EMBO/soda-nel huggingface dataset:
A role for Septin 2 in Drp1-mediated mitochondrial fission (2016)
Figure 3

Figure 3: Sept2 localizes to mitochondrial constrictions. (A) Immunofluorescence analysis of endogenous Sept2 (green) and mitochondria (Tom20, red) in HeLa cells. The insets show a twofold enlargement with arrowheads highlighting the presence of small Sept2 structures on mitochondria. Scale bar 10 µm. (B) 3D rendering of confocal sections showing Sept2 at mitochondrial constrictions (white arrowheads) and constricted mitochondria that are not marked by Sept2 (yellow arrowhead) Scale bar 2 µm. (C) CoDiM super-resolution imaging of endogenous Sept2. Arrowheads highlight the presence of Sept2 at mitochondrial constrictions. Insets show a threefold enlargement Scale bar 10 µm. (D) Still images from a live cell imaging experiment showing Sept2 (green) accumulation during two mitochondrial fission events (mitotracker orange, red), highlighted by yellow (first fission) or green arrowheads (second fission). Scale bar 2 µm.
A role for Septin 2 in Drp1-mediated mitochondrial fission (2016)
Figure 4

Figure 4: Sept2 interacts with Drp1. (A) Western blot analysis of endogenous Sept2 immunoprecipitation from HeLa cells showing that endogenous Drp1 specifically co-immunoprecipitates with Sept2. The asterisks mark the IgG heavy chains. (B) Elyra SIM image of U2OS cells stained for Sept2 (green), mitochondria (GFP targeted to the outer membrane (OM-GFP), shown in red), Drp1 (blue). Yellow arrows point at tetraspeck beads used for image alignment. Insets represent sevenfold enlargements. White arrows in the insets point to sites where Drp1 and Sept2 colocalize on mitochondria. Scale bar: 10 µm, insets are enlarged fourfold. (C) GST pulldown of recombinant Sept2 with GST-Drp1 or GST alone showing that Sept2 is able to interact directly with Drp1. (D) Sept2 immunoprecipitation from mock or CCCP-treated HeLa cells probed for Drp1 and Sept2. (E) Quantification of two independent experiments showing stimulated Drp1 co-immunoprecipitation with Sept2 in FCCP-treated cells. (F) Sept2 immunoprecipitation from mock or Mdivi-1-treated HeLa cells probed for Drp1 and Sept2. (G) Quantification of two independent experiments showing decreased Drp1 co-immunoprecipitation with Sept2 upon Mdivi-1 treatment. (H) Snapshots of a live cell imaging experiment in U2OS cells showing Sept2 (green, Sept2-YFP), Drp1 (red, Drp1-mCherry) and mitochondria (blue, mitotracker DeepRed) during mitochondrial fission. Merged and single channels show the dynamics of Sept2 and Drp1 during mitochondrial fission (yellow arrowheads). Scale bar: 2 µm.
A role for Septin 2 in Drp1-mediated mitochondrial fission (2016)
Figure 5

Figure 5: Sept2 is required for efficient recruitment of Drp1 to mitochondria. (A) Immunofluorescence of Sept2 (blue), mitochondria (Tom20, red) and Drp1 (green) in mock treated and Sept2 depleted cells. Scale bar: 10 µm, insets are enlarged fourfold. (B) Quantification of mitochondria-associated Drp1 in mock treated and Sept2 depleted cells (mean of 3 independent experiments, n=44-51 cells) (C) Average distance between mitochondria-associated Drp1 oligomers (representative experiment with n>25 individual mitochondria). (D) HeLa cytosol and crude mitochondrial fractions prepared from mock and Sept2 siRNA treated cells. The samples were analysed by western blotting with indicated antibodies, and show a decrease in mitochondria-associated Drp1 in Sept2 depleted cells.
A role for Septin 2 in Drp1-mediated mitochondrial fission (2016)
Figure 6

