EMBO/soda-nel huggingface dataset:
Ttyh1 regulates embryonic neural stem cell properties by enhancing the Notch signaling pathway (2018)
Figure 2

Figure 2. Ttyh1 increases Notch target gene expression.

A, B Two days after E14.5 primary neural progenitor cells were transduced with retroviral vectors expressing (A) Ttyh1 or (B) shTtyh1, mRNA expression levels of each indicated Notch target gene were measured by qPCR (n = 4 for A, n = 3 for B). To confirm specificity of the Ttyh1 knockdown, rescue experiments were performed by cotransducing retroviral vectors expressing shTtyh1 and Ttyh1res.

C Effects of dominant negative MAML1 (dnMAML1) on Ttyh1-enhanced Notch target gene expression were tested by qPCR using E14.5 primary neural progenitors at 2 days posttransduction (n = 4).

D The dnMAML1 plasmid was introduced with the Ttyh1 vector into embryonic brains at E13.5 and cell localization was analyzed at E15.5 by anti-GFP immunostaining. GFP and DAPI signals are shown in green and blue, respectively. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.

E Quantification of GFP+ cell positions in (D) (n = 3).

F The NICD plasmid was electroporated with the Ttyh1 knockdown vector (shTtyh1) into embryonic brains at E13.5 and cell localization was analyzed at E14.5 by anti-GFP immunostaining. Scale bar: 100 μm.

G Quantification of GFP+ cell positions in (F) (n = 3).

Data information: Error bars represent SD. Student's t-test was used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001.
Ttyh1 regulates embryonic neural stem cell properties by enhancing the Notch signaling pathway (2018)
Figure 3

Figure 3. Ttyh1 increases Notch intracellular domain (NICD) production by enhancing γ-secretase activity.

A-D Effects of Ttyh1 expression (A) or Ttyh1 knockdown (C) on the generation of NICD at 2 days posttransduction of E14.5 neural progenitor cells were analyzed by Western blotting using an anti-NICD antibody. (B, D) Quantification of band intensities in (A, C) (n = 4 for B, n = 3 for D).

E Schematic representation of the γ-secretase reporter assay. Following cleavage at the γ-secretase site in Notch∆E-Gal4-VP16 (Notch∆E-GVP), the intracellular domain containing the GVP domain translocates into the nucleus and triggers luciferase expression driven by the UAS promoter (Figure redrawn from Karlström et al [31]).

F, G (F) Ttyh1-transduced E14.5 neural progenitors were incubated with DAPT for 2 days, then cells were harvested and lysates were used for luciferase assay (n = 3). (G) Effects of Ttyh1-knockdown on γ-secretase activity was also assessed using E14.5 neural progenitor cells (n = 4 for F, n = 3 for G).

H, I Ttyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for Western blotting using anti-NICD antibody (H) or qPCR for Notch target genes (I) (n = 3).

J, K (J) The indicated plasmids were electroporated into E13.5 brains, and brain samples were harvested at E15.5 for anti-GFP immunolabeling. GFP and DAPI signals are shown in green and blue, respectively. (K) Quantification of GFP+ cell localization in (J) (n = 3). dnPS1, dominant negative presenilin 1; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.

Data information: Error bars represent SD. Student's t-test was used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001.
Ttyh1 regulates embryonic neural stem cell properties by enhancing the Notch signaling pathway (2018)
Figure 4

Figure 4. Cl- channel property of Ttyh1 is not associated with Notch pathway-enhancing activity.

A, B Effects of the ion selectivity-perturbing R371Q mutation in (A) Ttyh1 and (B) other Ttyh family members on Notch-dependent transcription. E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3).

C, D (C) Immunolabeling of E15.5 brain sections electroporated in utero with indicated plasmids at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. GFP immunofluorescence (green) was merged with DAPI-counterstained images (blue). (D) Quantification of GFP+ cell localization in (C) (n = 3). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.

E, F (E) Schematic representation of a series of Ttyh1 mutants with deletions in the C-terminal cytoplasmic tail region (Ttyh1 gene structure was not drawn in proportion to the actual domain size), and (F) their effects on cell distribution assessed by intraventricular in utero electroporation at E13.5 followed by immunolabeling at E15.5 using anti-GFP antibody. Scale bar: 100 μm.

