EMBO/soda-nel huggingface dataset:
Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3 (2020)
Figure 3

Figure 3: Reduced kainate-evoked inward currents in the absence of SEZ6.

(A) Left: Current trace resulting from whole-cell voltage-clamp recordings at a holding potential of -70 mV in a CA1 pyramidal neuron in a WT mouse hippocampal slice (P15). The current was recorded in the presence of 10 µM bicuculline, 20 µM GYKI53655 and 30 µM APV in the ACSF. Perfusion of the recording chamber with 10 µM kainate and 100 µM 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) are indicated with green and blue bars, respectively. Right: Analogous experiment in a SEZ6KO mouse (P15).

(B) Summary of the experiments in A. Bar graph illustrates mean charge with standard error of the mean carried by the inward currents (measured as area under the curve, AUC) for the two genotypes at P15 (WT 46.6 ± 0.5 nC (n=10 cells), SEZ6KO 30.8 ± 0.5 nC (n=9 cells), *p=0.0375 Mann-Whitney-U-test).
Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3 (2020)
Figure 4

Figure 4: Loss of SEZ6 impairs glycosylation and maturation of GluK2/3.

A) In WT neurons, GluK2/3 was seen as two closely comigrating bands, whereas the upper one of lower intensity was missing or running even more closely to the lower band of main intensity in SEZ6KO neurons (schematic representation on the right). When the total lysates of SEZ6KO and WT neurons were digested with endoglycosidase H (EndoH) - which removes immature, but not mature sugars - the two closely comigrating bands were converted to three bands of a lower apparent molecular weight, consistent with full deglycosylation of the lowest band and a partial deglycosylation of the upper two bands (marked in light blue, red and green in the right panel). In SEZ6KO neurons the uppermost (light blue) band was missing and a new band of lower apparent molecular weight was seen that was overlapping with the red labeled band and was indicated with the purple asterisk in the SEZ6KO. No difference in the glycosylation of GluA2 was detectable, pointing to a specific effect of SEZ6 on GluK2/3.

B) The running pattern of GluK2/3 in SEZ6KO and WT brains was compared upon EndoH digestion. Similar to primary neurons, GluK2/3 displayed an immature glycosylation pattern in SEZ6KO brains.

C) Total GluK2/3 levels were quantified and showed a significant but moderate reduction in SEZ6KO compared to WT neuronal lysates (plot shows mean ± S.E.M., 9 replicates in 3 independent biological experiments, Mann-Whitney **p-value=0.0012).

D) No significant change in GluK2/3 total levels was detectable in SEZ6KO and WT adult brains (plot shows mean ± S.E.M., 6 replicates, Mann-Whitney test was used).

E) Synaptosomes from knock-out (KO) brains of single SEZ6 family members (SEZ6, SEZ6L, SEZ6L2), triple knock-out (TKO) and respective controls (WT and TWT - triple WT) were digested with EndoH. GluK2/3 displayed the immature glycosylation in SEZ6KO and TKO brains, but not in SEZ6L and SEZ6L2 brains, demonstrating non-redundant functions for the SEZ6 family members. (n=4)

F) Brain homogenates from SEZ6KO and WT mice at different ages were digested with EndoH. GluK2/3 displayed the immature glycosylation at all developmental stages of SEZ6KO mice, suggesting that no compensation effect is occurring with aging.
Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3 (2020)
Figure 5

Figure 5: Loss of SEZ6 reduces HNK-1 epitope on GluK2/3.

A) The glycome fingerprint of GluK2/3 was analyzed by lectin chip microarray (LecChip). Lectin PHAL and PHAE (indicated with asterisks) detected reduced amounts of the oligosaccharide Galβ1-4GlcNAcβ1 on GluK2/3 in SEZ6KO neurons (plot shows mean ± S.E.M., 3 WT replicates and 4 SEZ6KO replicates were used, discoveries were determined using the FDR method of Benjamini and Hochberg, with Q = 2%).

