EMBO/SourceData huggingface dataset:
Figure 1Representative composite confocal image of single cultured hippocampal mouse neurons, labeled with MAP2 (green) on islands of glial cells, labeled with GFAP (red). Scale bar: 100 µm. Representative confocal image of single cultured hippocampal mouse neuron labeled with endogenous DCV cargo ChgB (green) and ß3-tubulin (red) as neurite marker. Scale bar: 20 µm.Analysis of hippocampal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. DCV pool size measured as number of ChgB puncta per neuron.Analysis of hippocampal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. Total neurite length (mm) per neuron.Analysis of hippocampal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. ChgB puncta per μm neurite.Analysis of striatal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. DCV pool size measured as number of ChgB puncta per neuron.Total neurite length (mm) per neuron.Analysis of striatal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. ChgB puncta per μm neurite.Correlation between ChgB puncta per neuron and total neurite length for hippocampal (green) and striatal (red) neurons.Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. Representative mouse brain slice with labeled axonal projections in cortical region (mCherry-filler, red) and immunostained for ChgA (green). Scale bar: 1 mm. (a-b) Zoom of axonal projections in (L), scale bars: 100 µm.Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. Representative confocal image and zoom of cortex tissue containing labeled axonal projections. Measurements of ChgA puncta (black) were performed within axonal projections (mask, yellow). Zoomed areas are indicated in top, right image (M). Scale bars: 10 µm (M), 5 µm (NAnalysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. Representative confocal image and zoom of cortex tissue containing labeled axonal projections. Measurements of ChgA puncta (black) were performed within axonal projections (mask, yellow). Zoomed areas Arrows (O) indicate ChgA staining measured as individual ChgA puncta. Scale bars: , 5 µmAnalysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. ChgA puncta per μm axon from mouse tissue, compared to the distribution of ChgB puncta in axons of cultured hippocampal neurons
Figure 2Representation of resolution limit in confocal imaging and dSTORM strategy to increase resolution. Electron micrograph of axon (green) and dendrite (red) containing DCVs, defined based on morphology. In conventional immunofluorescence confocal microscopy, individual DCVs cannot be separated (right zoom, green signal representing fluorescent signal using confocal). Super-resolution dSTORM imaging resolves the point-spread function (PSF) resulting in single DCV localizations. Scale bars: 100 nm.Representative confocal image and zoom of a single isolated hippocampal neuron (DIV14) immunostained for the endogenous DCV marker ChgB (green), axonal marker SMI312 (red) and dendritic marker MAP2 (blue). Areas of zoom (C) is indicated in (B). Scale bars: 20 µm (B), 5 µm (C).ChgB immunoreactivity of zoomed area indicated in (C) (top panel). Reconstructed ChgB localizations of dSTORM imaging (middle panel). Lower panel shows overlay of dSTORM ChgB localizations and ChgB immunoreactivity (confocal, 40x). Calibration bar represents no (0) to high (255) clustering of dSTORM localizations. Scale bar: 2 µm.Fluorescence intensity of ChgB immunoreactivity puncta (confocal) with corresponding number of dSTORM puncta for hippocampal (green) or striatal (red) cultured neurons. Squares show average ± SEM, dots represent individual observations. Hippocampus: N = 2 cells (89 puncta), striatum: N = 3 cells (37 puncta).Average histogram of fluorescence intensity of ChgB puncta (40x confocal) in neurons used for dSTORM imaging. Dotted lines indicate cut-off values of fluorescence intensity corresponding 1, 2, 3 or 4 dSTORM puncta. Data shows average ± SEM, Hippocampus: N = 2 cells, striatum: N = 3 cells.Percentage of ChgB puncta per neuron with corresponding fluorescence value of 1, 2, 3 or 4 dSTORM puncta. Data shows average ± SEM. Hippocampus: N = 2 cells, striatum: N = 3 cells.Analysis of STED microscopy of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. (H) Representative confocal (100x magnification) image of mCherry labeled axon (blue) with ChgA confocal signal (red) and corresponding super-resolution STED ChgA signal (green). Zoomed areas are indicated by arrows. Scale bar merged image (left): 1 µm, scale bars zooms: 100 nm. (I) Percentage of single ChgA puncta within mCherry labeled axons with corresponding number of overlaying STED puncta. Data shows average ± SEM. N = 2 mice (7 STED images, 70 single observations).