Data information: Error bars represent SD. Student's t-test was used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001.
Ttyh1 regulates embryonic neural stem cell properties by enhancing the Notch signaling pathway (2018)
Figure 5

Figure 5. Ttyh1 downregulates Rer1, and Rer1 expression counteracts Ttyh1-induced enhancement of neural stem cell properties.

A Representative Western blots showing coimmunoprecipitation (coIP) of HA-tagged Rer1 with Myc-tagged Ttyh1 using the HEK293T cell line. WCL, whole-cell lysates.

B Venus fluorescent protein-based bimolecular fluorescence complementation analysis of Ttyh1 and Rer1 interaction (green) in living COS-7 cells by fluorescence microscopy. Ttyh1 and Rer1 were tagged with N- and C-terminal fragments of the Venus fluorescent protein (VN and VC), respectively. DsRed2-ER was cotransfected to label the endoplasmic reticulum (ER, red). Scale bar: 25 μm.

C CoIP assays were performed with anti-Ttyh1 and anti-Rer1 antibodies to show endogenous interaction between Ttyh1 and Rer1 in U373MG cells.

D-G Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14.5 primary neural progenitor cells transduced with retroviral vectors expressing (D) Ttyh1 or (F) shTtyh1. (E, G) Quantification of relative band intensities in (D, F), respectively (n = 3).

H Cell extracts from primary neural progenitor cells transduced with Ttyh1 retroviruses in the presence of DMSO vehicle or a proteasome inhibitor lactacystin (10 μM), were immunoblotted with an antibody to Rer1.

I, J (I) HEK293T cells were transfected with Ttyh1-Myc and Rer1-HA expression plasmids, and after 24 h, cells were treated with cycloheximide (CHX) (100 μg/ml) and then harvested at the indicated time intervals. Cell lysates were analyzed by Western blotting using anti-HA antibody. (J) Quantification of band intensities in (I) (n = 3).

K γ-secretase-dependent luciferase activity was measured using primary neural progenitor cells transduced with Ttyh1 with or without Rer1 retroviral vectors at 2 days posttransduction (n = 3).

L, M (L) Effects of Rer1 expression on Ttyh1 activity in vivo were examined by in utero electroporation into E13.5 brains and subsequent GFP immunofluorescence of E15.5 brain sections. GFP and DAPI signals are shown in green and blue, respectively. (M) Distribution of GFP+ cells in each brain layer in (L) was quantified (n = 3). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.

Data information: Error bars represent SD. Student's t-test was used to determine statistical significance. *P < 0.05, **P < 0.01.

Expanded view figure legends
Molecular analysis of the ribosome recycling factor ABCE1 bound to the 30S post-splitting complex (2020)
Figure 1

Figure 1. In vitro assembly and cryo-EM structure of the archaeal post-splitting complex.

A, ABCE1IIEA efficiently splits 70S ribosomes in the presence of AMP-PNP and aRF1/aPelota. The 30S population contains a stoichiometric ratio of ABCE1 and ribosomal proteins, forming the post-splitting complex. rps: small subunit ribosomal proteins.

B, Cryo-EM density of the post-SC highlights the archaeal ribosomal protein eS21 and ABCE1. Domain architecture of ABCE1 including the mutation sites is shown below.

C, Molecular model of the archaeal post-SC, domain colors as in B.

Data information: In (A), the gradient profiles are representative for the respective nucleotide condition.
Molecular analysis of the ribosome recycling factor ABCE1 bound to the 30S post-splitting complex (2020)
Figure 2

Figure 2. The conserved ABCE1-30S interface is formed by essential interactions.

A, Zoom-ins into ABCE1-30S connections. Most interactions are salt-bridges or H‑bonds between ABCE1 residues and the rRNA phosphate backbone. The FeS cluster domain contacts rRNA h5 via R2 and K59, interacts with uS12 via S29 and R28, and contacts h44 by N17 and K15. The helix-loop-helix motif connects to rRNA h15 via R144 and E147. The positioning of the cantilever is stabilized by an interaction network of R2, I68 and N70 with N316 of hinge 1 and rRNA h5.

