EMBO/soda-nel huggingface dataset:
Cell-to-cell transmission of C9orf72 poly-(Gly-Ala) triggers key features of ALS/FTD (2020)
Figure 4

Figure 4. Rolipram rescues poly-GA dependent TDP-43 mislocalization and aggregation by boosting proteasome activity.

(A-E) Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. (A) Immunofluorescence reveals enhanced cytoplasmic TDP-43 levels in neurons with poly-GA aggregates or treated with MG132. Arrows mark punctate TDP-43 staining. Rolipram treatment reduced cytoplasmic TDP-43 in GA175-GFP neurons. Scale bar denotes 20 µm. (B) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced neurons. n=4 biological replicates. In total 462 GFP and 371 GA175-GFP cells treated with vehicle, and 386 GFP and 529 GA175-GFP cells treated with MG132, 513 GFP and 434 GA175-GFP cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. (C) Immunoblot to show effects of MG132 and rolipram on GA175-GFP and GFP expression. (D,E) Filter trap assay with quantification of SDS-insoluble aggregated GA175-GFP. n=5 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.

(F,G) HeLa cells were co-transfected with RFP-TDP-CTF and GFP or GA175-GFP for 2 days. For the final 16h, cells were treated with rolipram (30 µM) or MG132 (10 µM). Filter trap assay of SDS-insoluble TDP-CTF aggregates quantified by densitometry. n=4 biological replicates. Scatter dot plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. See also Appendix Fig S3.

(H,I) HeLa cells were co-transfected with TDP-43ΔNLS-GFP and iRFP or GA175-iRFP for 2 days. For the final 16h, cells were treated with either vehicle or rolipram (30 µM). Filter trap assay of SDS-insoluble TDP-43ΔNLS-GFP aggregates quantified by densitometry. n=3 biological replicates. Scatter dot plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.

Data information: ** denotes p<0.01, and *** denotes p<0.001. Red dashed line indicates the control's expression level.
Cell-to-cell transmission of C9orf72 poly-(Gly-Ala) triggers key features of ALS/FTD (2020)
Figure 5

Figure 5. Boosting proteasomal activity prevents poly-GA induced cytoplasmic accumulation of TDP-43.

(A-C) Hela cells were co-transfected with an RFP-based TDP-NLS reporter and GFP or GA175-GFP. 24 h after transfection, cells were treated with rolipram (30 µM) for 16h. In the immunofluorescence GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP-reporter. White arrows indicate cells with cytoplasmic TDP-43. (B) Automated quantification of cells with cytoplasmic TDP-NLS reporter in GFP and GA175-GFP positive cells. n=4 biological replicates. In total 345 GFP and 386 GA175-GFP cells treated with vehicle, 371 GFP and 404 GA175-GFP cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. (C) RFP-NLSTDPWT mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.

(D-F) Hela cells were co-transfected with the RFP-based TDP-NLS reporter, GFP or GA175-GFP, as well as PSMD11 or empty vector. Image analysis as in (A). n=4 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. 354 GFP and 330 GA175-GFP cells with vector, 367 GFP and 369 GA175-GFP cells with PSMD11 in total were analyzed. (F) RFP-NLSTDPWT mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.

(G-I) Immunofluorescence of Hela cells transfected with GFP or GA175-GFP showing reduced poly-GA aggregation upon rolipram treatment (30 µM, 16 h). (H) Automated quantification of poly-GA aggregate number per cell. n=3 biological replicates. In total 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction. (I) GA-GFP mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction.

Data information: Scale bars in immunofluorescent figures denote 20 µm. ** denotes p<0.01, *** denotes p<0.001.
Cell-to-cell transmission of C9orf72 poly-(Gly-Ala) triggers key features of ALS/FTD (2020)
Figure 6

Figure 6. Lysine 95 is critical for the inhibition of nuclear import of TDP-43 by poly-GA.

(A) Domain structure of TDP-43 and location of the bipartite NLS at position 78 to 99 (Winton et al, 2008). Known ubiquitination sites listed on www.phosphosite.org at K84 and K95 are highlighted.

(B) Immunoblot of HeLa cells transfected with RFP-based TDP-NLS wild-type (WT) or mutants (K84A, K84R, K95A, K95R).

(C, D) HeLa cells were co-transfected with the indicated TDP-NLS reporters as well as GFP or GA175-GFP, and treated with MG132 (10µM) or vehicle for 16h. (D) Automated quantification of RFP-NLS reporters in GFP positive cells. Note that K84A and K84R block overall import, while K95A and K95R allow import but are resistant to inhibition by poly-GA. n=4 biological replicates. The total number of cells analyzed per group was (from left to right): 667, 581, 789, 783, 809, 708, 628, 721, 938, 557, 857, 861, 886, 699,789, 539, 636, 577, 638 and 870. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001. Scale bar denotes 20 µm.