Figure 3Representative confocal images and zooms of a single isolated hippocampal neuron (DIV14) immunostained for the endogenous DCV marker ChgB (green), dendritic marker MAP2 (red) and ß3-tubulin (blue) as neurite marker. Areas of zooms are indicated in (A). Closed arrows: dendritic ChgB staining, open arrows: axonal ChgB staining. Scale bars: 20 µm (A), 5 µm (B, C). Zoom of immunostaining for the excitatory synaptic vesicle protein VGLUT1 and DCV marker ChgB. Closed arrows: synaptic ChgB staining, open arrows: extra-synaptic ChgB staining. Scale bar: 5 µm. Zoom of immunostaining for two synaptic proteins VGLUT1 and Synapsin I/II. Scale bar: 5 µm.Manders' coefficients of colocalization analysis between ChgB, ß3-tubulin and MAP2.Manders' coefficients of colocalization analysis between ChgB and VGLUT1, with VGLUT1-Synapsin I/II colocalization as positive control and VGLUT1-Synapsin I/II rotated 90° as negative control.Number of ChgB puncta per neuron in dendrites, axons and the total pool (dendrites + axons). Wilcoxon matched-pairs signed rank test: n.s. = not significant, p = 0.75.Total dendritic and axonal length (mm) per neuron. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001.ChgB puncta per μm dendrite and axon per neuron. Lines next to individual data points show mean ± SEM. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001.Mean ChgB fluorescence intensity per puncta in the total neuron (dendrites + axons) and in dendrites only. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001.Histogram of fluorescence intensity of ChgB puncta in the total neuron (dendrites + axons) and in dendrites only.ChgB puncta per μm dendrite and axon per neuron. Lines next to individual data points show mean ± SEM. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001. Mean ChgB fluorescence intensity per puncta in dendrites and axons. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001.Distribution of DCVs in dendrites and axons of sparse-labeled network cultures. (P) ChgB puncta per μm dendrite or axon. Lines next to individual data points show mean ± SEM. Mann-Whitney U test: **** p = < 0.0001. (Q) Mean ChgB fluorescence intensity per puncta in dendrites and axons. Unpaired t-test: *** p = < 0.0001.Analysis of DCVs in axons and dendrites of mCherry labeled mouse Dentate Gyrus (DG) granule cell tissue after stereotaxic viral injections and post-hoc immunostained for ChgA as DCV marker. (R) Representative mouse brain slice with labeled DG granule cells (mCherry-filler, red) and immunostained for ChgA (green). ChgA puncta were measured in dendrites and axons at indicated areas. Scale bar: 250 µm. (S) ChgA puncta per μm dendrite or axon in DG granule cells. N = 1 mouse (6 dendrite regions, 7 axonal regions).