B) Immunoblot analysis revealed reduction of HNK-1 epitope on GluK2/3 in SEZ6KO brains. Left panels: Comparable amounts of GluK2/3 were immunoprecipitated (IP) in WT and SEZ6KO brains using excess of brain homogenates compared to beads. Anti-HNK-1 antibody was used for detection. HNK-1 band on GluK2/3 was detected only in WT brains but not in SEZ6KO brains (n=6). As a control, HNK-1 modification of NCAM-1 was unaltered, as revealed after IP of NCAM-1 from WT and SEZ6KO brains (n = 3), followed by detection with anti-HNK-1 antibody. Different isoforms of NCAM-1 are annotated with arrowheads (NCAM 180, NCAM 140 and NCAM 120). Mid panel: To prove the specificity of the HNK-1 antibody, GluK2/3 was immunoprecipitated and digested with Peptide-N-Glycosidase F (PNGase F), which removes N-linked oligosaccharides. Upon PNGase F digestion, HNK-1 was not detectable in WT brains and the molecular weight of GluK2/3 was reduced (n=2). Right panels: In total brain homogenates (input), no difference of general HNK-1 epitope or NCAM-1 levels was detected.
Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3 (2020)
Figure 6

Figure 6: RUSH assay to analyze trafficking of SBP-GFP-GluK2 in HEK293T cells.

A) Schematic representation of the RUSH Cargo Sorting Assay using Confocal Microscopy. HEK293T cells were transfected with the SBP-GFP-GluK2 plasmid, that expresses a fusion protein consisting of streptavidin binding peptide (SBP) followed by GFP and GluK2, and a streptavidin-KDEL "anchor". The ER retention signal KDEL retains SBP-containing proteins in the ER. Upon addition of biotin to the cell culture medium, SBP-GFP-Gluk2 is released allowing its trafficking through the secretory pathway.

B,C) The constitutively ER-retained SEZ6 mutant SEZ6∆cytoER-HA (B) and full length wild-type SEZ6FL-HA (C) were co-transfected with SBP-GFP-GluK2. Biotin was added to elicit release of SBP-GFP-GluK2 from the ER (0 min) and cells were fixed at different time points (0, 20 and 40 min). HA-tagged SEZ6FL and SEZ6∆cytoER transgenes were labeled with anti-HA (mouse) and anti-mouse-Alexa594 antibodies. Size bars represent 5 µm.

D) Scatter dot plot represents number of vesicles per cell in SBP-GFP-GluK2 RUSH experiment with SEZ6∆cytoER-HA (control) or SEZ6FL-HA co-transfection. Vesicle counts from 15 to 28 cells per timepoint from 2 independent experiments with mean number of vesicles and error bars (SD) are shown. Mann-Whitney test was used to compare HA-tagged SEZ6FL and SEZ6∆cytoER at each time point. At 20 min ***p-value=0.0007, at 40 min ****p-value<0.0001.
Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3 (2020)
Figure 7

Figure 7. SEZ6 facilitates GluK2 trafficking in primary neurons.

A) Representative pictures of WT hippocampal neurons transfected with the SBP-mCherry-GluK2 RUSH construct, followed by treating neurons with biotin and anti-SBP antibodies (mouse, green) for 20 min or 40 min to live-label the GluK2 transported to the neuronal cell surface. As a control (no biotin), neurons were incubated only with SBP antibody for 40 min. The surface/total ratio of SBP-mCherry-GluK2 constructs was estimated by calculating the ratio of SBP (green)/ mCherry (red) fluorescence signals in the same area chosen for quantification.

B) Quantification results for A, indicating that incubation with biotin increases the surface/total ratio of SBP-mCherry-GluK2 over time. No Biotin n=16, 20 min n=13, 40 min n=15, all biological replicates. Graph shows mean±SEM, 1-way ANOVA p<0.0001 and Dunnett's post hoc test, No Biotin vs 20 min *p=0.0030; No Biotin vs 40 min ***p<0.0001.

C-D) SEZ6 flox/flox neurons were lentivirally infected with inactivated Cre (Y324F, Ctrl) or Cre recombinase (SEZ6KO), followed by transfection with the SBP-mCherry-GluK2 construct and treated with biotin and anti-SBP antibodies (mouse, green) for 20 min. Expression of inactivated Cre or Cre recombinase were confirmed by immunostaining with an anti-Cre antibody (rabbit, blue). SEZ6KO neurons showed a significantly reduced surface/total ratio of SBP-mCherry-GluK2 (ctl n=25, SEZ6KO n=23 biological replicates. Graph shows mean ± SEM, unpaired t-test, **p=0.0068).

Data information: Scale bars represent 20 µm.
Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3 (2020)
Figure 8

Figure 8: SEZ6 extracellular domain in the secretory pathway rescues GluK2/3 maturation in SEZ6KO neurons and binds to GluK2/3.

A) Schematic representation of the constructs used: SEZ6 full length (SEZ6FL), SEZ6 lacking the C-terminal domain (SEZ6Δcyto), SEZ6 ectodomain generated by BACE1 cleavage (SEZ6ecto), SEZ6 C-terminal fragment generated by BACE1 cleavage (SEZ6CTF) and SEZ6 lacking the C-terminal domain but fused to an ER retention signal (SEZ6ΔcytoER).

