EMBO/soda-nel huggingface dataset:
ESCRT machinery mediates micro-autophagy of the endoplasmic reticulum in yeast. (2019)
Figure 2

Figure 2 - ER whorls arise from ER stacks and undergo microautophagy.

A Individual frames from time-lapse imaging of a cell expressing GAL promoter-driven GFP-Pho8 and stained with the vacuole membrane dye FM4-64. Numbers indicate the time in minutes after the start of the image sequence. Scale bar: 2 µm.

B Electron micrograph of a cell expressing GPD promoter-driven GFP-Pho8. Arrows indicate the edges of ER cisternae that are part of a bent stack in a vacuole membrane invagination. Black arrowheads point to vacuole membrane that encases the bent stack and the whorl.

C Fraction of uptake intermediates that were converted into vacuolar whorls or stacks. n = 43.

D As in panel (A). The image sequence shows formation of a vacuolar whorl that becomes a cytosolic whorl. Scale bar: 2 µm.
ESCRT machinery mediates micro-autophagy of the endoplasmic reticulum in yeast. (2019)
Figure 3

Figure 3 - The Nem1-Spo7 complex and ESCRTs are key components for micro-ER-phagy.

A, B Fluorescence images of ∆atg7 and ∆atg8 cells (A) or ∆snf7 and ∆vps4 cells (B) expressing GAL promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. Scale bar: 2 µm.

C Relative activity of the ER-phagy reporter Yop1-Pho8∆60 in tunicamycin-treated cells. The dotted line indicates the activity in ∆atg7 cells. Mean + SEM, n ≥ 3.

D, E Relative activity of endogenous Pho8 in untreated cells (D) or after tunicamycin treatment (E). Background activity determined in ∆pho8 mutants was subtracted from the activity in all other strains. Mean + SEM, n = 3. WT, wild-type.

F Western blot of GFP from cells expressing Sec63-GFP and treated with tunicamycin (Tm) where indicated. The two Atg7-independent cleavage products of around 55 kDa were used as readout for micro-ER-phagy. Pgk1 served as a loading control.

G Quantification of Sec63-GFP cleavage normalized to the WT from western blots as shown in panel (F). Mean + SEM, n = 4.
ESCRT machinery mediates micro-autophagy of the endoplasmic reticulum in yeast. (2019)
Figure 4

Figure 4 - Macro- and micro-ER-phagy are parallel pathways with distinct morphologies and molecular requirements.

A Electron micrograph of wild-type yeast treated with DTT for 3 h. Inset 1 shows an ER whorl and inset 2 shows ER fragments in macroautophagic bodies (white arrows). Cy, cytosol; V, vacuole.

B Relative activity of cyto-Pho8∆60 after nitrogen starvation, which reports on non-selective macroautophagy of cytosol. Mean + SEM, n ≥ 3. WT, wild-type.

C-E Relative activity of the ER-phagy reporter Yop1-Pho8∆60 after nitrogen starvation (C) or after tunicamycin treatment (D, E). Mean + SEM, n ≥ 3.

F, G Growth assays of untreated and tunicamycin-treated WT, ∆atg39/40, ∆snf7 and ∆snf7 ∆atg39/40 cells. Numbers indicate growth relative to WT cells based on the areas under the curves. Mean ± SEM, n = 3.
ESCRT machinery mediates micro-autophagy of the endoplasmic reticulum in yeast. (2019)
Figure 5

Figure 5 - ESCRT machinery mediates vacuole membrane dynamics during micro-ER-phagy of GFP-Pho8 structures.

A Fraction of wild-type (WT), ∆vps23, ∆snf7 and ∆vps4 cells with GFP-Pho8 structures. Mean + SEM, n ≥ 3.

B Fraction of GFP-Pho8 structures classified as stacks, cytosolic whorls, uptake intermediates or vacuolar whorls. The number of GFP-Pho8 structures in WT, ∆vps23, ∆snf7 and ∆vps4 cells was 562, 345, 367 and 449, respectively. The table shows the results of Chi-Square tests for independence for all pairs of strains. ***, highly significant (P < 0.00001); ns, not significant (P > 0.05).

