EMBO/soda-nel huggingface dataset:
Hair follicle stem cell progeny heal blisters while pausing skin development (2021)
Figure 4

Figure 4. Effects of HF reduction on subepidermal blister healing.

A Type XVII collagen (COL17, arrowheads indicate the hair bulge) and laminin β1 (LAMB1) labeling in WT dorsal skin sections (P1). Scale bar: 100 μm.

B, C Blistered samples of Col17a1-/- mouse dorsal skin at P1. ITGA6 (indicated by arrowheads) and COL4 (arrows) labeling (B). L332 staining (C, arrows). Scale bar: 100 μm.

D H&E staining of blistered skin from Col17a1-/- mice at P1. HFs detached from the dermis in Col17a1-/- skin are indicated by arrowheads. Scale bar: 500 μm.

E Whole-mount AP staining of the blister roof epidermis from Col17a1-/- mice (right) and littermate controls (left) at P1. Scale bar: 500 μm.

F H&E (P2, left), ITGA6 (P2, middle) and LOR staining (P4, right) of Col17a1-/- mice (top) and littermate controls (bottom) . The regenerated epidermis is indicated by arrowheads. Scale bar: 200 μm.

G (Top) BrdU labeling of Col17a1-/- skin at P2. Scale bar: 100 μm. (Bottom) Quantification of BrdU-positive cells in the epidermis surrounding blisters (n= 3 (control) and 4 (Col17a1-/-) biological replicates). The data are shown as the mean ± SE. Student's t-test. NS, no significance.

H, I (H) Lineage tracing of K14CreER:R26R-mCherry:Col17a1-/- at P4. Scale bar: 100 μm. (I) Quantification of mCherry-positive cells in the regenerated epidermis (n=3 biological replicates). The data from individual mice are connected by lines. *0.01<p<0.05, Student's t-test.

Data information: Blisters are indicated by stars. Representative images are shown from three or more replicates in each group.
Hair follicle stem cell progeny heal blisters while pausing skin development (2021)
Figure 5

Figure 5. Involvement of keratinocyte shape transformation in subepidermal blister healing.

A Type VII collagen (COL7) labeling in WT dorsal skin sections (P1). Scale bar: 200 μm.

B ITGA6/COL4 (left, indicated by arrows) and L332 labeling (right, indicated by arrows) in the blistered skin of Col7a1-/- mice at P1. Scale bar: 100 μm.

C Schematic diagram of control, Col17a1-/-, and Col7a1-/- mouse skin splits. BM: basement membrane.

D H&E staining of blistered skin from Col7a1-/- mice (right) and their littermate controls (left) at P1. Scale bar: 200 μm.

E Whole-mount AP staining of the blister roof epidermis from Col7a1-/- mice (right) and their littermate controls (left) at P1. Scale bar: 500 μm.

F K10/K14 (low magnification, left) and K14 (high magnification, middle and right) labeling of Col7a1-/- mouse (bottom) and littermate control (top) blistered skin at P2. Scale bar: 30 μm.

G Quantification of BrdU-positive cells per μm HF length (n=55 HFs from three control and 143 HFs from four Col7a1-/- mice). The data are shown as violin plots. Student's t-test. NS, no significance.

H Length of the major axis of keratinocytes in the regenerated epidermis (n=244 (control, L), and 132 (Col7a1-/-, L) cells from four mice, respectively) and in the surrounding intact epidermis (basal cells; n=200 (control, NL), 299 (Col7a1-/-, NL), from four mice, respectively). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.

I K10/K14 (left) and K14 (high magnification, right) labeling of WT blistered skin treated with CaCl2 (middle and bottom) or PBS (top) at P2. Scale bar: 30 μm.

J Quantification of BrdU-positive cells per μm HF length (n=83 (PBS), 95 (1.8 mM CaCl2), and 97 (9.0 mM CaCl2) HFs from four mice). One-way ANOVA test, followed by Tukey's test. NS, no significance.

K Length of the major axis of keratinocytes in the regenerated epidermis (n=433 (PBS, L), 451 (1.8 mM CaCl2, L), and 425 (9.0 mM CaCl2, L) cells from four mice) and in the surrounding intact epidermis (basal cells; n=311 (PBS, NL), 279 (1.8 mM CaCl2, NL), 302 (9.0 mM CaCl2, NL) cells from four mice). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.