Figure 6: Sept2 does not affect mitochondrial Myo2 localization and does not require actin dynamics for interacting with Drp1. (A) Immunofluorescence analysis of P-MLC (green) recruitment to mitochondria (red, Tom20) in mock treated and Sept2 depleted HeLa cells. Insets show twofold enlargements. Scale bar: 10 µm. (B) Colocalization of P-MLC with mitochondria was assessed with Icy software and showed no difference between mock and Sept2 depleted cells. (C) Mock treated or Sept2 depleted Drp1 -/- MEF cells were labeled with mitotracker orange (red), treated for the indicated amount of time with 2µM FCCP and stained for actin with phalloidin (green). Insets are enlarged twofold. (D) Immunofluorescence analysis of the actin binding proteins Arp3 and cofilin (green), colocalized with cytocrome c (red) and cortactin (green), colocalized with Hsp60 (red) in mock treated or Sept2 depleted Drp1 -/- MEF cells. Scale bars: 10 µm (E) Cytosol and crude mitochondria fractions were prepared from mock treated or Sept2 depleted Drp1 -/- MEF cells and analyzed by western blotting with the indicated antibodies. (F) HeLa cells were mock treated or treated with cytochalasin D and subjected to Sept2 immunoprecipitation. Immunoprecipitates were analyzed by western blot for Drp1 and Sept2, showing that Drp1 co-immunoprecipitation with Sept2 is not affected by cytochalasin D treatment.
A role for Septin 2 in Drp1-mediated mitochondrial fission (2016)
Figure 7

Fig. 7 Mitochondrial structure in muscle cells is affected upon knockdown of UNC-59 and UNC-61.

(A) Live imaging of C. elegans expressing TOM70::GFP in body wall muscle cells. Depletion of the UNC-59 or UNC-61 led to an increase in mitochondrial network connectivity as shown by quantification in (B). Insets represent a twofold magnification. Scale bar: 10 µm. (C) Live imaging of C. elegans body wall muscle cells expressing TOM70::GFP. Depletion of DRP-1 or UNC-61 caused an increase in mitochondrial elongation and network connectivity, which did not increase upon combination of DRP-1 and UNC-61. Insets represent a twofold magnification. Scale bar: 10 µm.
PTX3 regulates synaptic function inducing AMPARs clustering via extracellular matrix and β1 integrin (2018)
Figure 1

Figure 1 - PTX3 expression is developmentally regulated and astrocyte dependent.

A, B (A) ELISA assay and (B) real time qPCR from brain lysates showing higher PTX3 expression in late embryonic and early postnatal brain, and lower in the adult brain. Data are presented as mean±SEM. (A) ng PTX3/mg of total proteins, E15=5,702±0.365, P0=4.904±0.865, P7=3.818±0.530, P14=2.140±0.107, P28=3.125±0.525, P90=2.212±0.324. One-way ANOVA analysis of variance followed by post hoc Tukey test: E15 vs P14 **p=0.001, E15 vs P28 *p=0.022, E15 vs P90 **p=0.002; P0 vs P14 $p=0.013, P0 vs P90 $p=0.016 ; 4 animals for each time point. (B) qPCR fold change normalized on E15, E15=1.037±0.154, P0=0.711±0.094, P7=0.472±0.0524, P14=0.135±0.007, P28=0.168±0.020, P90=0.183±0.029. One-way ANOVA analysis of variance followed by post hoc Tukey test: E15 vs P7 ***p=0.0001, E15 vs P14 **** p<0.0001, E15 vs P28 ****p<0.0001, E15 vs P90 ****p<0.0001; P0 vs P14 $$$p=0.0007, P0 vs P28 $$p=0.001, P0 vs P90 $$p=0.002; 4 animals for each time point.

C, D) Quantitation of PTX3 levels in pure astrocyte and neuronal cultures. (C) ELISA assay performed on astrocyte and neuronal culture medium and (D) real time qPCR performed on astrocyte and neuronal lysates. Only astrocytes release PTX3 (ELISA, astro=3.699±1.129, neu=N.D., not detectable. 3 independent astrocyte cultures evaluated. qPCR astro=1.167±0.288; neu=0.148±0.057; Mann-Whitney test **p=0.0095. 2 independent neuronal cultures evaluated. Data are presented as mean±SEM.
PTX3 regulates synaptic function inducing AMPARs clustering via extracellular matrix and β1 integrin (2018)
Figure 5

Figure 5- PTX3 activity is inhibited by thrombospondin 1 through direct interaction.

A) PTX3 binds TSP1 and TSP-2 but not TSP-4. Different amounts of human recombinant PTX3 were incubated in microplate wells coated with purified human TSP1 or recombinant TSP-2 and TSP-4. Binding is reported as Absorbance at 450 nm (Mean±SD). Data are from one experiment out of three performed.

B) N-terminal PTX3 binds TSP1. Binding of PTX3 N-term domain was performed on TSP1 immobilized on plastic wells. Data are reported as Absorbance at 450 nm (Mean±SD) and are representative of one out of two experiments performed.

