buffer (250 mM NaCl, 20 mM HEPES-NaOH, pH 7.0) and deposited
onto cleaned coverslips. After a 5-min incubation, excess proteins were
washed out with observation buffer. For observation of data presented
in Fig. 4b and Supplementary Video 8, the coverslip was treated with
0.02% 3-aminopropyltriethoxysilane for 5 min and then washed in
MilliQ-water before deposition of the protein mixture.
HS-AFM imaging
HS-AFM images were acquired in tapping mode using a tip-scan type
HS-AFM instrument^38 (Nano Explorer PS-NEX, Research Institute of Bio-
molecule Metrology Co.) equipped with a fluorescence microscope. We
used cantilevers measuring ~9 μm long, ~2 μm wide and ~0.13 μm thick
with a resonant frequency of ~1.5 MHz and a spring constant of 0.1–0.2
N m−1 (BL-AC10DS, Olympus). Scaffold droplets were selected for obser-
vation by fluorescence imaging using SNAP-Surface 549 labelled to
Atg13–SNAP and Alexa Fluor 488 labelled to SNAP–Atg17 and located
to the HS-AFM scanning area (~4 × ~6 μm^2 ) before nanoscale imaging
with HS-AFM. HS-AFM imaging conditions were as follows: scan size,
500 × 250 nm^2 (Fig. 4a and Supplementary Video 7) or 1000 × 500 nm^2
(Fig. 4b and Supplementary Video 8); pixel size, 120 × 60 pixels; imaging
rate, ~3.1 frames per s (fps). All imaging was performed at 23 °C. IGOR
Pro (WaveMetrics) based software for HS-AFM was used to process the
image, using features such as Gaussian filtering, automatic flattening
and fast Fourier transform-frequency filtering^39.
Preparation of GUVs
The natural swelling method is used to prepare GUVs containing PEMCC
in a buffer from a dry lipid film^40. To prepare GUVs using electrically
neutral lipids in a buffer of normal physiological ion concentration
(~150 mM NaCl), we used a very small fraction of PEG-lipid (that is, the
PEG-lipid method)^40 ,^41. We prepared 200 μl of a 1 mM phospholipid
mixture consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho-
line (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
(POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-
maleimidomethyl)cyclohexane-carboxamide] (PE MCC), and 1,2-dipal-
mitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000 (DPPE-PEG2000) (all lipids were purchased from Avanti
Polar Lipids) at a molar ratio of 69:20:10:1 in chloroform in a 5-ml glass
vial, and then produced a homogeneous thin film of lipid mixture by
evaporation of chloroform using the gentle application of nitrogen
gas. For the complete removal of chloroform, we then placed the glass
vial in a vacuum desiccator connected to a rotary pump overnight.
The following day, we prehydrated the thin lipid film on the bottom
of the glass vial using 20 μl of water at 60 °C for 7 min. Thereafter 1 ml
of HEPES buffer (20 mM HEPES, pH 7.0, 150 mM NaCl and 1 mM EGTA)
containing 0.1 M sucrose was added and samples were incubated for
2–3 h at 60 °C.
Observation of GUVs
For dilution, 200 μl of GUV solution was added to 800 μl of HEPES
containing 0.1 M glucose solution (external solution) into a hand-made
microchamber that was formed on a glass slide (Matsunami Glass) by
depositing (in parallel) two bar-shaped silicon-rubber (3-mm silicon
sheet) spacers between a cover slip (Micro cover glass, Muto Pure
Chemicals) and the glass slide^42. The microchamber was coated with
0.10% (w/v) BSA prepared in the same buffer used in experiments to
avoid strong contact of GUVs with the glass surface. To increase the
contrast of the GUVs for DIC observation, the interior and exterior
of GUVs were filled with 0.1 M sucrose or 0.1 M glucose, respectively.
Observation of GUVs was performed using a FV3000RS as described
above at room temperature (~23 °C).
