EMBO/soda-nel huggingface dataset:
Loss of neuronal Miro1 disrupts mitophagy and induces hyper-activation of the integrated stress response (2021)
Figure 1

Figure 1: Delayed Parkin recruitment to mitochondria in MiroDKO cells upon mitochondrial damage.

(A) Representative images (and details from boxed regions) of WT MEF cells transfected with YFPParkin (green) and treated with FCCP (10 μM) to induce mitophagy. Cells were immunostained with anti-Tom20 (magenta) to reveal the mitochondrial network. Each row represents one stage in the process of mitophagy that has been used to score the progression of mitophagy at different time-points for the different conditions tested (the specific time-point of each example is indicated in the image). From top to bottom: i) "Diffuse Parkin" defines cells in where Parkin expression is homogenously distributed throughout the cytoplasm (quantified in B). ii) Soon after mitochondrial damage Parkin appears enriched in small "puncta" throughout the cytoplasm localizing within isolated mitochondria (quantified in C). iii) Later in the process these puncta structures aggregate in bigger structures accumulating in perinuclear regions (quantified in D). iv) The process of Parkin translocation eventually affects all the mitochondrial population and usually collapses in few large structures possibly forming part of big autolysosomes in the perinuclear region (quantified in D). Scoring was performed from 3 independent experiments (n=3; one way ANOVA with Dunnett post test). (F) Representative images (and details from boxed regions) of WT MEFs cells transfected with GFPPINK1 (green) and treated with FCCP (10 μM) to induce mitophagy. Cells were immunostained with anti-Tom20 (magenta) to reveal the mitochondrial network. Each row represents one of the three categories of GFP signal used to describe PINK1 translocation to mitochondria after mitochondrial damage (cytoplasmic, intermediate and mitochondrial). The scoring of these categories was used to describe PINK1 translocation to mitochondria in WT and MiroDKO cells at different time-points shortly after mitochondrial insult (G). Data collected from 3 independent experiments (n=3).

Error bars represent s.e.m. Significance: *p<0.05 and **p<0.01
Loss of neuronal Miro1 disrupts mitophagy and induces hyper-activation of the integrated stress response (2021)
Figure 2

Figure 2: Miro1 is promiscuously ubiquitinated during the mitophagic process.

A) Schematic representation of hMiro1 sequence highlighting domains and lysine residues (Lys) in its sequence. Key lysine residues reported to be ubiquitinated upon mitochondria damage and mutated to generate the mycMiro15R mutant construct are highlighted. B) Representative images showing full length control and selected Miro1 lysine mutants (Miro15R, Miro1allR and Miro1R572K) expressing in MiroDKO MEF cells. All constructs (myc-tag, green) localise to mitochondria (Tom20, red). C) Immunoblot showing efficient expression of Miro1 lysine mutants in FlagParkin expressing SH-SY5Y cells. D) Ubiquitination assay showing that Miro1 lysine mutants have reduced ubiquitination upon FCCP treatment (1h, 10 μM) in FlagParkin overexpressing SH-SY5Y. The effect is quantified in (E) (n=4 independent experiments for Miro1WT and Miro15R and n=3 for Miro1allR and Miro1R572K, ANOVA with Sidak's post hoc test). F) Representative western blot showing a degradation assay in FlagParkin overexpressing SH-SY5Y cells transfected with Miro1WT, Miro15R or Miro1allR constructs and treated with FCCP (10 μM) for 3 or 6 hours. Quantification of Miro1 levels (G) the matrix protein PDH E1α in (H) and Parkin levels (I) (n=4 independent experiments; ANOVA with Sidak's post hoc test).

Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.
Loss of neuronal Miro1 disrupts mitophagy and induces hyper-activation of the integrated stress response (2021)
Figure 3

Figure 3: Miro1 ubiquitination and degradation is required for mitochondrial clearance through mitophagy.