B, C) SEZ6 constitutive KO neurons were transduced with SEZ6 constructs. SEZ6FL and SEZ6Δcyto were detectable as two bands corresponding to mature (higher) and immature (lower) forms of SEZ6. No difference in the apparent molecular weight was detected because the high percentage of the gel used (12%) and the small C-terminal deletion (39 amino acids). SEZ6ecto was present as the immature band in the neuronal lysates and as the mature, secreted band in the conditioned medium in panel C. SEZ6CTF (with HA and Flag-tags) was detected at the expected low molecular weight. Additionally, the C-terminal fragment generated from SEZ6FL by BACE1 cleavage was detected. Due to its lack of an HA-tag, it had a lower molecular weight than SEZ6CTF. The C-terminal fragment generated from SEZ6Δcyto upon BACE1 cleavage was not visible because of its low molecular weight. GluK2/3 glycosylation was rescued when SEZ6FL, SEZ6Δcyto and SEZ6ecto were expressed in the neurons, but SEZ6CTF was not able to rescue the phenotype. Color coding for asterisks is the same as in figure 4A. Representative blot of 3 independent experiments.

D) Sez6 constitutive KO neurons were transduced with SEZ6ΔcytoER and SEZ6Δcyto. SEZ6ΔcytoER presented only the band corresponding to the immature form of SEZ6, as expected for a protein retained in the ER. SEZ6Δcyto, but not SEZ6ΔcytoER, was able to rescue GluK2/3 glycosylation, showing that SEZ6 localized to the ER is not sufficient to rescue the GluK2/3 glycosylation (n=5). Color coding for asterisks is the same as in figure 4A.

E) HEK293T cells were co-transfected with a GluK2a plasmid and the SEZ6 constructs. Expression of SEZ6 constructs was analyzed in the total lysates ("Input") and revealed a similar expression of the different proteins. GluK2a was immunoprecipitated with GluK2/3 antibody and the SEZ6 mutants were detected with the HA.11 antibody ("IP"). SEZ6FL, SEZ6Δcyto and SEZ6ecto, but not SEZ6CTF were co-immunoprecipitated with GluK2a, suggesting that SEZ6 extracellular domain is necessary for GluK2a binding. Representative blot of 2 independent experiments. * indicates the light chain of the antibody used for immunoprecipitation.

Expanded View Figure Legends
Reversible HuR-microRNA Binding Controls Extracellular Export of miR-122 & Augments Stress Response (2016)
Figure 4

Figure 4. HuR driven miR-122 export from mouse liver enhances serum miR-122 content

(A) Schematic representation of starvation experiment in BALB/c mice.

(B) Change in miR-122 level in starved mouse liver. Endogenous miR-122 (left panel) and CAT-1 mRNA (middle panel) levels in mouse liver starved for 12h were quantified by qRT-PCR. miR-122 levels in the blood serum of corresponding animals were also measured (right panel). The Ct values of miR-122 and CAT-1 mRNA obtained for each individual animal liver and blood serum were plotted (mean+/- s.e.m., n=5).

(C)Relative level of HuR in both liver and lung of Fed and Starved mice were analysed by western blots, β-actin was used as loading control .

(D) Schematic representation of HA-HuR expression or Control plasmid injection experiment in BALB/c mice.

(E) Western Blot analysis to check expression of HA-HuR in liver samples of injected mice. β-Actin was used as loading control. HA-HuR expressing Huh7 cells lysate was used as positive control.

(F). Change in miR-122 level upon expression of HA-HuR in mouse liver. Endogenous miR-122 level in HA-HuR expression plasmid injected mouse liver were quantified by RT-PCR and compared against liver RNA samples collected from control (pCIneo injected) group of animals (mean+/- s.e.m., n=5).

(G) Endogenous miR-122 target CAT-1 (left panel) and Aldolase (right panel) mRNA levels in HA-HuR or pCIneo (control) injected mouse liver were quantified by RT-PCR (mean+/- s.e.m., n=4).

