EMBO/soda-nel huggingface dataset:
Proteostasis is essential during cochlear development for neuron survival and hair cell polarity (2019)
Figure 7

Figure 7: Amelioration of proteostasis by 4-PBA treatment improves the establishment of intrinsic polarity in cochlear HCs.

(A-C) Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (A) LGN and F-actin immunostainings of E16.5+1DIV OCs reveal that MG-132 perturbs LGN distribution at the apical surface of HCs, while co-treatment with 4-PBA ameliorates its enrichment at the lateral edge. Scale bars=5µm; magnified views=2µm. (B) Acetylated alpha-tubulin immunostaining reveals that the kinocilium is centrally shifted by MG-132 and partially restored by 4-PBA in E16.5+1DIV HCs (yellow arrowheads = kinocilium base; scale bar=2µm). (C) Kinocilium position plotted as a fraction of HC diameter for each HC row (DMSO/MG-132/MG-132+4PBA IHC: n=57/67/53, ***p<0.001, F=34.52, DF=2; OHC1: n=61/66/53, ***p<0.001, F=57.88, DF=2; OHC2: n=55/73/58, ***p<0.001, F=124.7, DF=2; OHC3: n=48/66/43, ***p<0.001, F=67.16, DF=2; pooled from 4 explants per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test).

(D-F) In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (D) The arc of lateral LGN enrichment remains in many Elp3cKO HCs after 4PBA administration. Scale bars=5µm; magnified views=2µm. (E) Acetylated alpha-tubulin immunostaining showing kinocilium position in WT or Elp3cKO OCs in the presence or absence of 4-PBA (yellow arrowheads = base of kinocilium; scale bar=2µm). (F) Kinocilium position plotted as a fraction of HC diameter for each HC row (WT/WT+4PBA/Elp3cKO/Elp3cKO+4PBA IHC: n=35/31/35/36, p=0.6812, F=4.510, DF=3; OHC1: n=36/32/37/40, ***p<0.001, **p<0.01, F=27.60, DF=3; OHC2: n=35/33/38/42, ***p<0.001, F=76.62, DF=3; OHC3: n=36/32/38/39, ***p<0.001, *p<0.05, F=27.86, DF=3; pooled from 3 animals per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test).

Data information: #IHC; *OHC1; **OHC2; ***OHC3
Transient upregulation of IRF1 during exit from naive pluripotency confers viral protection (2022)
Figure 2

Figure 2 - Formative pluripotency drives transient Irf1 expression

A Chromatin context of Irf1 in ESCs, EpiLC and BMDMs with and without IFNɣ stimulation. Shown are ChIP-seq tracks of histone mark H3K27ac, transcription factors OCT4, OTX2 and p300 and ATAC seq profiles. (data was generated by Data ref: Buecker et al. 2014, BMDM Data ref: Langlais et al. 2016). The putative enhancer that was knocked out is indicated.

B RT-qPCR analysis of Irf1 mRNA in Irf1+/+ and Irf1 enhancer KO in differentiation. Fold change is normalized against Rpl13a housekeeping mRNA expression and is calculated against ESC for each indicated cell line. This is shown as baseline with the dashed line at fold change = 1. Statistical tests were performed as homoscedastic one-sided t-tests. Shown are three biological replicates, datapoints from the same replicate are indicated with the same symbol. Black horizontal lines show the mean of the data.

C RT-qPCR analysis of Irf1 mRNA in Irf1+/+ and Irf1 enhancer KO in differentiation, treated with ruxolitinib. Fold change is normalized against Rpl13a housekeeping mRNA expression and is calculated against ESC for each indicated cell line. This is shown as baseline with the dashed line at fold change = 1. Statistical test was performed as homoscedastic one-sided t-tests. Shown are three biological replicates, datapoints from the same replicate are indicated with the same symbol. Note that each replicate contains two Irf1+/+ samples. Black horizontal lines show the mean of the data.

D Irf1 expression in murine blastocysts and ESCs. (Data Ref: Kinoshita et al. 2021). For E5.0, E5.5 and E6.0, entire single blastocysts were sequenced (n=3 biological replicates), for ESCs n=4 biological replicates.

E Irf1 expression in human blastocysts. Integrated single cell RNA-seq data from blastocysts collected at or cultured to indicated time points. Data is derived from a total of 16 embryos which passed quality control metrics. (Data Ref: Molè et al. 2021)
Transient upregulation of IRF1 during exit from naive pluripotency confers viral protection (2022)
Figure 3

Figure 3 - IRF1 regulates ISG expression in EpiLCs

A Western Blot analysis of Irf1-/- in ESC and EpiLC, probed with antibodies against IRF1 and VINCULIN as loading control. Shown are two independent clonal cell lines. See Figure EV3A for long exposure and size marker of the same blot.