C) Schematic representation of TSP1 monomer and the TSP1 proteolytic fragments (N-term, C-term) and recombinant domains (P123-1, E123-1, E123CaG-1, Ca-1) used in the study.

D) PTX3 binds the C-term proteolytic fragment of TSP1. TSP1 and its fragments C-term, N-term, and Ca-1 (type III repeats) were immobilized in plastic wells (5µg/ml) and binding of PTX3 is reported as Absorbance at 450 nm (Mean±SD). Data refers to one out of two experiments performed with similar results.

E) PTX3 and its N-terminal domain bind TSP1 C-terminal globular domain. 50 nM of P123-1 (type I "properdin" repeats), E123-1 (Type II EGF repeats), E123CaG-1 (type II repeats plus type III repeats and globular C terminus) and TSP1 were immobilized in plastic well. Binding with PTX3 or N-terminal domain (both at 220 nM) was analyzed. Data are reported as Absorbance at 450 nm (Mean±SD) and refers to one out of two experiments performed with similar results.

F) Representative images of 14DIV control and PTX3-treated cultures stained for the presynaptic marker bassoon (blue) and the microtubule protein tubulin (red). Scale bar: 10μm.

G) Quantification of synaptic density (Bsn/μm) in the different experimental conditions (Ctr=0.385±0.029, PTX3=0.407±0.032, TSP1=0.721±0.039, PTX3+TSP1=0.707±0.037, E123=0.764±0.047, PTX3+E123=0.719±0.041. Number of fields examined: 86, 80, 75, 72, 46, 55 respectively; Kruskall-Wallis test, p<0.0001 followed by Dunn's test as indicated in figure; at least 3 independent experiments, data are presented as mean values±SEM).

H) Representative images showing 14DIV neurons stained for surface AMPAR (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm.

I) Quantification of the synaptic surface GluA density (number of sGluA puncta/μm) in the different tested conditions showing that GluA density increases in neuronal cultures treated with PTX3 or E123+PTX3 but not in cultures treated with the full length TSP1+PTX3 (Ctr=1.000±0.055; PTX3=1.367±0.074; TSP1=1.04±0.061; PTX3+TSP1=0.968±0.076; E123=0.971±0.063; PTX3+E123=1.613±0.077. Number of dendrites examined: 103, 80, 88, 57, 51, 62 respectively; Kruskall-Wallis test, p<0.0001 followed by Dunn's test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM).
Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis (2012)
figf2

(a) Relative CK2α protein concentration over time following cytoplasmic microinjection of REF52 cells with the HA-tagged semisynthetic proteins bearing different modifications using immunocytochemistry with HA-specific antibody. Data represent mean values ± s.d. (b) Relative CK2α protein concentration following injection of β subunit together with CK2α proteins. Data represent mean values ± s.d. (c) Relative CK2α protein concentration following injection of proteasome inhibitor (1 μM MG132) together with unmodified CK2α protein. Data represent mean values ± s.d. (d) Western blot for total CK2α protein in HeLa cells following treatment with Cdk inhibitors. Numbers indicate relative quantification normalized for loading control. Mean values (n = 3) shown ± s.d. (e) Western blot for total CK2α protein in HeLa cells following treatment with nocodazole to arrest cells in mitosis. Full gels corresponding to those in panels d and e are in Supplementary Figure 8.
Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis (2012)
figf3

(a) Pull-down experiments with semisynthetic HA-CK2α protein and recombinant Pin1. CK2α was immobilized using HA-specific (anti-HA) antibodies bound to protein G beads. Pin1 was incubated with immobilized CK2α for 20 min at 4 °C. (b) Coimmunoprecipitation of endogenous Pin1 with CK2α. Pfa-containing and unmodified semisynthetic HA-CK2α proteins were spiked into REF52 cell lysates and immobilized using HA-specific antibodies. Input samples are shown for loading control. (c) Pull-down experiments with Pfa344 HA-CK2α protein and recombinant Pin1 in the presence of Pin1-specific or nonspecific IgY. (d) Relative CK2α protein concentration over time following microinjection of Pfa344 CK2α with Pin1-specific IgY or nonspecific IgY (control). Data represent mean values ± s.d. Full gels corresponding to those in panels a–c are in Supplementary Figures 10 and 11.
Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis (2012)
figf4