(A) Representative images at 3 hours of FCCP treatment (10 μM) of YFPParkin (green) expressing MiroDKO cells co-transfected with the specified myc-tagged versions of Miro1 (cyan). Tom20 (red) was used to reveal the mitochondrial network. A detail of YFPParkin translocation onto the mitochondrial network is also shown for each condition. (B and C) Quantification of the fraction of cells showing advanced stages of the mitophagic process (cells showing mitochondrial aggregation due to advanced Parkin translocation or the complete translocation of Parkin onto all the mitochondrial network) at 1 hour (B) or 3 hours (C) after FCCP treatment (data collected from at least 3 independent experiments: WT n=7, MiroDKO n=8, +mycMiro1WT=7, mycMiro15R=3, mycMiro1allR=3; mycMiro2=3; one way ANOVA with Dunnett post test). D) Western blots and quantification of Miro1 degradation in MiroDKO cells co-expressing YFPParkin and the indicated myc-tagged Miro1 constructs and treated with FCCP (10 μM) for the indicated times (n=3 different experiments; 2-way-ANOVA with Dunnett post test; comparisons between Miro1WT and Miro1allR are represented with yellow asteriscs, comparisons between Miro1WT and Miro15R are represented in green and comparisons between Miro15R and Miro1allR are represented in black). E). Representative images at 24 hours of FCCP treatment (10 μM) of YFPParkin (green) expressing MiroDKO cells co-transfected with the specified myc-tagged versions of Miro1 (cyan). Tom20 (red) was used to reveal the mitochondrial network. Expression of both ubiquitin mutants (Miro15R and Miro1allR - arrowheads) protects from the mitophagic clearance of mitochondria (arrows) after damage. (F) Quantification of the fraction of cells showing the complete loss of Tom20 signal of mitochondria after 24 hours of FCCP treatment (data collected from at least 3 independent experiments: MiroDKO n=8, mycMiro1WT=7, mycMiro15R=3, mycMiro1allR=3; one way ANOVA with Dunnett post test).

Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001
Loss of neuronal Miro1 disrupts mitophagy and induces hyper-activation of the integrated stress response (2021)
Figure 4

Figure 4: Miro1 and its ubiquitination are required for Parkin recruitment to damaged mitochondria in primary neurons.

(A) Representative confocal images of the soma of WT and Miro1KO cortical neurons expressing YFPParkin and MtDsRed after valinomycin treatment at the indicated time points (scale bars = 5 µm). (B) Airyscan confocal images of Parkin recruitment after 5 hours of valinomycin treatment in WT and Miro1KO neuronal processes (scale bars = 2.5 µm). (C) Fluorescent linescans of YFPParkin and MtDsRed signal in WT and Miro1KO neurons after 5 hours of valinomycin treatment (lines shown in A). (D) Quantification of Parkin recruitment to mitochondria (YFPParkin signal overlapping MtDsRed signal - intensity-adjusted and normalised to t=0) in WT and Miro1KO neurons following valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; two-way ANOVA). (E) Quantification of mitochondrial occupancy (area of MtDsRed signal in soma / entire area of soma) in the somas of WT and Miro1KO neurons following valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; two-way ANOVA). (F) Quantification of mitochondrial clearance in somas of WT and Miro1KO neurons between 2 and 5 hours of valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; Unpaired t-test). (G) Representative confocal images of WT, Miro1KO and PINK1KO cortical neurons immunostained with MAP2 (cyan) and s65-phospho-ubiquitin (green) after valinomycin treatment (Scale bars = 20µm). (H) Quantification of s65-phospho-ubiquitin signal intensity within MAP2 signal (normalised to MAP2 area and t=0) in Miro1WT, Miro1KO and PINK1KO neurons following valinomycin treatment n= 3, 4 and 3 embryos from WT, Miro1KO and PINK1KO embryos, respectively (6 ROIs per condition; two-way ANOVA). (I) Representative confocal images of the soma of Miro1KO cortical neurons expressing Miro1WT and mutant forms of Miro1, YFPParkin and MtDsRed without valinomycin treatment (Scale bars = 10 µm). Quantification of parkin colocalization with MtDsRed (YFPParkin signal overlapping MtDsRed signal - intensity-adjusted) in the soma of Miro1KO and PINK1KO cortical neurons (n=12 cells all conditions per genotype over 3 neuronal preparations; one-way ANOVA). (J) Representative confocal images of the soma of Miro1KO cortical neurons expressing WT and mutant forms of Miro1, YFPParkin and MtDsRed after 5 hours of valinomycin treatment (Scale bars = 10 µm). Quantification of Parkin recruitment to mitochondria (Normalised to t=0) in Miro1KO and PINK1KO cortical neurons following valinomycin treatment (n=12 cells all conditions per genotype over 3 neuronal preparations, two-way ANOVA).

Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001
Loss of neuronal Miro1 disrupts mitophagy and induces hyper-activation of the integrated stress response (2021)
Figure 5

Figure 5: Loss of Miro1 in mature neurons associates with age-dependent accumulation of defective mitochondria in neurons and an increase in mitophagy machinery.

(A) Representative blots of 4 and 12 months old hippocampal lysates from control, Miro1CKO and Miro2KO. B) Quantification of protein levels in 4 month lysates and 12 month lysates (n=3 animals / genotype at 4 months and n=4 animals / genotype at 12 months; one-way ANOVA with post-hoc Dunnett's test). C) Representative images of hippocampal regions from control and Miro1CKO crossed with MitoDendra animals. (D) Details from cortical regions of control, Miro1CKO and Miro2KO animals at 12 months of age showing large mitochondrial structures occurring only in the case of Miro1CKO animals. (E) Electron microcopy images of control and Miro1CKO brains showing the ultrastructure of the mitochondrial compartment. The mitochondrial units in Miro1CKO cells are enlarged and show altered cristae structure at 4 months of age. This process is progressive as mitochondrial structures appear larger and with reduced intramitochondrial complexity at 12 months of age. Representative images (F) and quantification (G) of Mfn1 and Mfn2 staining in cortical slices from 12 month WT and Miro1CKO mice (n=4 animals / genotype; student's t-test).

Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.
Loss of neuronal Miro1 disrupts mitophagy and induces hyper-activation of the integrated stress response (2021)
Figure 6

Figure 6: Long-term loss of Miro1 in mature neurons leads to the initiation of the integrated stress response.

(A) Representative western blot images of brain lysates from 12-month WT, Miro1CKO and Miro2KO mice immunoblotted with the antibodies stated. (B) Quantification of eIF2α and P-eIF2α band intensity (normalised to actin band intensity) from 12-month WT, Miro1CKO and Miro2KO brain lysates. Phosphorylation of eIF2α is defined as P-eIF2α/eIF2α (n=4 mice per genotype, unpaired t-test). (C) Representative confocal images of cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. (D) Quantification of P-eIF2α signal intensity from cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice (n=4 mice per genotype; unpaired t-test). (E) Representative confocal images (63x) of cortical regions of 12-month mitoDendra -crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. Arrows indicate the presence of megamitochondria (>2.5 µm2), stars indicate neuronal somas without megamitochondria. (F) Example zoom images of neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra -crossed Miro1CKO mice stained with P-eIF2α. (G) Quantification P-eIF2α signal intensity (a.u.) from neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra-crossed Miro1CKO (n=16 sections from 4 mice; paired t-test).

Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.
R3hdml regulates satellite cell proliferation and differentiation (2019)
Figure 1

Figure 1 - Expression of R3hdml during skeletal muscle development and satellite cell differentiation.

A Skeletal muscles in the lower legs were dissected and RNA was extracted on postnatal day 0, 7, and 21. The expression of R3hdml was examined by RT-PCR. GAPDH was used as an internal control. MM, 100 bp DNA marker.