B Immunofluorescence with IRF1 antibody in Irf1+/+ and Irf1-/- ESC and EpiLC. Nuclei are stained with DAPI. Scale bars are 10 µm.

C Dimension reduction (principal component analysis, PCA) of Irf1+/+ and Irf1-/- ESC and EpiLC, based on QuantSeq RNA data. PCA captures global transcriptional states of samples and similarity of these states is reflected in closeness of samples in the plot. n=2 biological replicates.

D Expression changes of selected pluripotency markers in Irf1+/+ and Irf1-/- ESC and EpiLC Quantseq RNA data. Data are shown as gene normalized z-score. n=2 biological replicates

E Expression changes of ISGs in Irf1+/+ and Irf1-/- ESC and EpiLC Quantseq RNA data. Shown are ISGs differentially expressed (p-value< 0.05) between Irf1+/+ and Irf1-/- EpiLCs. Data are shown as gene normalized z-score. See also Figure EV3E. Selected gene examples are indicated. n=2 biological replicates

F Expression changes of the same genes as in Figure 3E in dox-inducible SunTag-based IRF1 overexpression. Data are shown gene normalized as z-score. Selected gene examples are indicated. n=2 biological replicates.
Intra-membrane client recognition potentiates the chaperone functions of Calnexin (2022)
Figure 1

Figure 1. Calnexin interacts with non-glycosylated membrane proteins in a chaperone-like manner.

A Immunoblot analysis of overexpressed and ALFA-tagged CNX in CNX knockout cell lines clones #1 and #2, which were used for mass spectrometry. Endogenous CNX level of wildtype HEK293T cells is shown as a control.

B Schematic of CNX showing its lysine residues as blue dots. Lysines can be crosslinked by DSSO. The exact location of lysine residues is pictured in the crystal structure of the lumenal domain of CNX whereas the position of lysines in the cytoplasmic tail (C-tail) is an approximation.

C Volcano plots derived from LC-MS/MS analyses of ALFA-tagged CNX, immunoprecipitated in 1% NP-40 buffer from transfected CNX deficient HEK293T cells after DSSO crosslinking and compared to empty vector (EV) control co-IPs. Depicted is an enlarged section of the associated complete volcano plot in the upper right. Among the significantly enriched proteins, annotations are Uniprot gene names. CNX is shown in green. ER proteins with the GO-term annotation "endoplasmic reticulum", which are localized to the ER lumen, are highlighted in blue and among those several known CNX interaction partners are indicated with their names. Membrane proteins are shown in red and those which are non-glycosylated are additionally labeled with their gene names. Further details on the classification of the identified membrane proteins can be found in Appendix Fig S1B. Cut-off values (solid lines) in the volcano plot were defined as log2 = >1 (2-fold enrichment) and -log10 = (P-value) > 1.3 (P<0.05).

D Schematic of Cx32 structure and its orientation within the membrane. Positions of mutations analyzed in this study are indicated with stars. The overall topology and loop lengths were obtained from the UniProt server (human GJB1 gene).

E Co-immunoprecipitations of endogenous CNX with FLAG-tagged Cx32 WT and its mutants including quantification and statistical analysis. Constructs were expressed in HEK293T cells. One representative immunoblot is shown. Monomers and dimers of Cx32 are indicated with brackets. All samples were normalized to WT (mean ± SEM, N ≥ 4, *P value < 0.05, two-tailed Student's t tests).

F Immunofluorescence images of Cx32 and its mutants. COS-7 cells were transiently transfected with the indicated FLAG-tagged Cx32 constructs and immunofluorescence microscopy was performed using anti PDI (green) as an ER marker. Detection of Cx32 was performed using anti FLAG antibodies and suitable labeled secondary antibodies (red). Nuclei were stained with DAPI (blue). All three channels are overlayed. Images are representative of cells from at least three different biological replicates. GJ denotes gap junctions observed at cell-cell junctions of cells transfected with WT Cx32. Scale bars correspond to 20 µm.
Intra-membrane client recognition potentiates the chaperone functions of Calnexin (2022)
Figure 2

Fig 2. The transmembrane domain of Calnexin binds clients in the membrane.