C Fraction of stacks classified as peripheral, perinuclear or vacuole-associated. The number of stacks in WT and ∆snf7 cells was 252 and 351, respectively. The observed distributions in the two strains were analyzed with a Chi-Square test for independence. ***, highly significant (P < 0.00001).

D-F Fluorescence images of a cell expressing non-fluorescent GFP(Y66F)-Pho8, Snf7-mNeonGreen and the ER marker mCherry-Ubc6, and stained with the vacuole dye CMAC. Numbers indicate the time in minutes after the start of the image sequence. White arrows indicate a Snf7-mNeonGreen punctum adjacent to an ER whorl. Black arrow point to a vacuole membrane invagination. Panel (E) illustrates transient appearance of Snf7 at an ER whorl. Panel (F) illustrates orientation of a Snf7 punctum towards the neck of a vacuole membrane invagination. In this series, mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time. Scale bar: 2 µm.
ESCRT machinery mediates micro-autophagy of the endoplasmic reticulum in yeast. (2019)
Figure 6

Figure 6 - ESCRT machinery is required for completion of microautophagy of ER whorls during ER stress.

A Electron micrographs of wild-type and ∆snf7 cells treated with DTT for 3 h. Cy, cytosol; V, vacuole. Scale bars: 300 nm.

B Quantification of vacuolar whorls for which the uptake process was complete or incomplete. The observed distributions in the two strains were analyzed with a Fisher's exact test.

C, D Electron micrographs of serial thin sections of a ∆snf7 cell (C) or a wild-type cell (D) treated with DTT for 3 h. Note that the whorl appears separated from the cytosol in some sections (e.g. section 300 nm) but is still in contact with the cytosol (see sections 600 nm and 700 nm). In the wild-type cell (D), the vacuolar whorl is completely separated from the cytosol. Scale bar: 300 nm.
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy (2017)
Figure 3

Figure 3 Comparison of protein and RNA fixation by PFA and glyoxal.

A SDS-PAGE gel showing rat brain cytoplasm incubated for 60 min with different fixatives. The graph shows the summed intensity of the bands in each lane. Fixed proteins either no longer run into the gel or form only smears. To compare the efficiency of fixation, the bands that survive fixation were summed and were expressed as % of an unfixed control. The intensity of PFA-fixed samples was significantly higher than that of glyoxal-fixed samples (N = 5 independent experiments). Glut = 0.2% glutaraldehyde.

B Staining of nucleic acids after fixation. The propidium iodide signal in fibroblasts was significantly higher for samples fixed with glyoxal pH 4 (N = 6-8). To test whether the fixed nucleic acids were still available for specific detection, we performed FISH for GAPDH in cultured neurons, using a standard protocol provided by the company Affymetrix. The fluorescence signal of the samples fixed with glyoxal (pH 4) was significantly higher than for PFA-fixed samples (N = 5-6). Scale bar = 10 µm, * p < 0.05, ** p < 0.01
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy (2017)
Figure 4

Figure 4 STED imaging of primary hippocampal neurons fixed with either PFA or glyoxal.

Strong differences in labeling patterns can be observed. The images are brighter and less "spotty" for the glyoxal-fixed samples. Structures such as filaments or organelles are more easily detected. Quantification of the fluorescence signal (fold over background) shows that 16 out of 20 stainings are significantly brighter in glyoxal fixed samples compared to the PFA fixed samples. N = 35 - 132 objects. Scale bar = 6 µm for β-actin and α-tubulin, and 3 µm for the other proteins. ** p < 0.01, *** p < 0.001
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy (2017)
Figure 5

Figure 5 Comparison of immunostaining NUP160 after fixation with either PFA or glyoxal.

HeLa cells were stained for the nucleoporin complex protein NUP160 after fixation with either PFA, glyoxal pH 4 or glyoxal pH 5. Fluorescence intensities (fold over background) were compared and are shown in the graph. The quantification of fluorescence signals shows that glyoxal pH 4 fixation allows for significantly brighter stainings. N = 73 - 156 cells per condition analyzed. Scale bar = 10 µm. *** p < 0.001
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy (2017)
Figure 6

Figure 6 Comparison of immunostained AtT20 cells after fixation with either PFA or glyoxal.