Data information: Blisters are indicated by stars. Representative images are shown from three or more replicates in each group. The dashed and dotted lines in the violin plots show the median and quartiles, respectively.
Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement (2016)
Figure 1

Figure 1: Elongator is a two-lobed asymmetric complex.

A. Coomassie Blue stained SDS-PAGE gel of yeast Elongator isolated by FLAG affinity purification and glycerol gradient ultracentrifugation from a strain encoding C-terminally FLAG-tagged Elp1.

B. Representative segment of an electron micrograph of negatively stained Elongator after stabilization by gradient fixation (Lane 6 of Fig 1A). Circled particles correspond to intact Elongator. White scale bar corresponds to 50 nm.

C. Representative 2D class averages of the full Elongator complex from a total of 17,074 particles. All class averages shown in this figure have side width of 500 Å.

D. Representative 2D class averages of the Elp123 subcomplex prepared through a high salt dissociation procedure.

E. Six evenly-sized densities are arranged in one Elongator lobe. A projection of the Elp456 hexamer X-ray structure (PDB code: 4A8J) is shown for comparison.

F. Analysis of Elp2 localization using a C-terminal MBP tag. Class averages of untagged Elongator is shown for comparison. White arrowheads point towards additional densities in the tagged complex.

G. Elp3 localization analysis using a C-terminal MBP tag, denoted by the white arrowhead.

H. Mapping of Elp1 by incubating purified Elongator containing an Elp1 C-terminal FLAG tag with α-FLAG antibody. The additional antibody density is denoted by the white arrowhead.

I. Three dimensional reconstruction of the intact Elongator complex at 25Å.

J. Schematic of the approximate subunit locations based on 2D EM analysis.

K. Inset showing the cavity formed through the centre of the putative Elp456 density. Black scale bars in this figure correspond to 50 Å.
Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement (2016)
Figure 2

Figure 2: Mass spectrometry analysis of Elongator.

A. Number of peptides detected of each Elongator subunit purified through Elp4-FLAG pulldown from yeast lacking ELP1, ELP2, or ELP3 as a fraction of the number of Elp4 peptides. Individual data points are represented as circles, while the averages are represented as bars.

B. Purified Elongator was incubated with the primary amine crosslinker disuccinimidyl suberate (DSS) and analyzed by mass spectrometry. Schematic representation of the major crosslinking connectivity of Elp123 is shown.

C. Major crosslinking connectivity of the Elp456 complex.

D. Detailed locations of crosslinked residues plotted onto a domain representation of Elongator. Lysines from two residues found crosslinked together are denoted by black lines.
Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement (2016)
Figure 3

Figure 3: Conserved Elp2 loops are important for Elongator function.

A. Schematic of the conserved residues within four Elp2 loops that were mutated to alanine within an ELP2 plasmid.

B. Plasmids containing ELP2 and the four loop mutants were transformed into elp2Δ yeast. The strains were then plated onto –Ura media containing caffeine. The pRS416 vector is used for these experiments.

C. Growth curve analysis of loop mutants in –Ura media with and without K. lactis toxin zymocin. Shaded regions correspond to the SEM at each time point. N=3.

D. Lysates of Elp4-FLAG-containing yeast expressing the Elp2 loop mutants were incubated with α-FLAG resin and eluted with 3xFLAG peptides then analyzed by Western Blot using α-HA and α-FLAG antibody
Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement (2016)
Figure 4

Figure 4. Fitting of Elongator subunits in the 3D reconstruction.

A. Fitting of Elp123 structures into the 3D reconstruction of Elongator. The homology structure of the Elp1 N-terminal double WD40-domains, the homology structure of Elp3 based on the X-ray structure of D. mccartyi, the X-ray structure of Elp1 C-terminal dimer (5CQS), and the X-ray structure of Elp2 (4XFV). Scale bar corresponds to 50 Å, and applies to panels A-C.

B. Fitting of the Elp456 heterohexameric ATPase ring (4A8J) into the six-membered ring from the larger Elongator lobe.

C. Size comparison of a representative tRNA molecule (1VTQ) and the cavity observed through the middle of the Elp456 hexamer.

D. Alternate views of the Elongator model showing the subunit crosslinking patterns.
Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement (2016)
Figure 5

Figure 5: Model of Elongator subunit organization and Elp456 association.