(E,F) HeLa cells were co-transfected with the indicated GFP-TDP NLS reporters and iRFP or GA175-iRFP. Cell lysates were immunoprecipitated with anti-GFP and immunoblotted with indicated antibodies to detect co-immunoprecipitation of the TDP-43 NLS with importin-α5/KPNA1 nuclear import receptor. (F) Quantification of KPNA1 levels normalized to total GFP-NLSTDP reporter levels in anti-GFP immunoprecipitates. n=3 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001. See also Figure EV4. Red dashed line indicates the control's expression level.
Cell-to-cell transmission of C9orf72 poly-(Gly-Ala) triggers key features of ALS/FTD (2020)
Figure 7

Figure 7. Poly-GA induces poly-ubiquitination of TDP-43 within the NLS at lysine 95.

HeLa cells were co-transfected with wildtype or K95A GFP-TDP NLS, HA-ubiquitin and iRFP or GA175-iRFP. 24h after transfection, cells were treated with rolipram (30 µM), MG132 (10 µM) or DMSO (vehicle) for 16h. Lysates were immunoprecipitated with Protein G beads coupled with anti-GFP antibody.

(A) Immunoblotting of input (left panels) and anti-GFP immunoprecipitates (right panels) to show GFP-reporter levels and poly-ubiquitination.

(B) Quantification of HA-ubiquitin levels normalized to total GFP- NLSTDP reporter levels in anti-GFP immunoprecipitates. n=3 biological replicates. Scatter plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. * denotes p<0.05, and *** denotes p<0.001. Red dashed line indicates the control's expression level.
Endospore Appendages: A novel pilus superfamily from the endospores of pathogenic Bacilli (2021)
Figure 3

Figure 3. S-Ena structure determination and recombinant production.

(A) Representative area of the 3D cryoEM potential map for ex vivo S-Ena, at 3.2 Å resolution. An octameric peptide with sequence FCMTIRY was deduced de novo from the cryoEM potential map (shown in sticks) and used for a BLAST search of the B. cereus NVH 0075-95 genome. The red letters indicate amino acids that differ between KMP91697.1 and KMP91698.1 (see B).

(B) Multiple sequence alignment of 3 ORF's (KMP91697.1, KMP91698.1 and KMP91699.1) corresponding to DUF3992 containing proteins. Sequence motifs corresponding or similar to that deduced from the EM potential map are shaded in cyan. Secondary structure and structural elements as determined from the built model (see Fig 2) are shown schematically above the sequences (Ntc: N-terminal connecter; arrows correspond to β-strands, labelled as in Fig 2). Contiguous areas of >50% similarity are boxed blue.

(C) SDS-PAGE of recEna1B, treated with β-mercaptoethanol or TEV protease (to remove N-terminal 6xHis tag) as indicated. Bands with apparent MW of ~13 and ~15 kDa correspond recEna1B monomer with (Ena1B6His) and without (Ena1BC) the 6xHis tag and TEV recognition site, respectively. In the uncleaved sample, a band running at ~ 30 kDa (labeled *) corresponds to a non-physiological disulfide bound S-Ena1B dimer. Lower intensity in the cleaved recEna1B results from a loss of monomers to SDS-resistant high molecular weight complexes stuck in the stacking gel.

(D) Negative stain TEM images of rec1Ena1B oligomers formed after refolding, but prior to TEV removal of the N-terminal 6xHis tag.

(E) Close-up view that shows recEna1B oligomers form open crescents similar in dimensions and shape to single helical turns or arcs found in the S-Ena fiber (model - right). Steric hindrance by the 6xHis is thought to arrest recEna1B polymerization into single helical arcs.

(F) Negative stain image and 2D classification of S-Ena-like fibers formed after TEV digestion of recEna1B. Upon removal of the N-terminal 6xHis tag, recEna1B readily assembles into fibers with helical properties closely resembling those found for ex vivo S-Enas.
Endospore Appendages: A novel pilus superfamily from the endospores of pathogenic Bacilli (2021)
Figure 5

Figure 5. ena is bicistronic and expressed during sporulation.

(A) Chromosomal organization of the ena genes and primers used for transcript analysis (arrows).

(B) Agarose gel electrophoresis (1%) analysis of RT-PCR products using indicated primer pairs and cDNA made of mRNA isolated from NVH 0075-95 after 4 and 16 hours growth in liquid cultures or genomic DNA as control.