AtT20 cells stained for the SNARE proteins syntaxin1 and SNAP25 and the autophagy marker LC3B, were compared with regard to the fluorescence intensity (fold over background) of the stainings. Quantification of the intensity shows that glyoxal fixation allows for significantly brighter stainings of the membrane SNARE proteins. LC3B staining is brighter in PFA fixed cells. N = 9 - 20 cells per condition. Scale bar = 5 µm. * p < 0.05, *** p < 0.001
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy (2017)
Figure 7

Figure 7 Comparison of immunostained primary hippocampal neurons in STED resolution.

A Primary hippocampal neurons were stained for Actin and Ankyrin G. A comparison between PFA- and glyoxal-fixed samples shows that actin staining with phalloidin works as least as well in both, showing the prominent actin rings. Ankyrin G staining is brighter in PFA-fixed cells.

B Primary hippocampal neurons were stained for pan-Nav and Kv7.2. Both stainings seem to work at least as well for glyoxal fixed neurons as for PFA fixed neurons. Staining of K-channels shows a slightly more regular pattern in glyoxal fixed neurons.

C Primary hippocampal neurons were stained for Neurofilament L and betaII spectrin. While the spectrin staining seems to be equally well in both fixation conditions, neurofilament staining is brighter in glyoxal fixed cells.

D Growth cones of hippocampal neurons were stained for actin and βIII-tubulin after either glyoxal or PFA + glutaraldehyde fixation. The latter is a standard fixation used for the co-labeling of tubulin and actin, and is a stronger fixation than normal PFA fixation, which is incompatible with many organelle immunostainings (unlike glyoxal fixation). The filopodia and lamellipodia of the growth cones seem to be well stained for the samples fixed with glyoxal, whereas the samples fixed with PFA and glutaraldehyde seem to have lost some of the finer actin structures. Tubulin seems to be a bit better stained in samples fixed with PFA and glutaraldehyde. Scale bars = 1 µm.
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy (2017)
Figure 8

Figure 8 Comparison of immunostained sepia fin after fixation with either PFA or glyoxal.

Sepia fin was fixed with the respective fixative and stained for the neuropeptide FMRFamide. A clear change in morphology can be observed between samples fixed with PFA and samples fixed with glyoxal. The former appear broken and swollen, while the glyoxal-fixed ones appear complete. The effect is presumably due to the different speed of penetration into tissue and/or fixation. Scale bar = 5 µm.
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy (2017)
Figure 9

Figure 9 Comparison of immunostained ventricular myocytes fixed 10 min with either PFA or glyoxal.

Freshly isolated murine ventricular myocytes were either fixed with 4% PFA or 3% glyoxal and immunostained for caveolin-3 or ryanodine receptor type 2. Quantification of the fluorescence intensity of the stainings show significantly brighter stainings for glyoxal fixed myocytes. The graph shows mean values and error bars represent standard deviations. N = 10 (RyR2) and 12 (Cav3) myocytes per condition. Scale bar = 2 µm. *** p < 0.001
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy (2017)
Figure 10

Figure 10 Comparison of immunostained mouse inner hair cells after fixation with either PFA or glyoxal.

Acutely dissected organs of corti were fixed in the respective fixative and immunostained for inner hair cell proteins and synaptic proteins. The quantification of fluorescence intensity for each staining shows a significant increase in signal to noise ratio for three target proteins (CtBP2, Calretinin and Homer1) fixed with glyoxal. None of the stained proteins shows a significant decrease in fluorescence after glyoxal fixation. Representative images show maximum intensity projections from z-stacks of inner hair cell ribbon synapses. N = 5 independent stainings from two animals per condition (PSD95, CtBP2, Otoferlin, Calretinin) and 10 - 15 images per condition (CaV1.3 and Homer1). Scale bar = 2 µm. ** p < 0.01, *** p < 0.001
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy (2017)
Figure 11

Figure 11 Comparison of stained vimentin and α-synuclein in human neuroglioma cells after fixation with either PFA or glyoxal.

