EMBO/soda-nel huggingface dataset:
Cx43-mediated sorting of miRNAs into extracellular vesicles (2022)
Figure 1

Figure 1 - Cx43 modulates the selective sorting of miRNAs into extracellular vesicles.

A. EV produced by HEK293 cells expressing or not Cx43 (EVCx43+ or EVCx43-) were purified by differential ultracentrifugation. miRNAs expression profile analysis in EVCx43+ (represented as fold increase over EVCx43-).

B. Pathway enrichment analysis for the predicted targets of the over-represented miRNAs in Cx43-containing EV.

C. HEK293 cells were incubated with 5 µg of purified EVCx43- or EVCx43+ for 24 h, in the presence or absence of Bafilomycin A1 (Baf) in the last 6 h. The levels of LC3 were analyzed by WB and are depicted on graph (LC3-II; n=4 biological replicates).

D. miRNA-centered regulatory network established by the miRNAs enriched in Cx43-containing EV. The figure represents predicted targets that are simultaneously regulated by 3 or more of the selected miRNAs. The size of the symbols is proportional to the number of established functional connections. miRNA-regulatory are represented by grey edges. Protein-protein interactions obtained from STRING database are depicted in black lines.

E. HEK293 cells were incubated with EVCx43- or EVCx43+ for 24 h before WB analysis of ATP2A2/SERCA2, a predicted target of miRNAs enriched in Cx43-containing EV. Graph depicts quantification of ATP2A2/SERCA2 levels (n=4 biological replicates).

Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.
Cx43-mediated sorting of miRNAs into extracellular vesicles (2022)
Figure 3

Figure 3 - Cx43 interacts with hnRNPA2B1 and hnRNPQ.

A. Cx43 was immunoprecipitated (IP) from HEK293Cx43+ cells, after which interaction with hnRNPA2B1 and hnRNPQ was analyzed by WB.

B. Co-localization between Cx43 (green) and hnRNPA2B1 or hnRNPQ (red) in C33a cells was analyzed by confocal microscopy. Scale bars 10 μm. Arrowheads in the inset images highlight co-localization between Cx43 and hnRNPs.
Cx43-mediated sorting of miRNAs into extracellular vesicles (2022)
Figure 5

Figure 5 - Cx43 participates in selective sorting of miR-133b.

A. Cell free EV biogenesis was assessed by relative protection of Cx43-luciferase. Reactions were incubated at 4ºC or 30ºC in the presence of membranes derived from HEK293Cx43- transiently transfected with Cx43-luciferase and cytosol from HEK293Cx43- cells, except in the negative control. 1% TX100 was used to disrupt membranes, where indicated (n=3 biological replicates).

B. Crude membranes and cytosol from broken HEK293Cx43+ or HEK293Cx43- cells were mixed in an ATP regenerating system, supplemented with synthetic miR-133b or miR-509-3p, as indicated. Graph depicts the indicated miRNAs levels, assessed by RT-qPCR and represented as percent protected after RNAse I digestion of unincorporated miRNAs (n=7-8 biological replicates).

C. Co-immunoprecipitation of endogenous miR-133b and miR-509-3p following UV crosslinking immunoprecipitation (CLIP) of Cx43 (n=3-5 biological replicates).

D. WB analysis for Cx43 in samples derived by miRNAs pulldowns performed with whole lysates of HEK293Cx43+ cells and biotinylated miR-133b and miR-509-3p (n=5 biological replicates).

E-H. EMSA was performed using either Cy3-labeled miR-133b (E,F) or miR-509-3p (G,H), and Cx43 produced by in vitro translation inserted into lipid nanodiscs, in the presence or absence of recombinant GST-hnRNPA2B1 or GST-hnRNPQ, as indicated. For competition EMSA, an excess of unlabeled miR-133b or miR-509-3p was used (competitor). Empty nanodiscs were used to control unspecific binding.

I. EMSA was performed using either Cy3-labeled wild type or mutant miR-199a-3p, and Cx43-enriched membranes prepared from C33a parental cells (Cx43+). Cx43- membranes were used to control unspecific binding.

Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.
Cx43-mediated sorting of miRNAs into extracellular vesicles (2022)
Figure 6

Figure 6 - Interaction of Cx43 with miR-133b in vitro is increased in the presence of hnRNPA2B1 and hnRNPQ.