(H) miR-122 levels in the blood serum of pCIneo (control) or HA-HuR expression plasmid injected mice were measured. The Ct values of miR-122 and CAT-1 and Aldolase mRNAs obtained for each individual animal liver and blood serum were plotted (mean+/- s.e.m., n=5).

ns: non-significant, * P<0.05, **P<0.01, ***P<0.0001.P values were determined by paired t test. d.p.i: days post infection. Position of size markers in protein gels used for respective western blot analysis are shown against each panels. For estimations of relative level of either miRNAs or mRNAs Ct values only within the range of 20-32 were considered for analysis.
Direct interaction with DRP1 activates BAX and induces apoptosis (2021)
Figure 1

Figure 1. BAX and DRP1 specifically interact at mitochondria during apoptosis.

A-D) Dual-color SMLM shows co-localization of BAX and DRP1 at the nanoscale. A) Overview SMLM image of a HeLa cell transfected with GFP-BAX stained with anti-GFP-nanobody-AF647 and anti-DRP1 antibody probed with a CF680-labelled secondary antibody, 3 h after apoptosis induction. Scale bar 2 μm. B) Magnified SMLM images of GFP-BAX and DRP1 assemblies co-localizing up to 30 nm during apoptosis. Scale bar 100 nm. C) Co-localization control of DRP1 immunostaining with the same primary and different secondary antibodies (labelled with AF647 and CF680) in HeLa cells. Scale bar 100 nm. D) Quantification of the distance from GFP-BAX to DRP1 structures (distance measured at the center of each structure) from dual-color SMLM images. Data are quantified from n=4 independent experiments with a total of 720 BAX structure.

E,F) Representative confocal microscopy images of HeLa cells transfected with ddFP RA-BAX and GB-DRP1 (E) in untreated or apoptotic (STS) conditions, or with RA-BAX and GB-BAX during apoptosis (F). RA-BAX/GB-DRP1 and RA-BAX/GB-BAX complexes shown in green, mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm. Right panels are zoomed areas representing individual and merged channels. Scale bar 5 μm.

G) BAX/DRP1 interaction compared to BAX/BAX and BCL‑xL/DRP1 by ddFP. % mito-BFP transfected cells that show RA/GB signal was quantified in HeLa cells untreated (untr.) and 3 h after apoptosis induction (STS). Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, *** p < 0.001) compared to BAX/DRP1 after apoptotic induction (STS).
Direct interaction with DRP1 activates BAX and induces apoptosis (2021)
Figure 2

Figure 2. Dynamics of BAX and DRP1 interaction during apoptosis.

A) Confocal microscopy time-series images of the increase in cytosolic Smac-GFP (magenta) in relation to the detection of RA-BAX/GB-DRP1 complexes (green) during apoptosis induction in MEF DRP1 KO cells. Scale bar 10 μm. Images are representative for n=3 independent experiments.

B) Normalized fluorescence intensity of Smac-GFP in a region of interest in the cytosol and of RA-BAX/GB-DRP1 foci for individual cells (thin lines) and the average of all cells (thick lines, n=5). Time 0 min corresponds to the time point when RA-BAX/GB-DRP1 foci were detected.

C) Quantification of BAX/DRP1 interaction (% cells with RA-BAX/GB-DRP1 foci normalized to the mito-BFP positive cells, n=100 cells per experiment) during inhibition of effector caspases (Q-VD-OPh), the mitochondrial permeability transition pore (CsA) versus control (DMSO), with or without apoptosis induction (+/- STS). Box plots represent the interquartile (box), median (line) and SD (whiskers). Significance was determined from n=3 independent experiments (with n=100 cells each) by paired two-tailed Student´s t-test (n.s. indicating p > 0.05) compared to DMSO control after apoptotic induction (STS).
Direct interaction with DRP1 activates BAX and induces apoptosis (2021)
Figure 3

Figure 3. Direct interaction of BAX and DRP1 in the membrane affects their respective activities.

A-B) Representative auto- (green and violet curves) and cross-correlation (CC, BAX-DRP1, grey curves) curves of DRP1-AF488 and BAX-AF633 measured by FCCS in solution (A) and in the membrane of GUVs (B). Dash gray line depict raw data and solid lines correspond to data fitting.

C) Quantification of %CC between DRP1-AF488 and BAX-AF633 in solution (grey), in the membrane (violet), and in the membrane (beige) in presence of excess unlabeled cBID. Box plots represent the interquartile (outer box), mean (inner box), median (line) and range (whiskers). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, ** p < 0.01) from n=9 measurements in solution, n=46 individually measured GUVs in the membrane or n=17 GUVs in presence of cBID.

D) Effect of DRP1 on BAX-induced LUV permeabilization. BAX was activated by cBID (blue lies), or mild heat (42°C, grey lines). Data are presented as mean ± SD of n=3 individual experiments. Significance was determined at the end-point of the kinetic measurement (180 min) by paired two-tailed Student´s t-test (* p < 0.05, *** p < 0.001).