Figure 4Representative confocal image and zoom of a single isolated hippocampal neuron (DIV14) immunostained for the endogenous DCV marker ChgB (green), synaptic protein VGLUT1 (red) and dendritic marker MAP2 (blue). Area of zoom (B) is indicated in (A). Scale bars: 20 µm (A), 2 µm (B). Reconstructed ChgB localizations of dSTORM imaging of zoomed area in (B) (top). Middle panel shows overlay between dSTORM ChgB localizations and VGLUT1 immunofluorescence (confocal, 40x). Scale bar: 2 µm. Zooms of boxed areas containing synapses are visualized in lower panels (a-c). Arrows indicate extra-synaptic DCVs. Scale bar (a-c): 1 µm. Calibration bar represents n:o (0) to high (255) clustering of dSTORM localizations.Representative electron micrographs of synaptic sections containing DCVs. AZ indicates the active zone, dashed lines represent DCV diameter measurements. Arrow indicates extra-synaptic DCV. Scale bars: 100 nm.Histogram of number of DCVs in percentage of synapses. Inset: number of DCVs per synaptic section.Average diameter of DCVs (nm).Distance of DCVs to the active zone (nm), measured from center of DCV to the middle of the active zone. Histogram of distance of DCVs to the middle of the active zone (nm). Inset: shortest distance of DCVs to the active zone (nm).Histogram of normalized distance of percentage of synaptic vesicles (grey) to the active zone and corresponding distance of DCVs (black) to the active zone.Example electron micrograph zooms of synapses containing DCVs outside (top) or inside (bottom) the synaptic vesicle cluster. Scale bars: 100 nm. Percentage of DCVs outside or inside the synaptic vesicle cluster. Histogram of distance (nm) of DCVs to the border of the synaptic vesicle cluster, negative values are inside, positive values outside.
Figure 5Schematic representation of NPY-pHluorin as optical reporter for DCV fusion. Inset below shows F/F0 of single DCV fusion event. Scale bar: 1 µm.Zoomed confocal image of NPY-pHluorin expressing (green) hippocampal neuron (DIV14) immunostained for ChgB (red) and corresponding fluorescence intensity plot showing overlap between NPY-pHluorin and ChgB. Scale bar: 2 µm.Manders' coefficients of colocalization between NPY-pHluorin and ChgB per neuron.Total ChgB puncta per neuron in uninfected (control) and NPY-pHluorin infected neurons (NPY-pH). Student's t-test: n.s. = not significant, p = 0.90. Mean ChgB fluorescence intensity per puncta per neuron in uninfected (control) and NPY-pHluorin infected neurons (NPY-pH). Student's t-test: n.s. = not significant, p = 0.71.Correlation between ChgB puncta per neuron and total neurite length (mm) for uninfected control (grey) and NPY-pHluorin infected (green) neurons. Linear regression, inset shows goodness of fit (r2) and significance (p-value) per group. Slopes are not significantly different, ANCOVA: p = 0.19.Single isolated hippocampal neurons (DIV14) were labeled with NPY-pHluorin. DCV fusion events were quantified per neuron upon different stimulation paradigms. Histogram of DCV fusion events per stimulation per time point. Stimulations include 1 action potential (AP) (grey), 16 bursts of 50 AP at 50 Hz (light-green), superfusion with 60 mM KCl Tyrode solution for 30 s. (dark-green) and superfusion with 5 μM Ionomycin for 30 s. (blue). Inset: Typical ∆F/F0 trace of rise in intracellular calcium (Fluo5-AM) upon different stimulations. Traces are corrected for baseline (first 10 frames) and normalized.Single isolated hippocampal neurons (DIV14) were labeled with NPY-pHluorin. DCV fusion events were quantified per neuron upon different stimulation paradigms. Cumulative plot of fusion events per neuron for different stimulations.Single isolated hippocampal neurons (DIV14) were labeled with NPY-pHluorin. DCV fusion events were quantified per neuron upon different stimulation paradigms. DCV fusion events per neuron per stimulation. Kruskal-Wallis with Dunn's correction: *p < 0.05, **p < 0.01, ***p < 0.001. 1AP vs. 5 μM Ionomycin, 16x50 AP,50Hz vs. 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non-significant, p > 0.05. DCV release probability per neuron per stimulation. Kruskal-Wallis with Dunn's correction: **p < 0.01, ***p < 0.001. 1AP vs. 5 μM Ionomycin, 16x50 AP,50Hz vs. 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non-significant, p>0.05.DCV release probability per neuron in CaMKII-positive and negative hippocampus neurons (DIV14) upon repetitive electrical stimulation (16x50AP at 50 Hz). Mann-Whitney U test: n.s. = not significant, p = 0.12.