A. CXMS and EM data, combined with the X-ray structures of Elp1 C-terminus (5CQS), Elp2 (4XFV), Elp456 (4A8J), and homology models of the Elp1 tandem WD-40 domains and Elp3, were used to estimate the relative positions of each subunit to generate a multi-scale model of Elongator subunit organization.

B. Side view of the Elongator architectural model highlighting the cavity (red outline) spanning through the centre of the Elp456 hexamer.

C. Comparison of the Elp123 conformations of the intact Elongator complex (gold) and the Elp123 subcomplex (cyan). The Elp456-unloaded lobe of Elongator and one lobe of Elp123 were aligned.
Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement (2016)
Figure 6

Figure 6. Elongator can accommodate two copies of the Elp456 subcomplex

A. Silver-stained SDS-PAGE of Elongator glycerol gradient fractions purified using low salt (75 mM NaCl) buffer. The fraction containing the most Elp4, Elp5, and Elp6 is designated by an asterisk (*) and the corresponding fraction from a glutaraldehyde-containing glycerol gradient is used for EM analysis.

B. Comparison of class averages of Elongator purified in buffer containing regular or low salt concentrations (150 mM and 75 mM NaCl, respectively). Densities resembling a second copy of Elp456 is indicated by white triangles.

C. Scheme of the Elongator and rElp456 mixing experiment. Recombinant Elp456 subcomplex (rElp456) was purified from E. coli and mixed with native yeast Elongator in regular salt conditions. The mixture, as well as rElp456 alone, were then subjected to glycerol gradient ultracentrifugation.

D. Coomassie-stained SDS-PAGE of rElp456 glycerol gradient fractions.

E. Coomassie-stained SDS-PAGE of the Elongator mixed with rElp456 glycerol gradient fractions. The fraction containing the most intact complexes is indicated with an asterisk (*), and the corresponding fraction from a glutaraldehyde-containing glycerol is used for EM analysis.

F. Comparison of class averages of natively purified Elongator with and without rElp456 incubation. Densities resembling a second copy of Elp456 are indicated by white triangles.
Autophagy: a forty-year search for a missing membrane source. (2006)
Figure 1

The Dynamic Membrane Events Involved in Autophagy

(A) Upon induction of autophagy, a membrane sac called the isolation membrane (IM) forms and engulfs portions of the cytoplasm. Sealing of its edges gives rise to the double-membrane bound autophagosome. Fusion of the outer membrane with a lysosome results in formation of an autolysosome, in which the inner autophagosomal membrane and its contents are degraded.

(B) Starvation-induced autophagosomes (AP) and autolysosomes (AL) in the fat body (the functional analogue of the liver) of a fruit fly larva. Note that APs contain intact cytoplasm, whereas the contents of ALs show various stages of degradation.

(C) Liver cells of starved mice carrying a fluorescently tagged LC3 transgene, labeling cup-shaped and ring-shaped structures that correspond to IMs and autophagosomes, respectively. Images courtesy of Ryan Scott (B) and Dr. Noboru Mizushima (C).
SNARE-mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle (2018)
Figure 1

Fig.1. In vivo visualization of an expanding fusion pore.

A) z-stacks were acquired from FM4-64 labeled vacuoles in wildtype cells (BJ3505), using a spinning disc microscope (step size 200 nm). 3D reconstructions from these stacks reveal a peripherally located fusion pore. Scale bar 2 µm.

B) Wildtype (BJ3505) and isogenic pmc1∆/vcx1Δ mutant cells were labeled with FM4-64. The number of vacuoles per cell was determined in 3 independent experiments evaluating 200 cells each. Scale bar: 2 µm.

C) Frequency of visible fusion pores. Cells were grown as in (B). The percentage of cells showing visible fusion pore was determined from z-stacks. Means s.d. are shown for 100 stained vacuoles from 3 independent experiments.

D) Schematic view of Vph1-GFP and FM4-64 distribution over the vacuolar membranes in the presence of a nanoscopic fusion pore or of an extended hemifusion diaphragm.

E) Distribution of Vph1-GFP and FM4-64 in vacuole-vacuole contact sites. Vph1-GFP expressing cells, labeled with FM4-64, were analyzed under a spinning disk confocal microscope equipped with two cameras for the simultaneous acquisition of GFP and FM4-64 fluorescence signals. Separate channels are shown on the right. The entire contact surface, but not the pore, is accessible to both probes. Scale bar: 2 µm.