A Schematic of the reporter construct, where an antibody CL domain was fused to sequences of interest, to assess CNX interactions with individual Cx32 TMD segments. The four WT sequences of the Cx32-TMDs are shown. The Arg residue exchanged against Cys for BMH crosslinking is highlighted in TMD1. Predicted membrane integration energies are given using DGPred (Hessa et al., 2007). Sequences of TMD1 and TMD3 were inverted in the respective constructs to have the same amino acid composition and the same amino acids exposed to the lipid bilayer as in the corresponding Cx32 TMDs.

B Co-immunoprecipitations of endogenous CNX with the different CL-TMD constructs (NVT glycosylation site mutated to QVT) including quantification and statistical analysis. One representative immunoblot is shown. The asterisk (*) denotes a fraction of cleaved species as observed previously for similar constructs (Behnke et al., 2016). All samples were normalized to the construct containing TMD1 (mean ± SEM, N ≥ 3, *P value < 0.05, two-tailed Student's t tests).

C Interaction analysis of Calnexin with the first transmembrane domain of Cx32. Intracellular BMH crosslinking of transiently transfected V5-tagged CNX with the transiently transfected R26C mutant of Cx32-TMD1. In the AA mutant of CNX, the two cysteines within the CNX TMD were replaced by alanines. The species at approx. 250 kDa are possibly CNX dimers.

D Schematic of the minimal CNX construct (minCNXTMD). This consists of a preprolactin (PPL) ER import sequence, followed by an N-terminal MYC tag, the monomeric red fluorescent protein mScarlet-I, the TMD segment of CNX including an endogenous double lysine motif downstream of the TMD region and a C-terminal FLAG-tag. Flexible linker regions connect the individual components. Parts derived from CNX are shown in orange. A control construct, minCNXLECTIN contains the entire lumenal lectin domain of CNX whereas another control, ER-Scarlet, is lacking any CNX segments. A C-terminal KDEL sequence was included in both control constructs for ER retention.

E COS-7 cells were transiently transfected with the indicated constructs and immunofluorescence microscopy was performed using anti PDI (green) as an ER marker. Detection of minCNXTMD and both control constructs was carried out by mScarlet-I fluorescence (red). Nuclei were stained with DAPI (blue). Images are representative of cells from at least three different biological replicates. Scale bars correspond to 20 µm.

F Interaction of Cx32-TMD1 with the minCNX system. MinCNXTMD constructs and controls (see (D)) were co-transfected with Cx32-TMD1 into HEK293T cells. Interaction between the proteins was analyzed by co-immunoprecipitation experiments followed by immunoblotting.
Intra-membrane client recognition potentiates the chaperone functions of Calnexin (2022)
Figure 3

Fig 3. Design and validation of ConMem, a tool to query TM-domain recognition.

A Schematic of the the minimal consensus membrane protein ConMem which contains a preprolactin (PPL) ER import sequence, a superfolder GFP (sfGFP), a TMD flanked by two individual NVT glycosylation motifs (N1 and N2) and a C-terminal HA-tag. Individual construct components are connected by flexible linker regions. As illustrated, in the predicted topology of ConMem, only the first NVT glycosylation site (N1) is accessible to the ER glycosylation machinery (grey hexagon). The TMD of ConMem was designed on the basis of a multiple sequence alignment of 200 human single pass TMD sequences as illustrated in the sequence logo. Hydrophilic amino acid residues are depicted in blue, neutral ones in green and hydrophobic residues in black. The predicted amino acid sequence with the second highest score was selected as the TMD consensus sequence for ConMem (bold). Note that the orientation of the TMD was not included in this analysis, thus giving rise to a consensus sequence purely based on amino acid composition.

B HEK293T cells were transiently transfected with plasmids expressing ConMem or the indicated variants, where either the first (N1) or the second (N2) NVT glycosylation motif was ablated (N to Q mutation). Lysates were treated with or without EndoH or PNGaseF as indicated, and analyzed by immunoblotting. N-glycosylation occurs in the ER, complex glycosylation in the Golgi.

C COS-7 cells were transfected with the indicated constructs and fluorescence microscopy was performed using Sec61 mCherry (red) as an ER marker. ConMem, detected by sfGFP fluorescence (green), localizes to the plasma membrane. Nuclei were stained with DAPI (blue). Images are representative of cells from at least three different biological replicates. Scale bars correspond to 20 µm.
Intra-membrane client recognition potentiates the chaperone functions of Calnexin (2022)
Figure 4

Fig 4. Defining intramembrane recognition preferences for Calnexin.