B Quantification of RT-PCR results. Data are expressed as the means ± standard error of the mean (SEM). The experiments were repeated at least three times. **** p < 0.0001 vs. day 0 group; one-way ANOVA followed by Bonferroni post-hoc test.

C-E Satellite cells were isolated from skeletal muscles of adult mice by enzymatic digestion and placed in culture medium. Then, differentiation was induced in satellite cells (C). The proteins were extracted at the indicated time points. R3hdml expression was examined by western blotting using an anti-R3hdml antibody (D). β-actin was used as internal control. Quantification of western blotting results (E). Data are expressed as the means ± standard error of the mean (SEM). The experiments were repeated at least three times. Representative data are shown. * p < 0.05; Student's t-test. Scale bar = 500 µm.
R3hdml regulates satellite cell proliferation and differentiation (2019)
Figure 2

Figure 2 - Expression of R3hdml is controlled by the expression of MyoD.

A Cultured C2C12 cells were transfected with the firefly luciferase construct containing the 781 bp fragment upstream of the translational start codon of the R3hdml coding sequence (R3hdml-luc), together with Renilla luciferase vectors as controls; the cells were then differentiated in the presence of 2% horse serum. Cells were harvested at the indicated time points, after which luciferase activity of the cell lysate was measured.

B The table represents the schematic of each construct. All inserts in the constructs contain the same 3ʹ end. The 781-bp fragment of the 5ʹ flanking sequence from the murine genome, which contains several putative MyoD binding sites (black box), was subcloned into a luciferase reporter vector (Construct C). We also subcloned the genomic sequence containing one high affinity MyoD binding site CACCTG (Construct B) and one without a high affinity MyoD binding site (Construct A) into a luciferase reporter vector.

C Plasmids containing MyoD cDNA or empty vector were transfected into C2C12 cells. One day and 3 days after transfection, cells were lysed and subjected to SDS-PAGE and western blotting using anti-MyoD antibodies.

D Plasmids containing MyoD cDNA or empty vector together with R3hdml-luc and Renilla luciferase vectors were transfected into C2C12 cells. One day and 3 days after transfection, the luciferase activity of the cell lysate with or without MyoD overexpression was measured. Relative luciferase activity is expressed as the fold activity above the background conferred by the empty vector together with R3hdml-luc.

Data information: Data are expressed as the means ± standard error of the mean (SEM). The experiments were repeated at least six times. ** p < 0.01; **** p < 0.0001 (vs. day 0 group in A); one-way ANOVA followed by Bonferroni post-hoc test.
R3hdml regulates satellite cell proliferation and differentiation (2019)
Figure 3

Figure 3 - Silencing the R3hdml gene ameliorates C2C12 cell differentiation.

A R3hdml siRNA or non-targeting siRNA was transfected into C2C12 cells. Then, C2C12 cells were differentiated into myotubes. RNA was extracted from the cells after induction of differentiation at the indicated time points. Closed bars, control; open bars, silenced R3hdml gene; ** p < 0.01; * p < 0.05; Student's t-test.

B Immunofluorescence after C2C12 differentiation. MyHC is shown in green and the nuclei in blue (counterstained with Hoechst). Representative images of MyHC immunofluorescence of C2C12 myocytes transfected with the siRNA or after addition of R3hdml protein. Scale bar = 100 µm.

C The number of nuclei per MyHC+ cell were calculated on day 5 after differentiation. ** p < 0.01; one-way ANOVA followed by Bonferroni post-hoc test.

D The distribution of nuclei per myotube were calculated on day 5 after differentiation. ** p < 0.01; * p < 0.05; one-way ANOVA followed by Bonferroni post-hoc test.

Data information: Data are expressed as the means ± standard error of the mean (SEM). The experiments were repeated at least three times and at least three fields of view were evaluated.
R3hdml regulates satellite cell proliferation and differentiation (2019)
Figure 4

Figure 4 - R3hdml KO mice-derived satellite cells show reduced proliferation and differentiation compared with those from wild type controls.