Representative maximum projection images of microscopy-based miRNA-protein interaction using beads coated with GST-tagged NT/CT-Cx43, hnRNPA2B1 or hnRNPQ, as indicated, following the addition of Cy3-labelled miR-133b or miR-509-3p. Graph depicts quantification of total fluorescence/bead, as a measure of protein-miRNAs binding. Scale bars 50 μm (n=2 biological replicates; average of 23 beads analyzed/condition).

Data information: p-values were derived by Kruskal-Wallis (Dunn's post-hoc) test. Bars and error bars indicate mean ± SD in all graphs.
Cx43-mediated sorting of miRNAs into extracellular vesicles (2022)
Figure 7

Figure 7 - Cx43 facilitates the delivery of intraluminal RNAs to target cells.

A. Equal amounts of EVCx43+ and EVCx43- labelled with SYTO® RNASelect™ green fluorescent stain were incubated with HEK293Cx43+ cells for 30 min, at 37ºC. Scale bars 10 μm (n=5 biological replicates). p-values were derived by Mann-Whitney test.

B. Working model for the role of Cx43 in mediating the selective sorting of miRNAs into EV. Cx43 preferentially binds to miRNAs harboring a stable secondary structure, mediating its selective incorporation into EV. Although hnRNPs do not seem to directly participate in Cx43-dependent loading of EV-miRNAs, they strength the binding of miRNAs to cx43, suggesting that a multiprotein complex formed between RNA-binding proteins and Cx43 is important to select miRNAs.

Expanded View Figure Legends
A weakened interface in the P182L variant of HSP27 associated with severe Charcot-Marie-Tooth neuropathy causes aberrant binding to interacting proteins (2021)
Figure 1

Figure 1. The P182L mutation alters the IxI/V motif of HSP27 and disrupts its chaperone activity. (A) Domain architecture of human HSP27: N-terminal domain (N-term), α-crystallin domain (ACD), and C-terminal region (C-term). The conserved IxI/V motif in the C-terminal region is indicated. Missense, nonsense, and frameshift mutations associated with CMT disease and dHMN are shown. The P182L mutation in the IxI/V motif is blue. Residues with multiple mutations are italicized (e.g. P182L, P182S). The asterisk denotes a nonsense mutation and the X indicates a frameshift mutation. (B) Amino acid sequence alignment of IxI/V motifs and adjacent residues from vertebrate HSP27 orthologues: human (Homo sapiens), mouse (Mus musculus), alligator (Alligator mississippiensis), python (Python bivittatus), frog (Xenopus laevis), shark (Callorhinchus milii), gar (Lepisosteus oculatus), rice fish (Oryzias latipes), and lungfish (Protopterus annectens). (C) Domain boundaries of the ACD used in this study, cHSP27, and residues in the C-terminal IxI/V peptide. (D) Three-dimensional structure of the ACD dimer (black) bound to the IxI/V peptide from the C-terminal region (red). The β-strands of the ACD are numbered. (E) Zoomed-in region from D showing the contacts made between the IxI/V peptide and the ACD for the wild-type peptide. (F) Malate dehydrogenase (MDH; 0.2 µM) was incubated at 40 °C in the absence (black) or presence of 0.5 µM WT HSP27 (red) or 0.5 µM P182L HSP27 (blue). (G) Insulin (40 µM) was incubated at 40 °C in the absence (black) or presence of 20 µM WT HSP27 (red) or 20 µM P182L HSP27 (blue). The y-axes depict the absorbance at 340 nm due to the formation of large aggregates. The solid lines represent the average of three replicates with the filled area reflecting ± one standard deviation.
A weakened interface in the P182L variant of HSP27 associated with severe Charcot-Marie-Tooth neuropathy causes aberrant binding to interacting proteins (2021)
Figure 2