B, Yeast survival of ABCE1 variants (S. solfataricus colored, S. cerevisiae in gray). Most residues connecting to 30S in the post-SC show a growth defect when exchanged for a small one (alanine) or a negative charge (glutamate). ++ no effect, + growth defect, - lethal.

C, The cantilever link forms salt-bridges of E74 and E76 with NBD1 residues K89 and R293, respectively.

Data Information: In (B), data are representative for a set of two independent experiments.
Molecular analysis of the ribosome recycling factor ABCE1 bound to the 30S post-splitting complex (2020)
Figure 3

Figure 3. Structural and functional analysis of the hinge regions and NBSs.

A-D, Hinge 2 (emerald) residues interacting with the ribosome. R565E forms a conserved cation-π-stacking with A329 of h8, R574 forms a salt-bridge with the phosphate backbone of U328 in h14. Aromatic C-terminal residues Y592 and Y593 adopt a parallel coordination. R572 of hinge 2 and N305 of hinge 1 (light green) form an interaction that might be important for sensing. Essential S580 does not contact the ribosome, whereas Y581 and E588 form H-bonds to R69 and M1 of eS6 (blue), respectively.

E, Mutations in the α-helices of hinge 2 prevent 30S binding while the Y592A/Y593A (C-terminus) and L353Y (A-loop in NBSII) exchanges do not influence ribosome binding.

F, 70S splitting efficiency normalized to wildtype. Hinge 2 mutations Y592A/Y593A, R565E and S580E display strongly impaired splitting activity. Unspecific ribosome dissociation level as determined in control experiments in the absence of ABCE1 is marked by the dotted line.

G, ATP turnover per ABCE1 is not affected in all tested mutants.

H-J, Overview of ATP coordination in both NBSs and overlay of the two NBSs reveals only slight differences, which cannot elucidate the functional asymmetry. Residues of NBD1 and NBD2 involved in coordination are shown in gold and punch, respectively.

Data Information: In (F) and (G), the mean ±SD of assay triplicates and duplicates are plotted.
Integrative analysis of cell state changes in lung fibrosis with peripheral protein biomarkers (2021)
Figure 7

Figure 7. An SSTR2+/CFHR1+ pericyte state correlates with progression of fibrotic remodeling.

(a) The scatter plots show the positive correlation of CFHR1 in BALF (MS-intensity, x-axis) with meta lung function (y-axis).

(b) The UMAPs show the subset of COL1A2+ mesenchymal cells in the integrated scRNAseq dataset, with the pericytes highlighted by the dotted line. From left to right, the colors represent the cell type identities, disease status, expression of the pericyte markers PDGFRB and CFHR1, respectively.

(c) The volcano plot depicts the fold changes (x-axis) and the -log10 p-values (y-axis) derived from the differential gene expression analysis using diffxpy between ILD and controls within the subset of PDGFRB+ pericytes (outlier values truncated).

(d) The bar graphs show the top regulated gene categories after enrichment analysis using GO terms and Uniprot keywords.

(e) The heatmap shows the expression of genes most strongly associated with SSTR2 expression within the pericytes (left color bar indicates significant correlation or anti-correlation).

(f) Immunofluorescence analysis of SSTR2, the pericyte cell type marker PDGFRB and a marker for smooth muscle cells DESMIN in IPF (n=3) and control samples (n=3).

(g) Representative images of immunohistochemistry analysis of SSTR2 protein expression in tissue regions (n = 474) from ILD patients (n = 53) and control patients (n = 26).

(h) Correlation of immunohistochemistry signal of SSTR2 with Ashcroft scores.

(i) The bar graph shows the genes most strongly correlated with SSTR2 belonging to the GO category "transcription regulators". The dotted line marks a correlation coefficient of zero.

(j) The bar graph shows the top correlated transcriptional regulators, predicted by ingenuity Pathway Analysis (IPA) for the SSTR2 gene-gene correlations. The dotted line marks a correlation coefficient of zero.

(k) Diffusion map of pericytes colored by cell type and inferred pseudotime represents pericyte differentiation.