Figure 6Schematic representation of NPY-mCherry as optical reporter for DCV fusion with full cargo release (top). Zoom of axonal and dendritic areas of (B), with location of fusion events (middle). Average fluorescence decrease upon NPY-mCherry labeled DCV fusion in axons and dendrites (below).Representative neuron labeled with membrane-targeted EGFP (mEGFP) (black) and localization of axonal (blue dots) or dendritic (purple dots) events upon stimulation. Scale bar: 20 µm.Histogram of axonal (blue) and dendritic (purple) DCV fusion events upon repetitive electrical stimulation (16x50AP at 50 Hz).Cumulative plot of axonal or dendritic fusion events per neuron. Normalized cumulative plot of axonal and dendritic fusion events per neuron. Percentage of axonal fusion events per cell.
Figure 1A-B. Wild-type and ΔnlpI cells were heated at a range of temperatures and the soluble components were labelled by TMT, combined and quantified by LC-MS, using the published 2D-TPP protocol (Mateus et al., 2018). Shown are volcano plots of two replicates depicting changes in: protein abundance (A) and thermostability (B). A local FDR (false discovery rate) <0.01 was set as a threshold for significance. Highlighted proteins: outer membrane proteins (OMPs, light green), β-barrel assembly machinery (BAMs, red), PG synthases/regulators (green), PG hydrolases and regulators (blue) and the Tol-Pal complex (violet). All other proteins were colored grey and not labelled to increase the plot clarity.C-D. Affinity chromatography with immobilised NlpI. Membrane extracts from E. coli were incubated in low and high salt binding conditions (50 and 400 mM NaCl, respectively), and then eluted with 1 M NaCl or 2 M NaCl to identify possible interaction partners by label-free LC-MS analysis. The plot shows the log2 fold enriched proteins when compared to those eluted from a parallel empty column control, versus the log10 p-value, in low (4 replicates) (C) and high (2 replicates) (D) salt. Highlighted points are all interactions with PG enzymes and their regulators, as well as members of the divisome. All other proteins were colored grey and not labelled to increase the plot clarity; many were non-physiological interactions with abundant cytoplasmic proteins.
Figure 2A. Dissociation constants for interactions between NlpI with MepM, MepS, PBP4, PBP7 as determined by Microscale thermophoresis (MST). The values are mean ± standard deviation of three independent experiments. B. MepS-NlpI interaction by MST as an example plot for Fig 2A. MST curve plotted is the mean data +/- SD of three independent experiments. Fl, fluorescently labelled; FNorm, normalized fluorescence.NlpI has different binding sites for MepS and PBP4 as shown by the ability of labelled MepS to bind preformed NlpI-PBP4 (C) complexes by a fixed concentration MST assay. Values are mean ± standard deviation of 3-6 independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001)NlpI has different binding sites for MepS and PBP7 as shown by the ability of labelled MepS to bind preformed NlpI-PBP7 (D) complexes by a fixed concentration MST assay. Values are mean ± standard deviation of 3-6 independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001)
Figure 4A. Phase contrast image and corresponding fluorescence SIM image of BW25113 cells that have been grown in LB at 37°C and immunolabelled with specific antibodies against NlpI. Scale bar equals 2 µm.B. Genetic interactions of NlpI with PG machineries. An example of a 384 plate is shown. Bar plots shows the averaged values of 2 experiments (i=2, n=192). Error bars denote the 95% confidence interval of the mean.C. NlpI deletion exacerbates the morphological defects of the PBP1B/LpoB-mutant strains. The graph shows the cell width of single and double deletions strains (800 < n < 2000 cells). The box has a medium between 25 and 75%. The whiskers with the upper and lower vertical line indicating the 95 and 5%. The dots are individual points outside the 5 and 95% range. Representative images of the strains are shown above the graph. The scale bar equals 2 µm. Gene encoding protein legend: nlpI encodes NlpI, mrcA encodes PBP1A, lpoA encodes LpoA, mrcB encodes PBP1B, lpoB encodes LpoB.D. Dissociation constants for interactions between PBP1A and LpoA with NlpI, MepM, MepS, PBP4 and PBP7 as determined by MST. The values are mean ± standard deviation of three independent experiments. 1PBP1A was used as fluorescently labelled protein in all assays. 2LpoA was used as unlabelled ligand in all combinations, except with MepM and PBP4.