F) Expansion of a vacuolar fusion pore in real time. pmc1∆/vcx1Δ cells were labeled with FM4-64 and immobilized in a 50 µL flow chamber (Ibidi). The osmotic value of the medium was changed by perfusion with water, using a pump with a flow of 30 µL/s. A frame was acquired every 2 s for a total period of the 30 s, using the laser at minimal intensity.
SNARE-mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle (2018)
Figure 2

Fig. 2. In vivo assay for lipid and content mixing of vacuolar membranes.

A) Vacuolar membranes of wildtype (BY4741), nyv1∆ vam3∆, ypt7∆ or vma16F190Y cells were labeled with the vital dye FM4-64. A vacuole that was in a sufficiently peripheral location to be selectively photobleached was exposed to a laser pulse (white arrows indicate the bleaching area). Recovery of fluorescence in this area was assayed 10 seconds later by spinning disc confocal microscopy. Scale bar: 2 µm.

B) Kinetics of FM4-64 recovery in (A). Means and s.d. are shown for 20 vacuole clusters from 3 independent experiments.

C) In vivo lipid and content mixing. Vacuoles in living yeast were labeled with the indicated lumenal (CDCFDA) and membrane (FM4-64 or PX-GFP) probes. Note that CRY1 cells lack the ADE2 gene and hence naturally accumulate 5-amino-1-(5-phospho-D-ribosyl)imidazole as fluorescent fluid phase marker in the vacuolar lumen. FRAP-experiments were performed as in (A) (arrows indicate the bleaching area). 1. Non-bleached lumenal area. 2. Bleached lumenal area. 3. Bleached membrane area. Scale bar: 2 µm.

D and E) Kinetics of fluorescence recovery after photobleaching of the probes in (C). Numbers denote the areas shown in (C): 1. Non-bleached lumenal area. 2. Bleached lumenal area. 3. Bleached membrane area. Means and s.d. are shown for 20 vacuole clusters from 3 independent experiments.
SNARE-mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle (2018)
Figure 3

Fig. 3. FRET-based localization of FM4-64 at the inner membrane leaflet.

A) FM4-64 and GFP excitation/emission spectra.

B) FRET-based in vivo strategy to distinguish localization of FM4-64 in the inner or outer leaflets of vacuolar membranes. EGFP tags were added to the cytosolic C-terminus of Vph1 or to the lumenal C-terminus of Nyv1.

C) Assay by microscopy. Half of two cultures of cells expressing Vph1-EGFP or Nyv1-EGFP were labeled with FM4-64, washed and mixed with the other, non-labeled half of the same culture. Cells were analyzed by spinning disc confocal microscopy. The intensities of GFP fluorescence were tracked along the dashed lines and compared between neighboring FM4-64-labeled and unlabeled cells.

D) FACS analysis. The strains from (C) were labeled as described, washed and analyzes by FACS. Shown are side (SSC-A) and forward scatter (FSC-A), as well as the fluorescence intensity distributions for FM4-64 and for GFP in the absence or presence of FM4-64.

E) Comparison of the GFP mean fluorescence from FM4-64-stained cells and unstained cells (20'000 each).

F) Possibilities for FM4-64 transfer between the inner leaflets of two tethered vacuoles. Flipping between the trans-leaflets of a hemifusion structure; reversible flipping into the outer leaflet, establishing a minor pool that then transfers through the buffer or through the outer leaflet; or passage through a fusion pore.
SNARE-mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle (2018)
Figure 4

Fig. 4. Protein-based assay for a nanoscopic fusion pore

A) Synthetic reporter proteins carrying EGFP either at the lumenal or cytosolic end of the single-spanning transmembrane domain of Nyv1, which carries no hydrophilic extensions at its lumenal C-terminus.

B) The topology of these constructs in the vacuolar membrane is indicated.

C,D) FRAP assays for fusion pores. The indicated cells expressing (C) yeGFP-LT or (D) LT-yeGFP were labeled with FM4-64 and subjected to FRAP experiments as in Fig. 2. Cells were imaged before and 0 (bleached) or 30 seconds after photobleaching (recovery). The bleached areas are indicated by an arrow.

E,F) FRAP assays for fusion pores were performed as in (C, D), but with nyv1∆ cells. Scale bar: 2 µm.