A Satellite cells were isolated by enzymatic digestion from R3hdml KO (n = 6) and wild type control (n = 6) mice and placed in culture medium. Then, cell proliferation was evaluated by Ki67 staining. Cell nuclei were stained with DAPI.

B The ratio of Ki67 positive cells to total cells (DAPI-positive) was evaluated. The number of cells was counted under approximately 60 fields of view for each well. Closed columns, wild type controls; open columns, R3hdml KO mice. * p < 0.05; Student's t-test.

C-F Thirty freshly isolated myofibers from 8-12-week-old R3hdml KO (n = 7) and wild type mice (n = 7) were cultured for 72 h. Then, the isolated myofibers were fixed with 4% PFA followed by immunostaining using anti Pax-7, anti MyoD, and anti Myog antibodies. (C, D) Co-immunostaining parallel cultures for Pax7 and MyoD showed that there were fewer cells with the self-renewing Pax7+/MyoD− phenotype in muscles from R3hdml KO mice compared with control; Pax7+/MyoD− cells (arrowheads), Pax7−/MyoD+ cells (yellow arrows) and Pax7+/MyoD+ cells (white arrows). * p < 0.05; Student's t-test. (E, F) Co-immunostaining parallel cultures for MyoD and Myog showed that there were fewer cells with the differentiating MyoD−/Myog+ phenotype in muscles from R3hdml KO mice compared to control; MyoD−/Myog+ cells (arrowheads), MyoD-/Myog+ cells (yellow arrows), and MyoD+/Myog+ cells (white arrows). * p < 0.05; Student's t-test.

Data information: Data are expressed as the means ± standard error of the mean (SEM).
R3hdml regulates satellite cell proliferation and differentiation (2019)
Figure 5

Figure 5 - Expression of R3hdml increases during skeletal muscle regeneration, and R3hdml is important both for skeletal muscle regeneration and function.

A Seven days after CTX injection, skeletal muscles were dissected, fixed, and embedded in paraffin. Two consecutive sections were utilized, and the first section was subjected to in situ hybridization using an R3hdml sequence-specific RNA probe to detect Rh3dml, while the second section was used for IHC using the anti-MyoD antibody. MyoD protein was visualized with DAB stain (brown). R3hdml expression is highly detected in MyoD-positive cells. Scale bar in low magnification images = 100 µm; in high magnification images = 25µm.

B One hundred microliters of 10 µM CTX was intramuscularly injected into the forearm muscle of anesthetized R3hdml KO ([b], n = 10) and wild type control ([a], n = 10) mice. Subsequently, hand grip was evaluated 0, 3, 6, and 14 days after CTX injection. R3hdml genes were overexpressed in the forearm muscle of anesthetized R3hdml KO mice ([c], n = 10) and hand grip was also evaluated 0, 3, 6, and 14 days after CTX injection. Data are expressed as the means ± standard error of the mean (SEM). ** p < 0.01; **** p < 0.0001 vs. wild type control; # p < 0.05; ## p < 0.01 vs. R3hdml KO mice; two-way ANOVA followed by a Bonferroni post hoc comparison of the individual time points.

C Myofibers were freshly dissected from 8-12-week-old R3hdml KO (n = 4) and control mice (n = 4) 35 days after CTX injection, and the number of satellite cells was evaluated by Pax-7 staining. Arrowheads indicate Pax-7-positive cells. The number of Pax-7-positive cells per myofiber was counted. At least 50 myofibers in each animal were evaluated. Closed columns, wild type controls; open columns, R3hdml KO mice. Data are expressed as the means ± standard error of the mean (SEM). * p < 0.05; Student's t-test.
X-ray Structure of Main Protease of the Novel Coronavirus SARS-CoV-2 Enables Design of α-Ketoamide Inhibitors (2020)
Fig. 1:

Three-dimensional structure of 2019-nCoV Mpro, in two different views. One protomer of the dimer is shown in light blue, the other one in orange. Amino-acid residues of the catalytic site are indicated as yellow and blue spheres, for Cys145 and His41, respectively. Black spheres indicate the positions of Ala285 of each of the two domains III (see text). Chain termini are labeled N and C for molecule A (light blue) and N* and C* for molecule B (orange).
X-ray Structure of Main Protease of the Novel Coronavirus SARS-CoV-2 Enables Design of α-Ketoamide Inhibitors (2020)
Fig. 2:

Compound 13b in the substrate-binding cleft located between domains I and II of the Mpro, in the monoclinic crystal form (space group C2). 2Fo-Fc electron density is shown for the inhibitor (contouring level: 1σ). Carbon atoms of the inhibitor are magenta, oxygens red, nitrogens blue, and sulfur yellow. Note the interaction between the N-terminal residue of chain B, S1*, and E166 of chain A.
The Glycine Arginine Rich Domain of the RNA Binding Protein Nucleolin Regulates its Subcellular Localization (2021)
Figure 2

Figure 2. Nucleolin-kinesin interaction is directly mediated by the GAR domain.

A. GAR domain deletion or mutation perturbs binding to Kif5a. Co-immunoprecipitation analysis of Kif5a and HA-Dendra2-tagged nucleolin (both overexpressed in HEK-293 cells). IP was performed with HA antibody and probed in Western blot with anti-Kif5a and anti-HA antibodies. Ncl FL - HA-Dendra2- full length nucleolin; Ncl ΔGAR - HA-Dendra2- nucleolin with a GAR domain deletion; Ncl GAR(N) - HA-Dendra2- nucleolin with all 10 arginines in the GAR domain mutated to asparagines; DEN - Dendra2. Note that HA input blots for HA-Dendra2 (Dendra) and the other three constructs are from the same membrane, but shown discontinuously owing to the different migration of these proteins in PAGE.

B. Quantification of (A). n = 3 independent biological repeats; means ± SEM; **** p < 0.0001, ANOVA with Tukey's post-test.

C-E. R4, but not N4 peptide reduces Kif5a co-immunoprecipitation with nucleolin from mouse sciatic nerve axoplasm, assayed by automated capillary electrophoresis immunoassay for Kif5a. Schematic of the assay is shown in (C). Representative traces of the Kif5a immunoreactive peaks are shown in (D) and quantifications are shown in (E). Immunoprecipitated Kif5a levels are normalized with input levels and expressed relative to N4. n = 3 independent biological repeats; means ± SEM; * p<0.05, paired Student's t-test.

F. Surface plasmon resonance analysis of KLC2 binding to biotinylated R4 peptide Recombinant KLC2 was injected at different concentrations (5-160 nM) on biotinylated R4 or N4 peptides immobilized to a streptavidin sensor chip. The dissociation steady state constant KD, was determined by fitting the sensogram to a 1:1 model.

G. Kinetics analyses of photoconverted Dendra fusion proteins in cultured DRG neuron axons. Adult mouse DRG neurons were transfected by electroporation with constructs expressing HA-Dendra-GAR(WT) - wild-type nucleolin GAR domain N-terminally fused with HA-Dendra, or HA-Dendra-GAR(N) with 10 arginines in the GAR domain mutated to asparagines. 48 hr after plating and transfection, Dendra fluorescence was photoconverted using a DMD module to restrict conversion within the cell body. Time-lapse images were collected from the entire field of view, and red signal intensity was analyzed within axons at distances of 60-90 µm from the cell body. Photoconverted signal sampled at 10 sec intervals is shown, normalized to signal intensity before photoconversion. N = 3 biological repeats comprising 7 cells each. Means ± SEM; **** p<0.0001 (ANOVA).

See also Appendix Fig. S2.