A Schematic of ConMem N1Q where a central Val residue at position 13 within its TMD was replaced by all other 19 amino acids. Free energies for TMD insertion were predicted according to (Hessa et al., 2007).

B Representative blots from co-immunoprecipitation experiments from HEK293T cells transfected with the indicated ConMem constructs with or without ER-lumenal glycosylation site (N1) (mean ± SEM, N = 3, *P value < 0.05, two-tailed Student's t tests). Individual values were normalized to ConMem N1Q (Val) values that were set to 1.

C Interaction of ConMem N1Q variants as described in (A) with endogenous CNX (mean ± SEM, N ≥ 5, *P value < 0.05, two-tailed Student's t tests ). Individual values were normalized to ConMem N1Q (Val) values that were set to 1.

D Same as in (C) only that selected amino acids (proline and arginine) where shifted through the ConMem N1Q TMD segment to five different positions as shown in the illustration, revealing positional binding dependencies (mean ± SEM, N ≥ 4, *P value < 0.05, two-tailed Student's t tests). Individual values were normalized to ConMem N1Q (Val) values that were set to 1.
Intra-membrane client recognition potentiates the chaperone functions of Calnexin (2022)
Figure 5

Fig 5. A structural analysis of intramembrane Calnexin:client recognition.

A Meta analysis of MD simulations (N=8) where complex formation between CNX and Cx32-TMD1 or CNX and ConMem was observed. On the x-axis the CNX TMD residues obtained from UniProt are shown. The height of the bars corresponds to the number of simulations in which a specific CNX TMD residue interacted with the substrate helix CX32-TMD1 (turquoise) or ConMem (violet). Only CNX TMD residues Y6, T9 and L12 (highlighted in red in the helical wheel) interacted with CX32-TMD1 or ConMem in all sampled complexes.

B The YTL-motif of CNX (orange) interacts with ConMem (violet) mainly via hydrophobic interactions in MD simulations.

C Interactions between the CNX (orange) YTL-motif and Cx32-TMD1 (turquoise) include general hydrophobic interactions and hydrogen bonding between Y6 and the CX32-TMD1 backbone. The intramembrane arginine of Cx32 is involved in CNX interactions via cation-π stacking as well as polar interactions.

D Interaction of non-glycosylated ConMem N1Q constructs with V5-tagged CNX. In CNX variants, amino acids important for interaction as revealed in (A) were replaced by valine residues. HEK293T cells were transiently transfected with the indicated constructs and cell lysates and HA-immunoprecipitates were analyzed for HA-tagged ConMem N1Q and co-immunoprecipitating CNX mutants. One representative blot and quantifications are shown (mean ± SEM, N ≥ 9, *P value < 0.05, two-tailed Student's t tests).

E The same as in (D) for Cx32-TMD1 (mean ± SEM, N ≥ 6, *P value < 0.05, two-tailed Student's t tests).

F Analysis of mutations in the CNX TMD region and their effect on binding glycosylated ConMem. One representative blot and quantifications are shown (mean ± SEM, N ≥ 3, n.s.: not significant, two-tailed Student's t tests).

G The same as in (D) for Cx32 full-length (mean ± SEM, N ≥ 4, *P value < 0.05, two-tailed Student's t tests).

H Interaction of Cx32-TMD1 with CNX mutants, where amino acids not predicted to be important for interaction as shown in (A) were exchanged by valine residues. HEK293T cells were transiently transfected with the indicated constructs. One representative blot and quantifications are shown (mean ± SEM, N ≥ 9, n.s.: not significant, two-tailed Student's t tests).

I Shifting of the residues Y, T and L, most important vor CNX substrate interaction, and replacing these against valine residues, impacts interactions with Cx32-TMD1. HEK293T cells were transiently transfected with the indicated constructs. One representative blot and quantifications are shown (mean ± SEM, N ≥ 9, *P value < 0.05, two-tailed Student's t tests).
Intra-membrane client recognition potentiates the chaperone functions of Calnexin (2022)
Figure 6

Fig 6. Biological functions of intramembrane client recognition by Calnexin.

A Analysis of the interaction of the binding deficient CNX YTL TMD variant with Rhodopsin. CNX knockout HEK293T cells were transiently transfected with either N-terminal ALFA-tagged Rhodopsin alone or in combination with the indicated V5-tagged CNX constructs. In the absence of any CNX, Rho is hardly detectable (long exposure shown). Cell lysates and ALFA-immunoprecipitates were analyzed for Rhodopsin and co-immunoprecipitating CNX. One representative blot is shown on the left, quantifications on the right (mean ± SEM, N = 4, *P value < 0.05, two-tailed Student's t tests).

