mouse IgG1 secondary antibodies for 30 min. Analysis of fluorescence images and creation of

ratiometry images were performed using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of

3

Health, Bethesda) and MetaMorph (Molecular Devices). In zebrafish and mouse studies, mean intensities

of GFP and RFP signals in at least 125 μm2 of each tissue area were obtained using ImageJ. For the final

output, images were processed using Photoshop Elements 5.0 software.

Fluorescence measurement using a microplate reader

Cells were seeded in 96-well plates at 30,000 cells/well and grown overnight. After drug treatment, cells

were washed with PBS, fixed with 4% PFA in PBS for 10 min, and washed with 100 μl PBS.

Measurement of GFP and RFP fluorescence was performed using a microplate reader (Enspire,

PerkinElmer) with excitation/emission at 488/509 nm and 584/607 nm, respectively.

In vitro transcription and RNA microinjection

For in vitro mRNA synthesis, the pcDNA3-GFP-LC3-RFP-LC3ΔG plasmid was linearized with XhoI,

and capped mRNAs with poly(A) tails were synthesized using the mMESSAGE mMACHINE Ultra Kit

(Ambion) according to the manufacturer’s instructions. The synthesized mRNA was purified using a

MegaClear Kit (Ambion), and the mRNA concentration was determined using Qubit Assays (Thermo

Fisher Scientific) and e-spect (Malcom). Before microinjection, the mRNA was diluted in Tris-EDTA

buffer (pH 7.4) to 100 ng/µl and then filtered using Ultrafree-MC (Millipore). Microinjection was

performed as described above except that the mRNA was microinjected into the cytoplasm of metaphase

II (MII) oocytes or one-cell embryos.

Collection of oocytes and embryos and in vitro fertilization

MII oocytes were collected from superovulated female C57BL/6J mice (Japan SLC, Inc.) as previously

described (Tsukamoto et al., 2008). In vitro fertilization, embryo culture, and microinjection were

performed as previously described (Tsukamoto et al., 2014). In some experiments, MII oocytes were

treated with hyaluronidase (Sigma-Aldrich) to remove cumulus cells and used for microinjection.

Zebrafish

Wild-type RW (Riken, Japan) zebrafish were raised and maintained in 14-h light/10-h dark conditions at

28°C according to established protocols (Kimmel et al., 1995). Transgenic zebrafish were generated using

the Tol2 transposon system (Urasaki et al., 2006). Briefly, the GFP-LC3-RFP-LC3ΔG probe was inserted

into the pT2AL200R150G vector. Transposase mRNA was synthesized from pCS-TP using mMESSAGE

mMACHINE SP6 Transcription Kit (Ambion) and purified using RNeasy Mini Kit (Qiagen). Zebrafish

eggs were microinjected with 100 ng/µl of the Tol2 vector and TP mRNA at the one-cell stage using

FemtoJet (Eppendorf) equipped with Femtotip II (Eppendorf). The FIP200-/- zebrafish was produced by

deletion of exon 4 using the CRISPR/Cas9 system (Jao et al., 2013). The mutated allele harbored a 13-bp

deletion allele. Detailed description of the FIP200-/- zebrafish phenotype will be reported elsewhere.

4

Zebrafish embryos at 1 dpf were treated with 250 nM Torin1 (Wako), 500 nM wortmannin (Sigma), or 20

nM LysoTracker Red DND-99 (Thermo Fisher Scientific) by dipping into a solution containing each drug

(water as the vehicle).

Generation of transgenic mice

A 4.5-kb cDNA fragment was isolated from pCAGGS-GFP-LC3-RFP-LC3ΔG using SalI and Hind III,

purified using Wizard SV Gel and PCR Clean-Up System (Promega), and microinjected into the

pronucleus of one-cell embryos from C57BL/6J mice. The transgenic founders were confirmed by

genome PCR of EGFP using 5′-AAGTTCATCTGCACCACCG-3′ and 5′- TCCTTGAAGAAG

ATGGTGCG-3′. Sixteen mice were screened, of which five were positive for the transgene. One of the

transgenic lines, termed #2, showed the highest expression and was used in this study. All mouse

experiments were performed in accordance with the relevant guidelines and were approved by the Animal

Care and Use Committee of the University of Tokyo and the National Institute of Quantum and

Radiological Science and Technology.

5

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