(l) Diffusion map of pericytes colored by the gene expression of indicated genes.

(m) The line plot illustrates smoothed expression levels of the indicated genes across the pericyte pseudotime differentiation trajectory.
The crystal structure of MICU2 provides insight into Ca2+-binding and MICU1-MICU2 heterodimer formation (2019)
Figure 2

Fig 2

Interaction interface of the MICU2 homodimer in both states.

A Interaction interface of the MICU2 homodimer in the Ca2+-bound state. The left box (gray dotted line) shows residues involved in electrostatic interactions, the right box (gray line) residues involved in hydrophobic interactions. Molecule 1 and molecule 2 in the dimer are colored in purple and yellow, respectively. Side chains of residues that make crucial intreractions are shown in stick representation.

B Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of Ca2+ monitored at 280 nm absorption wavelength.

C Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of EGTA monitored at 280 nm absorption wavelength.
The crystal structure of MICU2 provides insight into Ca2+-binding and MICU1-MICU2 heterodimer formation (2019)
Figure 3

Fig 3

Pull-down experiments to test the contribution of individual MICU2 residues to the interaction with MICU1 in the apo and Ca2+-bound states.

A, B MICU2_WT (A), MICU2_K172A (B) were pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+.

C D MICU2_EF1mut was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+ (C), while MICU2_EF2mut was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (D).

E F MICU2_R352A was not pulled-down by GST-MICU1 in the absence of 2 mM Ca2+ (E), while MICU2_E329A was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (F).

G MICU2_D185A was partially pulled-down by GST-MICU1 in the presence of Ca2+, while they showed a strong interaction in the absence of 2 mM Ca2+.

H MICU2_E196K was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+, while a strong interaction was observed in the absence of 2 mM Ca2+.
The crystal structure of MICU2 provides insight into Ca2+-binding and MICU1-MICU2 heterodimer formation (2019)
Figure 4

Fig. 4

Characterization of MICU1 and MICU2 interactions in both apo and Ca2+-bound states.

A GST-MICU1_EF1mut failed to pull down MICU2_WT in the absence of 2 mM Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+.

B GST-MICU1_F383A & H385A failed to pull-down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+.

C GST-MICU1_E242K failed to pull-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+.

D GST-MICU1_D231A partly pulled-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+.

E GST-MICU1_F383A failed to pull down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+.

F GST-MICU1_H385A exhibited a strong interaction with MICU2 both in the absence and presence of 2 mM Ca2+.

G, H ITC analyses of MICU1 and MICU2 binding properties in the presence of 2 mM EGTA (G) and CaCl2 (H).
Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes (2016)
Figure 1

Figure 1. α-synuclein fibrils transfer efficiently between neuron-like CAD cells.

(A) Time course of rapid internalization of fluorescent α-synuclein fibrils by CAD cells (for characterization of α-synuclein assemblies, see Figure EV 1A). Internalization was measured by recording ATTO-550-positive neuron-like cells by flow cytometry. Percentage of ATTO-550-positive cells was quantified (mean ±s.e.m) (left panel) and representative histograms of ATTO-550-positive cells are showed on the right panel (a.u. arbitrary units). Similarly, α-synuclein fibrils internalization was also confirmed by fluorescent microscopy (see Figure EV 1B).

(B) Representative images of donor (upper panel) and acceptor cells (lower panel) after 24 h co-culture. Donor cells were loaded with α-synuclein fibrils prior to co-culture with GFP-transfected acceptor cells; in red: α-synuclein fibrils, in green: acceptor cells and in blue: nuclei. Scale bar represents 10 μm. (n = 3 independent experiments). A larger field where donor and acceptor cells are shown is presented in Figure EV 3A.

(C) Percentage of donor and acceptor cells containing α-synuclein fibrils after co-culture as in B: all acceptor cells received α-synuclein fibrils.

(D) Quantification of the number of α-synuclein fibrils in donor and acceptor cells after co-culture as in B. Donor cells contain around 70 α-synuclein fibrils puncta (median) while acceptor cells contain 38 α-synuclein fibrils puncta, respectively (****, p < 0.0001 by two-tailed Mann Whitney test).

