In situ structural analysis reveals membrane shape transitions during autophagosome formation.

Yeast Strains. A list of budding yeast ( S. cerevisiae ) strains used in this study is provided in SI Appendix , Table S5 . All of the yeast strains were based on the DF5 background. Standard protocols for transformation, mating, sporulation, and tetrad dissection were used for yeast manipulations ( 53 ). Chromosomally tagged strains and knockout strains were constructed using a PCR-based integration strategy ( 54 ). Standard cloning techniques were used. Live-Cell Fluorescence Microscopy. A detailed protocol of the live-cell fluorescence imaging is available under https://www.protocols.io/view/yeast-cells-live-fluorescence-imaging-n92ldzqjnv5b/v1 . For fluorescence microscopy, yeast cells were grown in synthetic growth medium supplemented with all essential amino acids and 2% glucose. The next day, cells were diluted to optical density (OD600) 0.1 and grown until midlog phase (0.5–0.8 OD600) before imaging. Microscopy slides were pretreated with 1 mg mL −1 concanavalin A solution. Widefield imaging was performed at the Imaging Facility of the Max Planck Institute of Biophysics using a Nikon Ti2 Eclipse microscope comprising an Olympus Apo total internal reflection fluorescence 100× 1.49 oil objective and a Hamamatsu ORCA-Flash 4.0 LT+ Digital complementary metal oxide semiconductor camera. The images were deconvolved using the Nikon NIS Elements Batch Deconvolution Tool (automatic function). Image analysis was performed using the CellCounter plugin ( https://imagej.nih.gov/ij/plugins/cell-counter.html ) in ImageJ 1.53 ( https://imagej.nih.gov/ij/ , RRID: SCR_003070). Cryo-ET Sample Preparation. A detailed protocol of the correlative cryo-ET workflow is available under https://www.protocols.io/view/3d-correlative-fib-milling-and-cryo-et-of-autophag-e6nvwkz4wvmk/v1 . Starvation and plunge freezing. Yeast cultures were inoculated from overnight cultures in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) to an OD600 of 0.15 and grown at 30 °C to an OD600 of 0.8. At this point, medium was switched to SD-N (synthetic minimal medium lacking nitrogen; 0.17% yeast nitrogen base without amino acids and ammonium sulfate, supplemented with 2% glucose) and cells were incubated for a time span of 0.5–3 h at 30 °C. For 3D correlation on the grid, 1 μm Dynabeads (Dynabeads MyOne carboxylic acid No. 65011, Thermo Fisher Scientific) were added to the cells at a dilution of 1:20. Grids (200 Mesh Cu SiO 2 R1/4, Quantifoil) were plasma cleaned for 30 s before plunging. Four microliters of starved cell solution with beads was applied on the grid, blotted, and plunged in ethane–propane with a Vitrobot Mark IV (settings: blot force = 8, blot time = 10 s, room temperature).