Figure 2. The P182L variant of HSP27 forms larger oligomers both in vitro and in vivo. (A) SEC-MALS data from WT HSP27 and the P182L variant, with cHSP27 included for comparison. All samples were injected at 40 µM concentration (monomer). The respective average molecular masses are 14 MDa, 470 kDa, and 18 kDa for P182L, WT HSP27, and cHSP27. The normalized UV signals are plotted on the left y-axis as solid lines and the molecular masses are plotted on the right y-axis as circles. (B) Fractionation of HeLa cells overexpressing either HSP27 or the P182L mutant by a 10% to 80% sucrose gradient. Western blot against anti-V5 is shown. (C) Expression of V5-epitope-tagged HSP27 (WT or P182L mutant) in HSP27 knock-out HeLa cells and immunostaining of HSP27. The scale bars correspond to the non-expanded dimensions, derived by dividing by the sample expansion factor of 4.3. (D) The P182L mutation decreases the density of HSP27 spots upon detection of local fluorescence intensity maxima and quantification of the number of detected spots in expanded samples (n = 30). The error bar is the standard deviation for the different cytoplasmic regions. The asterisks correspond to a P value < 0.0001 as obtained from a t-test. (E) Distribution of the intensity of individual spots (n = 3342) after Gaussian fitting of fluorescence intensity in the neighborhood of detected spots. The asterisks correspond to a P value < 0.0001 as obtained from a Mann-Whitney U test. Data were plotted with a square-root transformation of the y-axis to improve the visualization of the complete distribution of intensities. (F) Immunostaining of HSP27 (white) in motor neurons differentiated from CMT patient-derived iPSCs that harbor the heterozygous P182L mutation reveals large, cytoplasmic aggregates (center and right panels). The control motor neurons that contain WT HSP27 do not show evidence of any aggregates. The nucleus is stained with Hoechst (blue). Scale bar, 10 µm.
A weakened interface in the P182L variant of HSP27 associated with severe Charcot-Marie-Tooth neuropathy causes aberrant binding to interacting proteins (2021)
Figure 3

Figure 3. The P182L mutation diminishes the affinity of the IxI/V motif for cHSP27. (A) Primary sequences of the WT peptide (red) and P182L peptide (blue). The IxI/V motif is indicated with white text. Below: regions of 2D 1H-15N HSQC spectra of 15N-labeled cHSP27 (black) in the presence of increasing amounts of WT (red) or P182L peptide (blue). Resonances in the β4 and β8 strands broaden and disappear upon peptide binding, indicative of intermediate exchange. (B) Zoomed-in regions of resonances during the peptide titrations. The color bars indicate the amount of added WT or P182L peptide. (C) The combined 1HN and 15N chemical shift changes, denoted as chemical shift perturbation (CSP), are plotted as a function of added peptide concentration. Lines indicate the best fit to equation 2. The global Kd ± one standard deviation is shown in the upper left. Equation 2 only applies to resonances that are in fast exchange; the resonances in panel A therefore cannot be fit in this manner. (D) CSPs shown as a function of residue number for the WT (red) and P182L peptide (blue). (E) X-ray structure of cHSP27 bound to the WT peptide (PDB: 4mjh), shown in white sticks, with the results from (D) plotted onto the structure.
A weakened interface in the P182L variant of HSP27 associated with severe Charcot-Marie-Tooth neuropathy causes aberrant binding to interacting proteins (2021)
Figure 4

Figure 4. The P182L mutation attenuates the IxI/V association rate constant. (A, B) i. 15N CPMG RD data for residues in the β4 strand (T113) and the β8 strand (S154) in the absence of added peptide. CPMG data are shown for the same residues in the presence of ii. ca. 2% cHSP27-WT peptide complex or iii. ca. 2% of the cHSP27-P182L peptide complex. Uncertainties in R2,eff values are derived from the standard deviations of fitted peak intensities obtained from duplicated νCPMG values. (C) i. The association (kon) and dissociation (koff) rates for WT (red) and P182L (blue) IxI/V peptide binding to the ACD, calculated directly from the CPMG RD data. Residues that are impacted by the presence of a small amount of peptide are indicated by red (ii., WT) and blue (iii., P182L) spheres. In both cases, residues in similar ACD regions show enhanced Rex, indicative of chemical exchange at similar sites. Error bars were obtained using standard error propagation and represent one standard deviation from the calculated value. (D) Free energy landscape of the WT (left) and P182L (right) peptides calculated from the MD trajectories as a function of the P182 or L182 φ/ϕ angles. A lower value of ΔG corresponds to a higher relative population. Select IxI/V conformations extracted from the MD simulations are shown for the indicated φ/ϕ angle pairs in red (WT MD) and blue (P182L MD). The conformation of the IxI/V motif in the crystal structure is shown in grey (X-ray) and the P182L reference structure in grey (Reference), as obtained with in silico mutagenesis using PyMol. The black arrows indicate rotations about the φ or ϕ angles. The green circle denotes the φ/ϕ angles of P182 when bound to cHSP27, measured from the crystal structure. (E) Contour plot showing the first and second principal components of a PCA for the MD trajectories for the WT (left) and P182L peptide (right). The larger spread in the P182L peptide suggests a larger sampling of the conformational landscape.
A weakened interface in the P182L variant of HSP27 associated with severe Charcot-Marie-Tooth neuropathy causes aberrant binding to interacting proteins (2021)
Figure 5