(a) Phosphorylation of C-terminal tail CK2α peptides (residues 337–352) by Cdk1–cyclin B. In vitro Cdk1–cyclin B kinase assays were performed using the following peptide substrate: SSMPGGSTPVXSANMMK(ɛBiotin), where X = serine or S-GlcNAc serine. After reactions were quenched, peptides were separated from the reaction mixture on Tris-tricine gels. Data represent mean values ± s.d. (b) Immunoprecipitation and western blot for O-GlcNAc CK2α in HeLa cells after treatment with the O-GlcNAcase inhibitor TMG for 12 h. Numbers indicate relative quantification normalized for total CK2α concentrations. (c) Western blot for total CK2α protein in HeLa cells following treatment with TMG for 12 h or 16 h. Numbers indicate relative quantification normalized for loading control, mean values (n = 4) shown ± s.d. Full gels corresponding to those in panels b and c are in Supplementary Figures 14 and 15.
Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis (2012)
figf5

Phosphorimage analysis of in vitro CK2 kinase assays with protein substrates. All reactions were performed at 30 °C. Numbers indicate the concentration (nM) of product for each kinase reaction. (a) AHCYL2 substrate (25 ng μl−1) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min. (b) NAP1L3 substrate (15 ng μl−1) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min. (c) NIPBL substrate (50 ng μl−1) with 100 μM ATP and 5 nM CK2α ± 11 nM Pin1 for 5 min. Full gels corresponding to those in panels a–c are in Supplementary Figures 19 and 25.
Pool size estimations for dense-core vesicles in mammalian CNS neurons (2018)
Figure 1

Figure 1 - Number and distribution of DCV puncta in neurons in vitro and in vivo.

A Representative 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.

B 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.

C-F Analysis of hippocampal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. (C) Percentage of glutamatergic and GABAergic neurons in hippocampal cultures determined by immunostainings for VGLUT1 and VGAT, respectively. (D) DCV pool size measured as number of ChgB puncta per neuron. (E) Total neurite length (mm) per neuron. (F) ChgB puncta per μm neurite.

G-J Analysis of striatal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. (G) Percentage of glutamatergic and GABAergic neurons in striatal cultures determined by immunostainings for VGLUT1 and VGAT, respectively. (H) DCV pool size measured as number of ChgB puncta per neuron. (I) Total neurite length (mm) per neuron. (J) ChgB puncta per μm neurite.

K Correlation between ChgB puncta per neuron and total neurite length for hippocampal (green) and striatal (red) neurons. Linear regression, inset shows goodness of fit (r2) and significance (p-value) per group. Slopes are not significantly different, ANCOVA: p = 0.97.

L-P 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. (L) 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. (M-O) 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 (N, O) are indicated in top, right image (M). Arrows (O) indicate ChgA staining measured as individual ChgA puncta. Scale bars: 10 µm (M), 5 µm (N, O). (P) ChgA puncta per μm axon from mouse tissue, compared to the distribution of ChgB puncta in axons of cultured hippocampal neurons (Fig. 3K). Mann-Whitney U test: *** p = < 0.0008. N = 3 mice (52 images of 2 cortical regions per mouse).

Data information: Bars show mean ± SEM, grey dots represent measurements of individual single neurons. N numbers represent number of independent experiments and number of single neuron observations are in brackets.
Pool size estimations for dense-core vesicles in mammalian CNS neurons (2018)
Figure 2

Figure 2 - Quantification of total DCV pool in vitro and in vivo using super-resolution microscopy.

A Representation 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.

B-C 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).

D 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.

E 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).

F 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.

G 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.

H - I 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).
Pool size estimations for dense-core vesicles in mammalian CNS neurons (2018)
Figure 4

Figure 4 - Synaptic DCVs localize to the border of the synaptic vesicle cluster.

A-B Representative 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).

C 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.

D-E 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.

F Histogram of number of DCVs in percentage of synapses. Inset: number of DCVs per synaptic section.

G Average diameter of DCVs (nm).

H Distance of DCVs to the active zone (nm), measured from center of DCV to the middle of the active zone.

I Histogram of distance of DCVs to the middle of the active zone (nm). Inset: shortest distance of DCVs to the active zone (nm).

J Histogram of normalized distance of percentage of synaptic vesicles (grey) to the active zone and corresponding distance of DCVs (black) to the active zone.

K Example electron micrograph zooms of synapses containing DCVs outside (top) or inside (bottom) the synaptic vesicle cluster. Scale bars: 100 nm.

L Percentage of DCVs outside or inside the synaptic vesicle cluster.

M Histogram of distance (nm) of DCVs to the border of the synaptic vesicle cluster, negative values are inside, positive values outside.

Data information: Bars show mean ± SEM. N numbers represent number of experiments and number of single observations are in brackets. N= 2 (110 synaptic sections with in total 54 DCVs).