E) Effect of DRP1 on BAX-induced GUV permeabilization. Left: % GUVs permeabilized to Cytochrome c488 (Cyt c, 12 kDa, beige) and allophycocianin (APC, 104 kDa, blue) in the absence or presence of cBID, BAX and DRP1 combined as indicated. Data are presented as mean ± SD of n=4 independent experiments. * p < 0.05 (paired two-tailed Student´s t-test). Right: representative confocal microscopy images showing GUVs (grey) in a solution of Cyt c (green) and APC (magenta). Scale bar 10 µm.

F-G) Effect of BAX on DRP1 membrane density and DRP1-induced shape alterations of liposomes measured by flow cytometry. F) Representative flow cytometry plots outlining DRP1 (Alexa Fluor 488 signal) binding to LUVs (Rhodamine signal) in the absence or presence of BAX. % DRP1-positive LUVs indicated in green. G) Membrane density of DRP1 (corrected fluorescence units, cFU, left graph) and DRP1-induced membrane tethering (indicated by a shape index > 1, right graph) in LUVs in the absence or presence of different concentrations of BAX. Data are presented as mean ± SD of n=3 independent experiments. * p < 0.05 (paired two-tailed Student´s t-test) vs. DRP1 without BAX.
Direct interaction with DRP1 activates BAX and induces apoptosis (2021)
Figure 4

Figure 4. Interaction surfaces between BAX and DRP1.

A-D) BAX peptide array to define interaction-site with DRP1. A) Schematic representation of the peptide array corresponding to BAX secondary structure with boxes indicating BAX α-helices 1-9. BAX sequence was divided in peptides of 15 amino acids (aa) preceded by a biotin head. The last 5 aa of each peptide overlapped with the first 5 aa of the next peptide. B) Graphical representation of the DRP1 binding assay. Biotin-containing BAX peptides were attached to GUVs using biotinylated lipids and streptavidin. Binding between BAX peptides and DRP1-AF488 results in a recruitment of DRP1-AF488 to the GUV membrane indicated by AF488 fluoresce detected at the rim of the GUV. C) Representative images of GUVs incubated with DRP1-AF488, streptavidin and the corresponding BAX peptides (#3, #4, #12, #15). Scale bar 10 µm. D) Quantification of the relative fluorescence intensity of DRP1-AF488 at the rim of the GUVs for each BAX peptide of n=3 individual experiments (with n=100 vesicles each). Black lines indicate the mean value. Interacting BAX peptides are highlighted in beige.

E-F) Protein crosslinking coupled with mass spectrometry of BAX and DRP1 in liposomes. E) BAX/DRP1 crosslinking analysis workflow. After incubation with crosslinking reagents, proteins were digested using trypsin subjected to LC-MS/MS analysis. F) Schematic representation of BAX and DRP1 with detected cross-linked residues connected by dash lines using DSS and EDC cross-linkers as indicated.
Direct interaction with DRP1 activates BAX and induces apoptosis (2021)
Figure 5

Figure 5. Regions required for interaction between BAX and DRP1 in apoptotic cells.

A) Schematic representation of RA-BAX mutant and deletion variants analyzed for the interaction with GB-DRP1. The variants are displayed in the corresponding region of BAX secondary structure (boxes correspond to BAX helices 1-9 as indicated).

B) Representative confocal microscopy images of U2OS BAX/BAK DKO cells of the interactions between RA-BAX variants and GB-DRP1, shown as green signal, 3 h after apoptosis induction. Mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm.

C-E) Quantification of the interaction of DRP1 with variants of BAX based on the % cells with detectable RA-BAX/GB-DRP1 fluorescence signal (interacting cells) normalized to the number of mito-BFP positive cells. Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) compared to BAX wild type (WT).

F) Schematic representation of GB-DRP1 mutant variants analyzed for the interaction with RA-BAX. The variants are displayed in the domain distribution of DRP1 (amino acid positions correspond to DRP1 isoform 3).

G) Representative confocal microscopy images of U2OS BAX/BAK DKO cells of the interactions between RA-BAX and GB-DRP1 variants, shown as green signal, 3 h after apoptosis induction. Mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm.

H) Quantification of the interaction of DRP1 variants with BAX based on the % cells with detectable RA-BAX/GB-DRP1 fluorescence signal (interacting cells) normalized to the number of mito-BFP positive cells. Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Significance was tested using paired two-tailed Student´s t-test (n.s. p > 0.05) compared to DRP1 WT.