Figure 5. The P182L variant binds with enhanced affinity to IxI/V-containing proteins. (A) Percentage of [V/I]X[V/I] tripeptides among (top) the total number of all tripeptides (% tripeptides) or (middle) the total number of [V/I]X[V/I] tripeptides in the structured (black) or disordered regions (red) ([V/I]X[V/I] %) shown as a function of the X residue type. (Bottom) The difference between [V/I]X[V/I] % for disordered and structured regions shows the enrichment (positive) or depletion (negative) of specific [V/I]X[V/I] motifs in disordered regions. The grey bars indicate the [V/I]L[V/I] and [V/I]P[V/I] motifs studied in this work, i.e. ILV and IPV. (B) Volcano plot representing the interactors that are enriched for the P182L mutant versus GFP as a negative control, obtained with affinity-enrichment mass spectrometry. Proteins that co-immunoprecipitated significantly more (p<0.05) with the P182L variant are shown in the upper right quadrant. Interactors with one or more [I/V]-x-[I/V] motifs are displayed in red while other significantly enriched interactors are displayed in black. (C-D) Co-immunoprecipitation from HeLa cells stably overexpressing V5-epitope-tagged HSP27 (WT or P182L mutant) using anti-V5 beads. Co-immunoprecipitation was quantified and presented as a percentage relative to the P182L variant (n=3). The mean ± one standard deviation (n=3) is shown. Right: the location of Pro182 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the P182L mutation (blue). The peptide is shown in white sticks. P182L specifically weakens binding of the HSP27 IxI/V motif. (E) Co-immunoprecipitation from HeLa cells stably overexpressing V5-epitope-tagged HSP27 (WT or P182L mutant) and transiently transfected with BAG3-eGFP (WT or IPV-mutant) using anti-GFP beads. Western blots for anti-GFP (BAG3), V5 (HSPB1), and α-tubulin. NS indicates a non-specific band. Note that detection of BAG3 (anti-GFP) and HSP27 (anti-V5) occurs with different antibodies. The locations of the IPV-to-GPG mutations in BAG3 are depicted below. (F) Co-immunoprecipitation from HeLa cells transiently transfected with V5-epitope-tagged HSP27 (P182L or S155Q/P182L mutant) using anti-V5 beads (n=2). Right: the location of Ser155 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the S155Q mutation (purple) showing a clash with the bound peptide. The peptide is shown in white sticks.S155Q disrupts binding of all IxI/V motifs.
SCHENGEN receptor module drives localized ROS production and lignification in plant roots (2020)
Figure 1

Fig. 1 Apolar SGN1 leads to ectopic lignin accumulation in endodermal cells

(A) Schematic of Casparian strip development (magenta). Casparian strips start to appear as centrally aligned discontinuous dots in the endodermal cell layer, progressing into a network of fused rings functioning as a root apoplastic barrier.

(B) Lignin accumulation patterns at endodermal surface or median positions with or without the 100 nM CIF2 (Casparian strip integrity factor 2) ligand treatment. Lignin and cellulosic (unmodified) cell walls are stained with Basic Fuchsin and Calcofluor White, shown in magenta and white respectively. Schematics are indicating the position of optical sections in a 3D illustration. For each condition, at least 10 roots were tested and showed similar results in two independent experiments. White arrows indicate sites of excess lignification on the cortex facing (outer) side. Scale Bar = 5 µm.

(C,D) Localization of SGN1-Citrine and lignin deposition patterns in pCASP1::SGN1-Citrine lines in wild-type (Col) and different mutant backgrounds (sgn1, sgn3, cif1 cif2) (C). myrpalm-SGN1-Citrine localization and lignin deposition patterns in pCASP1::myrpalm-SGN1-Citrine lines (D). Lignin (Basic Fuchsin) and cell walls (Calcoflour White) are shown in magenta and white respectively. For this experiment, two or three independent lines were tested. From each transgenic line, 2 positions from 12 roots were observed and representative pictures are shown in the figure. Schematics are indicating the position of optical sections in a 3D illustration. White arrows in (D) highlight excess lignification on the pericycle-facing (inner) side. Scale bars = 5 µm.

(E) Schematic illustrating how signal activation can be governed by SGN1 localization and peptide ligand diffusion from the stele.