Single GUV method for observation of GUV–protein interactions
The single-giant unilamellar vesicle (GUV) method, by which proteins
can be precisely added one by one in the vicinity of a GUV, was used for
observation of GUV–protein interactions^43. Purified proteins in HEPES
buffer containing 0.1 M glucose were added slowly one by one into
the vicinity of a single GUV through a 12–15 μm diameter glass micro-
pipette, the position of which was controlled by a micromanipulator
(Narishige) at room temperature^42. The distance between the GUV
and the tip of the micropipette was maintained at ~50 μm. The glass
micropipette was prepared as follows: first we pulled a glass tube of
1.0-mm diameter to a needle point using a puller, and the needle point
was then microforged to the desired tip diameter (all equipment from
Narishige). Proteins in the external solution of GUVs were filled in the
micropipette by aspiration using a vacuum pump (ULVAC KIKO), and
then the micropipette was held by a micromanipulator, enabling us fine
control over tip positioning in the vicinity of the GUV. Protein applica-
tion pressure in the vicinity of the GUV was controlled by changing the
height of a vertical column of water to which the micropipette was
hydraulically connected^43. The application pressure was measured
using a differential pressure transducer (Validyne), pressure amplifier
(Karone), and a digital multimeter. For the constant application of pro-
teins in the vicinity of GUVs using the micropipette, we first determined
the equilibrium pressure by bringing the tip of the micropipette near to
a small vesicle and adjusting the pressure to keep the vesicle at the tip
of the micropipette. After fixing equilibrium pressures, an additional
~300 mV pressure was added to constantly apply protein solution in
the vicinity of GUVs.Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.Data availability
All relevant data are available from the authors. Source data for gels
and blots are provided as Supplementary Information. Source Data
for graphs are provided with the paper.- Adams, A., Gottschling, D. E., Kaiser, C. A. & Stearns, T. Methods in Yeast Genetics (Cold
Spring Harbor Laboratory Press, 1998). - Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat.
Methods 9 , 676–682 (2012). - Ries, J. & Schwille, P. Studying slow membrane dynamics with continuous wave scanning
fluorescence correlation spectroscopy. Biophys. J. 91 , 1915–1924 (2006). - Ries, J., Chiantia, S. & Schwille, P. Accurate determination of membrane dynamics with
line-scan FCS. Biophys. J. 96 , 1999–2008 (2009). - Kolaczyk, E. D. & Dixon, D. D. Non-parametric estimation of intensity maps using Haar
wavelets and Poisson noise characteristics. Astrophys. J. 534 , 490–505 (2000). - Kolaczyk, E. D. Non-parametric estimation of gamma-ray burst intensities using Haar
wavelets. Astrophys. J. 483 , 340–349 (1997). - Rossum, G. V. Python tutorial. Technical Report CS-R9526 (Centrum voor Wiskunde en
Informatica, 1995). - Magatti, D. & Ferri, F. Fast multi-tau real-time software correlator for dynamic light
scattering. Appl. Opt. 40 , 4011–4021 (2001). - Fukuda, S. et al. High-speed atomic force microscope combined with single-molecule
fluorescence microscope. Rev. Sci. Instrum. 84 , 073706 (2013). - Imamura, M. et al. Probing structural dynamics of an artificial protein cage using high-
speed atomic force microscopy. Nano Lett. 15 , 1331–1335 (2015). - Alam, J. M., Kobayashi, T. & Yamazaki, M. The single-giant unilamellar vesicle method
reveals lysenin-induced pore formation in lipid membranes containing sphingomyelin.
Biochemistry 51 , 5160–5172 (2012). - Alam, J. M. & Yamazaki, M. Spontaneous insertion of lipopolysaccharide into lipid
membranes from aqueous solution. Chem. Phys. Lipids 164 , 166–174 (2011). - Karal, M. A., Alam, J. M., Takahashi, T., Levadny, V. & Yamazaki, M. Stretch-activated pore
of the antimicrobial peptide, magainin 2. Langmuir 31 , 3391–3401 (2015). - Yamazaki, M. The single GUV method to reveal elementary processes of leakage of
internal contents from liposomes induced by antimicrobial substances. Adv. Planar Lipid
Bilayers Liposomes 7 , 121–142 (2008). - Noda, T., Matsuura, A., Wada, Y. & Ohsumi, Y. Novel system for monitoring autophagy in
the yeast Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 210 , 126–132
(1995).
Acknowledgements We thank H. Yamamoto, T. Kawamata, Y. Kamada and D. S. Goldfarb for
providing plasmids and strains for yeast experiments, Y. Ishii for assistance with protein
preparation and H. Tochio for critical advice. This work was supported in part by JSPS
KAKENHI grant number 25111004, 18H03989, 19H05707 (to N.N.N.), 15H01651, 17H05894,
17K07319 (to Y.F.), 19K16344 (to D.N.), 16H06375 (to Y. Ohsumi), 26119003, 17H06121 (to