Cells were fixed with PFA or glyoxal for 10 minutes, and were stained for endogenous vimentin, or for expressed alpha-synuclein. A quantification of the staining intensities indicates that glyoxal fixation allows for significantly brighter stainings for alpha-synuclein, but that PFA was superior for endogenous vimentin (leftmost graph). The fluorescence intensity of vimentin expressed with an mOrange2 tag was also analyzed after fixation with PFA or with glyoxal; the latter allowed more mOrange2 fluorescence to be detected (rightmost graph). N = 29 - 81 cell regions per condition. * p < 0.05, *** p < 0.001 Scale bar: 10 µm.
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy (2017)
Figure 12

Figure 12 Comparison of immunostainings for mitochondrial proteins after fixation with either PFA or glyoxal.

Cells were stained with MitoTracker Orange prior to fixation with the respective fixative and immunostained for the mitochondrial proteins ATP5B, COA6, NDUFA9 and TIM23. Quantification of the staining intensity shows a significant increase of fluorescence (signal over background) for 2 markers (ATP5B and Mitotracker) after fixation with glyoxal, whereas for the remaining 3 proteins immunostainings seem to be more efficient after fixation with PFA, albeit the differences are small. N = 18 - 128 cells per condition. Scale bar = 5 µm. *** p < 0.001
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy (2017)
Figure 13

Figure 13 Comparison of immunostained HeLa cells after fixation with either PFA or glyoxal.

The fluorescence intensity of a variety of proteins was compared between cells fixed with PFA or glyoxal. Quantification is shown as percentage of signal derived from PFA fixed cells. 8 out of the 18 target proteins which were stained show significantly brighter signal when fixed with glyoxal. Only 4 proteins show significantly reduced staining intensity. For LC3B the intensity of less than 5 cells was quantified, therefore single data points were plotted in addition to the bars. N = 3 - 44 cells per condition. Scale bar = 10 µm. ** p < 0.01, *** p < 0.001
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy (2017)
Figure 14

Figure 14 Comparison of immunostained U2OS cells and primary hippocampal neurons after either PFA or glyoxal fixation.

Immunostaining of the Na/K ATPase in primary hippocampal neurons shows a different distribution of the protein between PFA fixed and glyoxal fixed samples. While in PFA-fixed neurons the antibody falsely stains the nucleus as well as the cytoplasm (100% of the 82 cells we analyzed), in glyoxal-fixed neurons the nucleus is devoid of signal, and the membrane appears to be correctly labeled (arrowheads; 100% of the 60 cells we analyzed). U2OS cells were immunostained for mitochondria (Tom20) and ER (EGFP-KDEL). The fixation and/or staining of mitochondria seems to be comparable in glyoxal and in PFA fixed cells. The staining of the ER shows an improved signal to noise ratio. The signal appears de-localized from the ER for multiple PFA-stained cells (25% of 36 analyzed cells, see arrows), while this is rare for the glyoxal-stained cells (3.4% of 58 analyzed cells). Scale bar = 10 µm.
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy (2017)
Figure 15

Figure 15 Comparison of mouse tissue staining following either PFA or glyoxal fixation.

A - B Confocal images showing staining of Olfactory Marker Protein (OMP) and ß3-tubulin along the dorsal aspect of the mouse olfactory epithelium. While sections from both types of fixative show OMP signal in the olfactory sensory neuron somata, their dendrites, and axons, the axon bundles (green arrow) located above the olfactory epithelium exemplify the clear signal-to-noise ratio benefits of glyoxal fixation versus that of the PFA-fixative. Immunostaining with the ß3-tubulin antibody stains the dendrites and axons in both PFA and glyoxal-fixed tissue, but strong staining of the cilia can only be observed in the glyoxal-fixed sections (B).

C - D Confocal images depicting bundles of axons belonging to olfactory sensory neurons on the path toward the olfactory bulb. Identities of the axons are in part defined by the neuropilin-1 (Nrp1) and neuropilin-2 (Nrp2) expression levels, visualized here with antibodies raised against the two proteins. While complementary expression of the two molecules can be seen in the PFA-fixed sections (C), the glyoxal-fixed sections (D) exhibit profoundly improved signal-to-noise ratios for Nrp-1 (red arrows), and in the case of Nrp-2, also the segmentation of the axon bundle into varying levels of Nrp-2 (green arrows).