(C) Transcription level of ena1A (x), ena1B (▲), ena1C (○) and dedA (♦) relative to rpoB determined by RT-qPCR during 16 hours of growth of B. cereus strain NVH 0075-95. The dotted line represents the bacterial growth measured by increase in OD600. Of note, the transcription of ena1C was surprisingly higher than ena1A and ena1B, the major components of the isolated S-Ena (Fig EV2).

(D) Expression analysis of Ena1 subunits shown as normalized ELISA signal for α−Ena1A, α−Ena1B, or α−Ena1C serum binding to 2,000 ng total protein of B. cereus strain NVH 0075-95 cells lysed after 4, 8, 12 or 16 hours of growth in sporulation medium.

Data information: Error bars represent standard deviation of three independent experiments, with two technical replicates each.
Extracellular interface between APP and Nicastrin regulates Aβ length and response to γ-secretase modulators (2019)
Figure 2

Figure 2. NCT ectodomain regulates GSEC processivity towards APPC99.

(A) Ala/Phe substitutions of aa 239-244 were introduced in the NCT ectodomain to evaluate the role of this region in the regulation of GSEC processivity. Aβ38, Aβ40, and Aβ42 levels present in the conditioned medium collected from KO NCT MEF cells rescued with WT or respective mutant NCT GSECs and transiently expressing with APPC99 were quantified by ELISA. Aβ(38+40)/42 ratio was calculated to determine GSEC processivity towards APPC99. DKO PSEN1/PSEN2 MEFs rescued with the indicated FAD PSEN1 mutant and transduced with APPC99 were used as references.

(B) Representative SDS-PAGE/western blot analysis of CHAPSO solubilized membranes from KO NCT MEF cell lines stably expressing WT or mutant NCT subunits. The presence of mature, glycosylated NCT, N-terminal and C-terminal fragments of the endoproteolysed PSEN1 and PEN-2, compared to NCT knock-out (KO) cells, indicate that WT and mutated NCTs reconstitute GSEC complexes. Arrowheads indicate the position of molecular weight markers.

(C) Quantification of the effects of the indicated substitutions of aa 241, 242, and 244 in NCT on GSEC processivity of APPC99, estimated as indicated above by the Aβ(38+40)/42 ratio. Figure EV1B shows the corresponding Aβ42/40 ratios.

Data information: Data is presented as mean ± SD, N ≥ 4 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance of all tested NCT mutants; *p > 0.05, **p > 0.01, ***p > 0.001, ****p > 0.0001, F(DFn, DFd): F(27, 230) = 84,22.
Extracellular interface between APP and Nicastrin regulates Aβ length and response to γ-secretase modulators (2019)
Figure 3

Figure 3. The NCT ectodomain contributes to the stability of GSEC-APP/Aβn complexes and accordingly modulate Aβ product length

(A) (Top) Representative bright field microscopy images show MEF cells stably expressing WT or NCT mutant GSECs after 24h incubation at 37°C and 42°C. 25µm scale bar is shown. (Bottom) Cell viability determined at the indicated time points is not affected by prolonged (≥ 24h) incubation at 42°C. Data are presented as mean ± SD of 4 replicates.

(B) Relative GSEC-APP/Aβn stabilities were assessed in cell-based GSEC thermoactivity assays, which evaluate changes in GSEC processivity (Aβ(38+40)/42 ratio) upon incubation at 42°C for 24h, relative to 37°C. Aβ peptides in the conditioned medium collected from the indicated MEF cell lines (transiently expressing APPC99) were quantified and the Aβ(38+40)/42 ratios determined. The data are shown as mean ± 95% CI, as % of the WT GSEC cell line incubated at 37°C for 24h, N = 6 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine the statistical significance (F(DFn, DFd): F(6, 46) = 100); ****p > 0.0001 compared to WT at 37°C, #p > 0.05, ##p>0.01, ####p>0.0001 compared to WT at 42°C. Note: Aβ profiles (including Aβ43 levels) normalized to total Aβ quantified in the cell-based thermoactivity assays with the different mNCT WT/mutants are shown in Figure EV2.
Extracellular interface between APP and Nicastrin regulates Aβ length and response to γ-secretase modulators (2019)
Figure 4

Figure 4. Direct, short-distance interaction between positions hNCT-242 and APP/Aβ-28 regulates GSEC processivity towards APPC99.