(E) Quantification of the average size of α-synuclein fibrillar foci in donor and acceptor cells after co-culture as in B. (****, p < 0.0001 by two-tailed Mann Whitney test). In Figure EV 3 is shown an example of α-synuclein fibrillar puncta detection in an acceptor cell. After detection, the number and the size of foci were determined using the ICY software.
Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes (2016)
Figure 2

Figure 2. α-synuclein fibrils induce the aggregation of the soluble α-synuclein protein after transfer in acceptor cells.

(A) Representative confocal images showing ChFP-α-syn transfected cells (in red) cultured alone (upper) or co-cultured with donor cells containing fluorescent Alexa-488 α-synuclein fibrils (in green) for 72 h (bottom). The arrow is pointing out representative α-synuclein fibrils Alexa-488 co-localized with ChFP-α-syn puncta. Scale bar represents 10 μm. Nuclei are stained with DAPI (blue) (n=3 independent experiments).

(B) Quantification of the number of ChFP-α-synuclein puncta in ChFP-α-syn acceptor cells showed a significant increase in ChFP-α-synuclein puncta number when cells were co-cultured with donor cells containing α-synuclein fibrils (***, p < 0.001 by two-tailed Mann Whitney test).

(C) Quantification of the percentage of co-localization of α-synuclein fibrils Alexa-488 and ChFP-α-syn puncta. Data shows mean ± s.e.m. After co-culture, 11% of the transferred α-synuclein fibrils Alexa-488 co-localized with ChFP-α-syn puncta
Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes (2016)
Figure 3

Figure 3. α-synuclein fibrils transfer is favoured by cell-to-cell contact.

(A) Representative images of donor cells (upper panel) and acceptor cells (bottom panel) of the co-culture system explained in Figure EV 2E; in red: α-synuclein fibrils ATTO-550, in green: α-synuclein fibrils Alexa-488 and in blue: nuclei. Scale bar represents 10 μm (n=3 independent experiments). In insets, arrows point to co-localized ATTO-550 and Alexa-488 α-synuclein fibrils puncta whereas arrowheads point to puncta of α-synuclein Alexa-488 fibrils that do not co-localize with fibrillar ATTO-550 α-synuclein.

(B) Quantification of the percentage of Alexa-488 α-synuclein fibrils co-localizing with α- ATTO-550 α-synuclein fibrils and the reverse co-localization in discrete puncta in donor and acceptor cells as in A. Both measurements revealed high co-localization of the two fluorophores in donor cells (white bar), but less in acceptor cells.

(C) The amount of fibrils taken up by CAD cells exposed to 1µM fibrils, of fibrils remaining in the cells and exported into the medium after 24h incubation were quantified by a filter trap assay. Data are mean ± s.d (n=3 independent measurements, filtered in duplicate). The standard fluorescence curve for increasing ATTO550-α-synuclein fibrils concentrations is given.

(D) Representative images of GFP-transfected acceptor cells that were either (i) co-cultured with donor cells (upper panel, Co-culture), (ii) cultured with the conditioned medium of donor cells (middle panel, CM) or (iii) physically separated from donor cells using a filter (bottom panel, Filter). Prior to culture, donor cells were loaded with ATTO-550 α-synuclein fibrils. In red: α-synuclein fibrils, in green: acceptor cells and in blue: nuclei. Scale bar represents 10 μm (n = 3 independent experiments).

(E) Quantification of the percentage of acceptor cells containing α-synuclein fibrils from images such as those presented in C. When acceptor cells were cultured with the conditioned medium from donor cells previously treated with -synuclein fibrils (not diluted, concentrated or diluted) or co-cultured with a filter, the percentage of acceptor cells containing α-synuclein puncta was low. Data shows mean ± s.e.m (***, p < 0.001; by two-tailed Student’s t-test).

(F) Quantification of the number of puncta of α-synuclein fibrils per acceptor cell from D. While in co-culture condition the number of α-synuclein puncta in acceptor cells is on average 35 (median), this number was on average 1 puncta per acceptor cells in CM and filter conditions, respectively (***, p < 0.001 by two-tailed Mann Whitney test).