E - F Confocal images of olfactory sensory neuron axons coalescing into glomeruli where they synapse with dendrites of olfactory bulb neurons. The axons of olfactory sensory neurons can be readily visualized with OMP staining (green) in the superficial olfactory nerve layer and terminating in glomeruli located below (green arrows). While sections fixed with either PFA or glyoxal display adequate staining levels, the signal distribution of the PFA-fixed tissue appears more irregular (E), seemingly lacking the neurofilimentary morphology that appears preserved in the glyoxal-fixed sections (F). The glomeruli themselves are neuropil structures comprised primarily of olfactory sensory neurons forming synapses with dendrites of mitral/tufted cells as well as dendrites of periglomerular neurons. Immunostaining with Vesicular Glutamate Transporter 2 (VGLUT2) allows visualization of these structures and is easily seen in the glyoxal-fixed section (F), while it appears the antigen was masked by PFA fixation as no signal above background can be seen in the PFA-fixed panel (red arrows in E). Note that a different polyclonal antibody for VGLUT2 from the same provider does provide signal with PFA, albeit weaker versus Glyoxal (inset). Staining with ß3-tubulin touts the benefits of glyoxal both due to the signal improvements in the case of the mild staining in the axons of the olfactory nerve layer that is only visible in the glyoxal-fixed tissue, but also in preserving tissue morphology as demonstrated by the dendritic processes inside glomeruli (blue arrows) and in the external plexiform layer located below the glomeruli.
Complex-centric proteome profiling by SEC-SWATH-MS (2018)
Figure 5

Figure 5: Complex-centric detection of COP9 Signalosome variant CSN1/3/8.

A For nearly half the CORUM complex hypotheses queried, two or more distinct subunit co-elution signals were detected (See methods and Appendix information on CCprofiler).

B Among the four distinct co-elution signals detected from the eight canonical CSN subunits' chromatograms (here with CSN7A, not CSN7B) are two distinct signals indicating distinct co-elution of two different complex variants.

C Distinct co-elution of holo-CSN (observed at the expected fraction 35) and Mini-CSN CSN1/3/8 (observed eluting offset only one fraction late, F45, of the expected fraction, F44). Expected fractions are estimated from the cumulative sum of one copy per component and external size calibration. Coloring adapted to highlight subversion components and their partitioning across holo- and sub-complex. CSN1/3/8 interact and form a submodule within the CSN holo-complex structure (PDB accession 4D10). The observations are consistent between the two whole workflow replicates (see Appendix Figure S6A). Lower right panel, validation of distinct elution behavior of holo-CSN exclusive (CSN7A) and shared subunit (CSN8) by immunoblotting. For full immunoblotting data (CSN1, CSN3, CSN4, CSN5, CSN7A and CSN8) see Appendix Figure S7C).

D CSN1/3/8 display distinct fractionation patterns in co-fractionation experiments performed in other laboratories, specifically in orthogonal ion exchange fractionation of HEK293 lysates (Wan et al. 2015, upper panels) and size exclusion chromatographic fractionation of U2OS lysates (Kirkwood et al. 2013, lower panel), in line with the CSN1/3/8 as distinct entity.

E Baculoviral co-expression of human CSN1, CSN3 and CSN8 in Sf21VM insect cells, with CSN8 N-terminally Strep(II)- and CSN1 & CSN3 N-terminally His6-tagged, followed by affinity purification and SDS-PAGE displays banding pattern in line with the formation of a stable trimer CSN1/3/8.
Complex-centric proteome profiling by SEC-SWATH-MS (2018)
Figure 7

Figure 7: SECexplorer tool for customized interrogation of SEC-SWATH elution profiles.

A SECexplorer web-interface for querying custom protein sets for co-elution behavior in the SEC-SWATH-MS data, viewing chromatograms for interpretation and with algorithmic assistance.

B Zoom in to high MW peak shoulder of holo-COP9 Signalosome (compare Figure 5), where defined co-elution signals of CSN substrate components CUL4A and DDB2 suggest the partial resolution of substrate-bound and free pools of CSN holo-complex.

C Estimation of the fraction of holo-CSN in the likely substrate-bound pool versus the free pool, with eight measurements along the eight subunits and based on Gaussian deconvolution of two signals underlying the observed peak and shoulder (also see Appendix Figure S7).