(A) Molecular dynamics simulations superimposing the cryo-EM-determined GSEC structure (PDB: 5FN3) with an NMR Aβ structure (PDB: 1IYT) on the co-purifying helical peptide (I), present hNCT- I242 and APPC99 -K28 at the NCT ectodomain-APP interface and modelling of the NCT I242E mutant suggests a direct (II) or indirect (bridged by H2O, III) interaction between the NCT -E242 and the APPC99 -K28 side chains.

(B) Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from WT or mutant (NCT) MEF cells lines co-expressing WT or mutant APPC99 substrates were assayed by ELISA. Schematic models of the tested GSEC-APP complexes are shown.

(C) To account for the increased production of Aβ37 and Aβ38 at the expense of Aβ40 and Aβ42 in the tested conditions, the ratio of ((Aβ 37/40) + (Aβ 38/42))/2 was determined and used to estimate GSEC processivity of APPC99. All the data are presented as mean ± SD, N ≥ 3, Unpaired, two-tailed Student's t-test was used to determine the statistical significance. **p > 0.01, ****p > 0.0001. Calculated T- and Df values were: T = 11,63; Df = 16 (I241-K28 vs I241E-K28), T = 11,37; Df = 14 (I241-K28 vs I241-K28E), T = 4,572; Df = 8 (I241E-K28 vs I241-K28E), T = 4,497; Df = 5 (I241-K28E vs I241K-K28E), T = 2,561; Df = 7.
Extracellular interface between APP and Nicastrin regulates Aβ length and response to γ-secretase modulators (2019)
Figure 5

Figure 5. Short distance interaction between NCT 241 and APPC99 K28.

(A) Chemical structure of the BMPS hetero-bifunctional (amine- and thiol-reactive groups) cross-linker.

(B) Introduction of Cys at position 241 in NCT allows chemical cross-linking of NCT and APPC99 with BMPS. Representative SDS-PAGE/western blot show cross-linked NCT-APPC99, NCT and APPC99 bands in detergent-extracted membrane proteins from BMPS cross-linker-treated membranes prepared from MEF cell lines expressing the indicated WT or mutant NCT GSEC complexes together with APPC99 or in the absence of the substrate. Orange, black, and white arrows indicate the NCT-APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers.

(C) Analysis of the western blot density profiles in panel (B) shows that co-migration of NCT (orange arrow) with APPC99 shifts the cross-linked band towards a higher molecular weight than the observed for mature NCT (black arrow). Immature NCT is indicated with an open (white) arrow. Full-range molecular weight density profiles are shown in Figure EV3C.

(D) Schematic representation of the GSEC-APPC99 complex modified at NCT- I241 and APP-K28 by the introduction of Cys substitutions.

(E) Representative SDS-PAGE/western blot presents NCT-APPC99, NCT and APPC99 expression in detergent-extracted membranes prepared from MEF cell lines co-expressing the mutant NCT- I241C GSEC complex with the APPC99-K28C substrate. As negative control, the same sample was supplemented with β-mercaptoethanol (BME) to reduce all disulfide bridges. A full-range molecular weight western blot is shown in Figure EV3B. Orange, black, and white arrows indicate the NCT-APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers.

(F) Analysis of the integrated density profiles of the respective western blot bands supports the spontaneous formation of a disulfide bond between NCT- I241C andAPPC99 K28C (mature and immature NCT are indicated with black and white arrows, respectively. The filled orange arrow points at the high molecular weight APPC99/Aβ density (crosslinked NCT-APPC99/Aβ band). Note that peak amplitudes for the cross-linked NCT-APPC99 and the mature NCT band were normalized to similar values for clarity purposes. Full-range molecular weight density profiles are shown in Figure EV3D.
Extracellular interface between APP and Nicastrin regulates Aβ length and response to γ-secretase modulators (2019)
Figure 6

Figure 6. GSECs destabilized by the N242F and N242Y NCT mutations do not respond to imidazole-based GSEC modulators.

(A) Chemical structures of acidic-GSEC modulator (GSM I) and imidazole-based-GSM II and GSM III (GSM B in (Szaruga et al, 2017)).

(B) Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from vehicle (DMSO) or GSM-treated (1 µM final concentration) MEF cells co-expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA. Data generated using 0.3 µM GSM-treated MEF cells is shown in Figure EV4. Data are presented as mean ± SD, N ≥ 3 independent experiments.

(C) Aβ 37/40 and Aβ 38/42 ratios were calculated to evaluate the efficiency of the GSEC-mediated cleavage of APPC99 (Figure EV4) and statistical analysis (P values) (two-way ANOVA with Sidak's post hoc test) for the effect of GSMs on WT and mutant complexes is presented (response to GSM normalized to the corresponding vehicle condition).
Localized incorporation of outer membrane components in the pathogen Brucella abortus (2018)
Figure 3

Figure 3 - Co-localization of R-LPS and the essential porin Omp2b.