Cryo-fluorescence microscopy and correlative

FIB milling. Grids were mounted on modified autogrids with cut-out for FIB milling, and fluorescence image stacks were acquired on a cryo-confocal microscope (Leica SP8 with Cryo-Stage) equipped with a 50×/0.9 numerical aperture objective (Leica Objective No. 506520) and two HyD detectors. Stacks (step size 300 nm, x–y pixel size 85 nm) were acquired using 488-nm and 552-nm laser excitation for eGFP- and mCherry-labeled proteins, respectively. In the case of eGFP-only strains (eGFP-Atg8 and eGFP-Ede1/ ypt7Δ ), signal from autofluorescent Dynabeads was acquired as second channel corresponding to red emission wavelengths to easily distinguish fiducial beads from cellular signal. Stacks were deconvolved using Huygens Essential (20.10.0, Scientific Volume Imaging, https://svi.nl/Huygens-Software , RRID: SCR_014237). Target sites corresponding to Atg8 puncta or Ede1 END cargo were 3D correlated to SEM/IB images in the FIB/SEM microscope (FIB Scios and Aquilos, Thermo Fisher Scientific) using the 3D-Correlation Toolbox (3DCT) ( https://3dct.semper.space/ ) ( 23 ). Lamellae were milled in correlated sites as described in a previously published protocol ( 24 ). In a few cases (e.g., SI Appendix , Fig. 1 A ), a widefield microscope integrated in the FIB/SEM chamber (METEOR, delmic) was used to confirm the presence of fluorescence signal in the lamella, as previously published ( 55 ). Cryo-EM Data Acquisition. Tomograms were acquired on a TEM (Titan Krios, field emission gun 300 kV, Thermo Fisher Scientific) equipped with an energy filter (Quantum K2, Gatan) and a direct detection camera (K2 Summit, Gatan) at a magnification of 42,000× (pixel size 3.52 Å) and defocus ranging from −5 to −3.5 µm. Positions for tomogram acquisition were determined by correlation of fluorescence data to TEM images of the grid squares containing lamellae (3DCT), followed by inspection of low-magnification lamella images. Frames were recorded in dose-fractionation mode, with a total dose of 120 e − /A 2 per tilt series using SerialEM 3.9.0 (RRID: SCR_017293, https://bio3d.colorado.edu/SerialEM/ ) ( 56 ). A dose-symmetric tilt scheme was used with an increment of 2° in a total range of ±60° from a starting angle of 10° (+ or −) to compensate for lamella pretilt (mostly around 11°). Frames were aligned using MotionCorr2 (v.1.4.0, https://emcore.ucsf.edu/ucsf-software ) ( 57 ), and reconstruction was performed in IMOD (v.4.10.49, RRID:SCR_003297, https://bio3d.colorado.edu/imod/ ) by using the TomoMAN wrapper scripts ( 58 ). Tomogram Analysis. Here, we give a brief overview of the analysis workflows used in this study. Detailed descriptions of all analyses are provided in the SI Appendix , SI Methods . Segmentation and visualization. Tomograms at 2× binning (IMOD bin 4) with a nominal pixel size of 1.408 nm were denoised using cryo-CARE on tomograms reconstructed from odd/even frames ( 59 ). Membrane middles (middle of phospholipid bilayer) were detected automatically using TomoSegMemTV (04/2014, https://sites.google.com/site/3demimageprocessing/tomosegmemtv ) ( 40 ) and selected in Amira 2019 (Thermo Fisher Scientific, https://www.thermofisher.com/de/de/home/electron-microscopy/products/software-em-3d-vis/amira-software.html ). Segmentations for display purposes ( Fig. 1 I – L ) were manually refined in Amira, Gaussian filtered, and displayed in ChimeraX 1.2.5 ( https://www.cgl.ucsf.edu/chimerax/ , RRID: SCR_015872) ( 60 ). For analyses of membrane curvature (phagophore rims and contact sites), the automatic segmentations were refined manually in Amira. Mesh generation from the filled segmentation and curvature determination was done using PyCurv (09/2020, https://github.com/kalemaria/pycurv ) ( 61 ) using a radius hit of 8 nm. Visualizations of different parameters on segmented membranes ( Fig. 4 and SI Appendix , Fig. 3 D and E and 4 A ) were produced with PyVista 0.27.4 ( https://docs.pyvista.org/ ) ( 62 ). Cargo analysis. For each captured autophagic structure, the general type of cargo (ribosomes, ribosomes + other cargo, and selective) was annotated from visual inspection of the tomograms. Ribosome positions were determined by template matching with StopGAP 0.7.0 ( 63 ), followed by subtomogram averaging and classification using Warp/M ( 64 ) and Relion 3.1.2 ( 65 ). The positions in the refined particle list were combined with tomogram segmentations to calculate ribosome densities and nearest neighbor distances in- and outside of the autophagic structures ( SI Appendix , SI Methods ). Contact sites. For all contact site analyses, tomograms from the ypt7Δ strain were excluded since the overall cellular architecture in this strain was disturbed by accumulation of medium-sized vacuoles ( 66 ) ( SI Appendix , Fig. 1 I ). Nearest distances, interaction areas, and deformations were measured and determined in IMOD as described in the SI Appendix , SI Methods . To analyze phagophore–vacuole and phagophore–ER contact sites in detail, we used PyCurv to determine local membrane curvatures and analyzed the resulting meshes with custom python scripts ( SI Appendix , SI Methods ). Membrane morphology of autophagic structures. Intermembrane distances were determined from the automatically generated membrane segmentations using the refined minimum distance algorithm ( SI Appendix , SI Methods ). For size and sphericity measurements, ellipsoid fits into the segmented membranes were performed with an iterative algorithm adapted from Kovac et al. ( 67 ). The ellipsoid fits and mean intermembrane distances were further used to estimate the area-to-lumen ratios of autophagosomes. To assess the completeness of phagophores, we calculated for each structure its “rim opening angle φ”, defined as the angle between a plane through the phagophore rim and tangential planes to the phagophore membrane close to the rim. Meshes generated from refined segmentations of phagophore rim segments were analyzed with custom python scripts to quantify rim swelling, report site-specific curvatures, and estimate bending energies. A detailed description of all algorithms and discussion on different completeness parameters is given in the SI Appendix , SI Methods . Statistics. Statistical analyses were performed with the statistical analysis package in scipy 1.6.2 (scipy.stats) and the pingouin package (v.0.3.11, https://pingouin-stats.org/ ) ( 68 ), using the tests indicated in each respective analysis. In general, statistical analysis of differences between two groups was performed using the Mann–Whitney U test for independent and the Wilcoxon signed-rank test for dependent samples. Comparison of more groups was performed with the Kruskal–Wallis H test and pairwise Games–Howell post hoc test. Correlation between variables was assessed with Spearman’s rank correlation coefficient.

Supplementary Material Supplementary File

Data, Materials, and Software Availability All tilt series of autophagic structures analyzed in this study are available at EMPIAR-11166 ( 69 ). Representative tomograms are additionally available in the Electron Microscopy Data Bank under the following accession codes: EMD-15526 ( 70 ) ( Fig. 1 E ), EMD-15545 ( 71 ) ( Fig. 1 F ), EMD-15546 ( 72 ) ( Fig. 1 G ), EMD-15547 ( 73 ) ( Fig. 1 H ), EMD-15549 ( 74 ) ( Fig. 4 A ), and EMD-15548 ( 75 ) ( Fig. 4 D and F ). Source data for all plots are deposited under https://zenodo.org/record/6607443 ( 76 ). Custom python code written for this study is available on GitHub ( https://github.com/Anna-Bieber/autophagy-tomo-analysis ) ( 77 ), and published on Zenodo ( 78 ).