G) The histogram reports the fraction of cells showing FRAP. Means and s.d. are shown from 3 independent experiments, with 100 cells analyzed in each.
SNARE-mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle (2018)
Figure 5

Fig. 5. Discrimination of inner and outer leaflet mixing.

A-F) The indicated yeast strains were transformed with expression plasmids for FYVE2-GFP and their vacuolar membranes were labeled with FM4-64. FRAP was performed as in Fig. 2, using a spinning disk microscope equipped with an emission beam splitter and two synchronized cameras, allowing to simultaneously analyze FM4-64 and GFP fluorescence. The histograms show the kinetics of signal recovery for (A) wildtype (WT), (B) vam3tsf, (C) ypt7T22N, (D) vma16F190Y, (E) vps33P184L, and (F) nyv1∆ cells. Scale bar: 2 µm. Arrows indicate the bleaching area. Means and s.d. are shown for 20 stained vacuoles from 3 independent experiments.
SNARE-mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle (2018)
Figure 6

Fig. 6. Rapid re-establishment of membrane continuity after membrane fission.

A-D) Wild-type (A, B) or nyv1∆ cells (C,D), which lack the vacuolar R-SNARE Nyv1, were labeled with FM4-64. Cells were immobilized on a chambered slide and subjected to FRAP experiments as in Fig. 2A. (C) Vacuole fission induced by hypertonic shift. FRAP experiments were performed before (0') or 15 min after addition of NaCl to 0.5 mol/l. The fluorescence images show the vacuoles inside a single cell. (B, D) Cells in S-phase were selected, which grow a daughter cell and therefore fission their vacuoles into a chain of tubulo-vesicular structures, part of which are transported into the daughter cell. FRAP experiment on vesicles in the inheritance structure were performed. Arrows indicate the bleaching area, dashed orange lines the cell cortex. Scale bar: 2 µm.
Inhibition of O-GlcNAcylation protects from Shiga toxin-mediated cell injury and lethality in host (2021)
Figure 1

Figure 1. An acute increase of the cellular O-GlcNAc level by the induction of ER stress upon Stx2a-exposure causes apoptosis of THP-1 cells

A. Representative western blot showing changes in O-GlcNAcylation in THP-1 cells treated with Stx2a or Stx2amut (10 ng/mL each) for 0-12 h.

B. Quantification of the band intensities in (A). Data are presented as mean ± S.D. (n = 3 biological replicates) normalized against β-actin, which was used as a loading control.

C. Representative western blot showing changes in various ER stress markers and O-GlcNAcylation-related protein expression in THP-1 cells treated with Stx2a (10 ng/mL) for 3 h in the presence or absence of O-linked N-acetylglucosamine transferase (OGT) inhibitor OSMI-1 (10 µM final) or serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1 a (IRE1a) inhibitor MKC-3946 (10 µM final).

D. Quantification of the band intensities in (C). Data are presented as mean ± S.E.M. (n = 3 biological replicates) normalized against β-actin, which was used as a loading control.

E. Representative western blot of O-GlcNAcylation and pro-caspase-3 cleavage in THP-1 cells treated with Stx2a (10 ng/mL) in the presence or absence of OGT inhibitor OSMI-1 (10 µM final).

F. Quantification of the band intensities for O-GlcNAcylation (RL-2) in (E). Data are presented as mean ± S.E.M. (n = 3 biological replicates) normalized against β-actin, which was used as a loading control. The effects of Stx2a-mediated induction for O-GlcNAc levels at each time-point were compared to 0 h (left panel), and OSMI-1 treatment was compared with that of the vehicle (DMSO) control at each time-point (right panel).

G. Quantification of the relative band intensities of cleaved caspase-3 in (E) (Left panel: upper band; Right panel: lower band). Data are presented as mean ± S.D. (n = 3 biological replicates) normalized against β-actin, which was used as a loading control. The effects of OSMI-1 were compared with those of the vehicle (DMSO) control at each time-point. (n.d. = not detected).

H. Left panel: Representative flow cytometric plots showing apoptosis of THP-1 cells detected by the TUNEL assay upon treatment with Stx2a (10 ng/mL) in the presence (blue) or absence (red) of OSMI-1 (10 µM, final). Right panel: Quantification of the percentage of apoptotic cells at each time-point derived from the plots presented in the upper panel. Data are presented as mean ± S.D. (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) control at each time-point.

Data information: Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.