A Co-localization of Omp2b (red) and R-LPS (green) in exponential phase bacteria. Scale bars: 2 μm.

B Co-detection of Omp2b (red) and R-LPS by AFM. R-LPS was detected using AFM tips functionalized with mAb against R-LPS. AFM images of whole bacteria and of the separated areas (red squares in first images, 0.4 x 0.4 μm2) are shown. The percentages of adhesion Padh are indicated below each area. Scale bars: 1 μm.

C Adhesion ratio between the Omp2b positive area compared to the Omp2b negative area. Grey dashed line shows ratio = 1 corresponding to homogeneity in surface structure. nWT = 13 bacteria, nΔgmd = 13 bacteria. Statistical analysis with Mann-Whitney U test showed **P < 0.01.
Localized incorporation of outer membrane components in the pathogen Brucella abortus (2018)
Figure 4

Figure 4 - Unipolar insertion of Omp25 and low mobility of Omp25 and Omp2b.

A Localization of initially labeled Omp25 (green) on TRSE-labeled (red) bacteria before (0 h) and after 2 h of growth in the absence of both labelings (2 h). As a control, Omp25 localization after pulse-chase labeling with TRSE is shown at the bottom panel. Scale bars: 2 μm.

B Model of TRSE (red) and Omp25 (green) labeling corresponding to (A). Incorporation of new material at the pole and/or at the constriction site is shown as black areas.

C Quantification of localization of newly inserted Omp25 represented by negative Omp25 areas from (A). Numbers are indicating mean percentages. n = 279 bacteria (5 biological replicates).

D Localization of initially labeled Omp2b (green) on TRSE-labeled (red) bacteria before (0 h) and after 2 h of growth in the absence of both labelings (2 h). Scale bars: 2 μm.

E Co-localization of Omp25 and Omp2b positive pixels with TRSE positive or negative pixels, respectively. Error bars correspond to s.d. from independent experiments. Differences were statistically analyzed by t-test. ***P < 0.001. nOmp25 = 279 bacteria, 85666 pixels (5 biological replicates). nOmp2b = 252 bacteria, 74630 pixels (3 biological replicates).
Localized incorporation of outer membrane components in the pathogen Brucella abortus (2018)
Figure 5

Figure 5 - Unipolar insertion of PG.

A Short pulse labeling of exponential phase bacteria with the fluorescent D-amino acid HADA. Merge is showing old pole marker PdhS-mCherry (red) and PG insertion sites (white). Scale bars: 2 μm.

B Demographic representation of HADA labeling. Bacteria were sorted according to their cell length and oriented with the old pole at the bottom of the demograph by PdhS-mCherry fluorescence signal. NFI: normalized fluorescence intensity. n = 393 bacteria (3 biological replicates).

C Quantification of localization of PG insertion sites from (A). Numbers are mean percentages. n = 393 bacteria (3 biological replicates).
Localized incorporation of outer membrane components in the pathogen Brucella abortus (2018)
Figure 6

Figure 6 - Unipolar insertion and low mobility of LPS.

A Localization of S-LPS (green) in the eFluor-labeled (magenta) inducible rough strain Δgmd plac-gmd possessing the old pole marker PdhS-mCherry (red). The strains Δgmd and Δgmd plac-gmd -IPTG were used as negative controls. Scale bars: 2 μm.

B Quantification of S-LPS insertion from (A). Numbers are indicating mean percentages. n = 468 bacteria (3 biological replicates).

C Demograph represents fluorescence profile of S-LPS labeling from (A). Cells were oriented with the old pole at the bottom of the graph by PdhS-mCherry and sorted by cell length. NFI: normalized fluorescence intensity. n = 468 bacteria (3 biological replicates).

D Short pulse labeling of LPS (2 h) by Kdo-N3 (green) on eFluor-labeled (magenta) bacteria expressing the old pole marker PdhS-mCherry (red). Scale bars: 2 μm.

E Quantification of LPS insertion sites from (D). Numbers are indicating mean percentages. n = 566 bacteria (3 biological replicates).

F Demographs represent fluorescence intensities of eFluor (OM, left) and Kdo-N3 (LPS, right) labeling. Cells were sorted by cell length and aligned with the old pole at the bottom of the graph by PdhS-mCherry localization. NFI: normalized fluorescence intensity. n = 566 bacteria (3 biological replicates).