Data information: "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.
SCHENGEN receptor module drives localized ROS production and lignification in plant roots (2020)
Figure 2

Fig. 2 SGN1 acts as a transducer of CIF2 signaling and is phosphorylated by the SGN3 receptor

(A) Quantification of defects in CSD formation as number of holes per 100 µm in the CASP1-GFP domain at around 10 cells after onset of CASP1-GFP expression in 5-day-old seedlings. In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to maximum or minimum values no further than 1.5 times IQR (interquartile range, the distance between the first and third quartiles). One-way ANOVA was performed followed by Tukey's test. Different letters show significant statistical differences. (p<0.05, One-way ANOVA and Tukey's test, 12 roots in total were observed for each condition in two independent assays).

(B) Propidium iodide (PI) penetration assay in the presence or the absence of CIF2. CS barrier function was scored as exclusion of PI signal from the inner side of endodermal cells. In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters show significant statistical differences. (p<0.05, One-way ANOVA and Tukey's test. During two independent experiments, 10 roots in total were tested for each condition).

(C) [γ-32P]ATP radioactive in vitro kinase assay of SGN3 kinase domain against SGN1. Autoradiograph is shown on top. Coomassie stained-gel below illustrates presence and equal loading of recombinant proteins. Note that a kinase-dead SGN1 variant was used to avoid auto-phosphorylation activity of SGN1. Also note that trigger factor represents a very big tag protein, accounting for the high migration of TF-SGN1. Representative result of three independent experiments is shown.
SCHENGEN receptor module drives localized ROS production and lignification in plant roots (2020)
Figure 3

Fig. 3 Both RBOHD and F are required for CIF2-induced excess lignin accumulation

(A) Localization of Citrine-RBOHD (left) or Citrine-RBOHF (right) in endodermal (en) cells. Both proteins were expressed under the control of pCASP1, an endodermis-specific promoter. Representative pictures are shown, 2 positions from 10 roots for each transgenic line were inspected. Scale bar = 5 µm.

(B) Lignin accumulation in WT and rbohD and rbohF single mutants and a double mutant with or without 2-hour 100 nM CIF2 peptide treatment. Arrowheads indicate excess lignification. Pictures are shown as overviews (maximum projection) or median sections. Lignin and cell walls are shown with magenta (stained with Basic Fuchsin) and gray (stained with Calcofluor White) respectively. Representative pictures are shown, 12 roots (overview) and 2 positions in 12 roots (median section) were inspected. Scale bars = 20 µm (lignin overviews), 5 µm (median sections). "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.
SCHENGEN receptor module drives localized ROS production and lignification in plant roots (2020)
Figure 4

Fig. 4 ROS production is enhanced by SGN3/CIFs and requires RBOHD and F

(A) Co-visualization of pCASP1::SGN1-mCherry and pCASP1::SGN3-GFP. Note that their respective localization at the PM has only restricted overlap at the cortical side of the CS domain (Arrow). Representative picture is shown, 3-6 roots in 4 different transgenic lines were inspected. Scale bar = 10 µm. "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.

(B,C) Overview of endodermal cells after with or without 1 µM CIF2 treatment for 24 hours. Red arrowheads are indicating ROS production sites and boxes in dotted lines are corresponding to the regions in (sky blue and creme boxes in C and black boxes in D). Similar patterns were obtained in 39 or 41 cells from 5 roots with or without the peptide treatment. Scale bars = 500 nm.

(D) In situ H2O2 detection at Casparian strips in WT, sgn3, sgn1, rbohF, rbohD and rbohDF with or without 24-hour treatment of 1 µM CIF2. Brackets and arrowheads indicate Casparian strips (seen as uniformly whitish cell wall areas) and H2O2 production sites (black area) respectively. Scale bar = 1000 nm.

(E) Quantification of ROS production as a number of dark pixels area (n = 23 - 44 sites from 5 roots of each condition in one experiment.). For quantification methods, see fig. EV 3. In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters mean significant statistical differences. (p<0.01, One-way ANOVA and Tukey's test.)
SCHENGEN receptor module drives localized ROS production and lignification in plant roots (2020)
Figure 5

Fig. 5 SGN1 directly activates NADPH oxidases in a cellular context

(A) [γ-32P]ATP radioactive in vitro kinase assay of TF-SGN1 against GST-N-terminal cytoplasmic domains of RBOHD or F. Autoradiograph is shown on top. Coomassie stained-gel below illustrates presence and equal loading of recombinant proteins. Experiments were done independently three times with similar results.

(B) HEK293Tcell based NOX activation assay. Cells were transfected with the indicated plasmid combinations. The phosphatase inhibitor Calyculin A was added directly before the start of the measurements. Each data point represents the mean of six wells analyzed in parallel, bars indicate S.D.. Experiments were repeated three times (another set of results is shown in Fig. EV 4B).