Figure 5A-B. PBP4 has different interaction sites for PBP1A/LpoA and NlpI as shown by a single concentration MST assay. Plots show the FNorm or fluorescence values of fluorescently labelled PBP1A or LpoA with or without PBP4, NlpI or PBP4-NlpI. Values are mean ± standard deviation of three independent experiments.
Figure 6A. Estimated number of molecules and molarity of PBP1a/LpoA and EPases. 1Numbers obtained by ribosomal profiling in rich growth medium (Li et al., 2014). 2Concentration of monomer. 3Decreases in the presence of NlpI (Singh et al., 2015). 4"no change detected". The periplasmic concentrations of proteins were estimated for a cell with periplasmic volume of 3.33 ×10-16 L, where 1 molecule corresponds to 5 nM.
Figure 1(B) Islet graft survival of mice receiving vehicle (PBS) or IL-33 was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001 vs islet+vehicle. (C) Blood glucose level of mice treated with IL-33 (the horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection). Each line represents one mouse.(D) Intraperitoneal glucose tolerance test (IPGTT) was assessed in normal mice, islet transplant mice receiving vehicle (on the day when grafts were considered rejected), and islet transplant mice treated with IL-33 (at day 30 and day 80 post-islet transplantation). Data shown are the mean ± SEM (n=6-9 per group). (E) Area under the curve (AUC) for IPGTT was assessed. Data shown are the mean ± SEM (n=6-9 per group) and a one-way ANOVA was performed, ***P<0.001. (F) Representative immunohistochemical staining for insulin in graft samples from mice receiving vehicle or IL-33. Scale bar = 100 µm.
Figure 2C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days. (A, B) Representative FACS analysis showing the proportion of Tregs (CD4+Foxp3+) in the CD4+ T cell compartment from the spleens (A) and kidneys (B) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6). C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days. (C, D) Representative FACS analysis showing the proportion of ILC2s (Lin-GATA-3+) in the CD45+ leukocyte compartment from the spleens (C) and kidneys (D) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6).(E, F) proportion of Tregs (E) and ILC2s (F) in the spleen and kidney in PBS-injected controls (n=4) and at weeks 1-12 after IL-33 treatment (n=4-6 per IL-33-treated group). Data information: Data shown are the mean ± SEM; Statistical analysis was performed with an unpaired t-test, **P<0.01, ***P<0.001.
Figure 3(B-D) Proportion or numbers of CD4+Foxp3+ Tregs in the spleens, kidneys and islet grafts of normal, islet transplant mice receiving vehicle and islet transplant mice with IL-33 treatment (at day 7, 30 and 80 post-islet transplantation). Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001.(E) Representative confocal microscopy images of immunostaining for CD4, Foxp3 and insulin in islet grafts. Scale bar = 50 µm.(F-H) Proportion or numbers of ILC2s in the spleens, kidneys and islet graft of mice with or without IL-33 treatment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.(I) Representative confocal microscopy images of immunostaining for CD127, ST2, CD3 and insulin in islet grafts. Scale bar = 50 µm.(J) The mRNA expression of IL-25, IL-33 and TSLP in islet grafts of mice with or without IL-33 treatment was examined by qPCR, and expressed relative to the control of each experiment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.
Figure 4Proportion of CD4+Foxp3+ Tregs (B) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001.Proportion of Lin-GATA-3+ ILC2s (C) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001.(D) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. *P<0.05, **P<0.01.(E) Data are shown as blood glucose measurement in islet transplant mice treated with IL-33/DT, IL-33/PC61, or IL-33/DT/PC61. The horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection. Each line represents one mouse.