B Analysis of Rhodopsin degradation and its dependency on CNX. CNX knockout HEK293T cells that were transfected with the indicated constructs were incubated with cycloheximide and lysates were collected at different timepoints. CNX WT and CNX YTL immunoblots are from the same blot (dashed line), showing similar expression levels. Hsc70 was used as a loading control (also from the same blot, dashed line). Quantifications are shown below representative immunoblots (mean ± SEM, N ≥ 8, *P value < 0.05, two-tailed Student's t tests).
Defective ribosomal products (DRiPs) challenge nuclear function by impairing nuclear condensate dynamics and immobilizing ubiquitin (2019)
Figure 1

Figure 1: DRiPs accumulate in nucleoli of normally growing HeLa cells (see also Figure EV1).

(A) DRiP labelling in GFP-NCL HeLa Kyoto cells that were either left untreated or treated with OP-puro (25 µM) for 2 h. Where indicated cells were allowed to recover in drug-free medium (control) or in presence of MG132 (10 µM) for 5 h. Scale bars: 5 µm.

(B) GFP-NCL HeLa Kyoto cells were treated as described in A, with the exception that a lower concentration of OP-puro was used (5 µM). The distribution of DRiPs and GFP-NCL was analyzed by STED super-resolution microscopy. Scale bars: 5 µm.

(C) Isolation of nucleoli from HeLa Kyoto cells untreated or treated with puromycin (25 µM) for 2 h. Puromycylated proteins were detected by immunoblotting (right panel). The left panel shows the purity of isolated nucleoli by western blotting, where fibrillarin was used as nucleolar marker, FUS as nucleoplasmic marker and TUBA4A as cytoplasmic marker.
Defective ribosomal products (DRiPs) challenge nuclear function by impairing nuclear condensate dynamics and immobilizing ubiquitin (2019)
Figure 2

Figure 2: Upon heat shock or proteasome inhibition, DRiPs promote amyloidogenesis in NoABs (see also Figure EV2 and Appendix Figure S1).

(A) HeLa cells were left untreated or treated with heat shock (HS) at 42˚C for 3 h, alone or with cycloheximide (CHX; 50 μg/ml), or Actinomycin D (Act. D; 3 µM) followed by staining with the dye Amylo-glo.

(B) DRiP labelling in GFP-NCL HeLa Kyoto cells that were either left untreated or treated with HS at 42˚C and OP-puro (25 µM) for 2 h. Where indicated, cells were cotreated with CHX (50 μg/ml).

(C) HSPA8 subcellular distribution and recruitment inside nucleoli following exposure to HS at 42˚C for 2 h, alone or combined with CHX, in GFP-NCL HeLa Kyoto cells.

(D) DRiP labelling in HeLa cells treated with MG132 (10 µM) alone or with CHX (50 μg/ml) for 8 h; where indicated, OP-puro (25 µM) was added during the last 45 min, prior to cell fixation.

(E) Amylo-glo staining of HeLa cells left untreated or exposed to MG132 (10 µM) alone or combined with CHX (50 μg/ml) for 8 or 13 h.

(F) Amylo-glo staining of HeLa cells left untreated or subjected to transcriptional stress (MG132 10 µM and Act. D 4 µM) for 6 hrs. Where indicated, translation was concomitantly inhibited with CHX (50 μg/ml). Amylo-Glo staining is also shown in HeLa cells treated with Act.D alone or with CHX for 6 hrs, as control.

(G) Amylo-glo staining of HeLa cells exposed to HS at 42˚C for 3 h and let to recover for 3 h in drug-free medium (+ rec. control) or with VER (40 µM; + rec. VER). Where indicated cells were treated with VER alone (VER 3 h).

(H) Amylo-glo staining of HeLa cells treated with MG132 (10 µM) for 13 h and let to recover for 5 h in drug-free medium (+ rec. control) or with VER (40 µM; + rec. VER). Where indicated cells were treated with VER alone (VER 5 h).

(I) HeLa cells were transfected with vectors encoding for GFP, GFP-GR50 or RPL23a-GFP. 24 hrs post-transfection cells were fixed and stained with Amylo-glo. The percentage of transfected cells with Amylo-Glo positive nucleoli is shown. Number of cells counted/condition: GFP, 412; RPL23a-GFP, 482; GFP-GR50, 359, in three independent experiments; statistical significance via One-way ANOVA; p < 10-7, ± s.e.m.

(A-I): Scale bars: 10 µm.