EMBO/soda-nel huggingface dataset:
Reduced proteasome activity in the aging brain results in ribosome stoichiometry loss and aggregation (2020)
Figure 5

Figure 5. Partial pharmacological inhibition of proteasome activity in adult killifish brain affects aging protein networks and induces stoichiometry changes in a subset of protein complexes.

(A) Study design. Adult (12-14 wph) killifish were treated with different proteasome inhibitors or vehicle control for 4 days. Proteasome activity and proteome changes were analysed in brains from treated and control fish.

(B) Chymotrypsin-like (CT-L) proteasome activity in brain extracts of killifish treated with different proteasome inhibitors. The activity was measured at day 4 after the beginning of treatment. n=8 per sample group; error bars indicate standard deviation. ** p < 0.01, **** p < 0.0001, one-way ANOVA followed by Holm-Sidak's multiple comparisons test.

(C) Principal component analysis (PCA) of brain samples based on proteome profiles obtained by Data Independent Acquisition (DIA) quantification treated with Bortezomib or vehicle control (DMSO). n=10 per sample group. The smaller dots represent individual samples and the larger dots the centroids of each age-matched group. Ellipses represent 95% confidence intervals. The percentage of variance explained by the first two PC axes is reported in the axis titles.

(D) Proteasome inhibition induces proteasome activators (PSME4), assembly factors (POMP) ubiquitin ligases (UBE3C), mediators of autophagosome formation (ATG2B), heat shock proteins and chaperones in killifish brain. Protein abundances were quantified by DIA mass spectrometry and they are shown relative to the mean value of vehicle control samples (DMSO) set to 1, n=10 per sample group. Adj. p < 0.05 for all the displayed proteins. Error bars indicate mean +/-SD.

(E) Overlap between proteins affected by Bortezomib treatment and aging in killifish brain. For all comparisons, only significantly affected proteins adj. p < 0.05 were considered. A significant overlap between Bortezomib and aging-affected proteins is detected (Fisher test, as indicated in the figure panel). Significantly enriched GO biological process terms in the subset of overlapping proteins are indicated (FDR < 0.05).

(F) Bortezomib treatment affects the stoichiometry of a subset of protein complexes. The number of affected proteins (adj. p < 0.25) for each protein complex is indicated. Only protein complexes that had at least two members affected are shown.

(G) Members of the mitochondrial and cytoplasmic ribosomes affected by Bortezomib treatment. Relative protein abundances (normalized to the mean of the protein complex to which they belong) are shown. The mean value of vehicle control samples (DMSO) was set to 1. n=10 per sample group. Adj. p < 0.25 for all the displayed proteins. Error bars indicate mean +/-SD.

Data information: Related to Dataset EV8.
Wnt regulates amino acid transporter Slc7a5 constraining integrated stress response in mouse embryos (2019)
Figure 2

Figure 2 Slc7a5-null embryos exhibit overt neural tube closure and limb-bud outgrowth defects, reduced Fgf8 expression domains and aberrant neuronal and neural crest differentiation

(A-H) Live wildtype littermate and Slc7a5-null embryos imaged shortly after dissection. (A-D) Wildtype and (E-H) Slc7a5-null E9.75 embryos from lateral (A, D, E, H), frontal (B, F) or dorsal (C, G) views; (e1-h1) higher magnification images of the Slc7a5-null embryo. White arrowheads indicate the smaller limb bud (compare B and F), open/reduced forebrain (compare D and H), open neural tube at posterior (compare C and E, e1) and anterior (compare B and F, f1) regions. Black arrowheads indicate kinked neural tube (compare C and G, g1) and apparently missing optic and smaller otic vesicles (ov) (compare D and h1).

(I-P) mRNA in situ hybridization and immunofluorescence in E9.5 or E10.5 wildtype and Slc7a5-null embryos for key marker genes.

(I-L) Fgf8 mRNA transcripts were detected in wildtype (I-j1) and in Slc7a5-null (K-l1) E10.5 embryos (n=4 each) (white arrowheads indicate limb buds in K and the isthmus in L) with (j1, l1) sections through isthmus at midbrain/hindbrain border and (i1, k1) lateral views of forelimb buds.

(M-N) Wholemount Tubulin-β-III (Tuj1) immunofluorescence performed on (M, m1) wildtype and (N, n1) Slc7a5-null E9.5-E10.5 embryos (n=5 each condition). Dorsal root ganglia (DRG) and Sympathetic Chain Ganglia (SCG) are indicated with open arrowheads together with cranial ganglia IX, X and XI. Images in (m1, n1) show higher magnification of the cranial ganglia V, VII/VIII (open arrowheads).

(O-P) Neurog2 expression in wildtype (O-o4) and Slc7a5-null embryos (P-p4) (n= 5 and n=6 each), showing reduced expression in forebrain (o2, p2), cranial ganglia (o1, o3, p1, p3, arrowheads) and in spinal cord at level of forelimb (o4, p4, arrowheads indicate position of neural crest).

Data information: Scale bars 200 µm, except sections 100 µm.

­
Wnt regulates amino acid transporter Slc7a5 constraining integrated stress response in mouse embryos (2019)
Figure 3

Figure 3 Slc7a5-null embryos exhibit altered expression of neural crest genes

(A-B) Sox10 in situ hybridization in (A) wildtype and (B) Slc7a5-null E9.5 embryos (n=4 each) with TSs of (a1, b1) the hindbrain at level of trigeminal ganglion V, (a2, b2) otic vesicles and the neural tube at more posterior levels (a3, a4, b3, b4). Scale bars 200 µm, except sections a1 - b4, 100 µm. Closed white arrowheads in (a3, a4) indicate neural crest, open white arrowheads indicate depleted neural crest in (b3) and position where neural crest should be in (b4).

(C-G) Immunofluorescence on TSs caudal spinal cord of E9.5 wildtype littermates and Slc7a5-null embryos; Pax3 and FoxA2 were used as indicators for dorso-ventral organisation in (C, F) wildtype, n=2 (for FoxA2) and n= 4 (For Pax3) and (D, G) Slc7a5-null, n=3 (for FoxA2 ) and n = 3 for Pax3) neural tube. Arrowheads indicate border of Pax3 expression domain. Scale bars 50 µm.

(E) Percentage of Pax3 expressing cells was determined by counting these cells and all DAPI labelled nuclei in the neural tube and comparison made between wildtype (4 embryos, 6 sections each) and Slc7a5-null (3 embryos, 6 sections each), each dot represents the average for one embryo, unpaired t-test, *p = 0.018 (see original source data). Error bars indicate SEM.
Wnt regulates amino acid transporter Slc7a5 constraining integrated stress response in mouse embryos (2019)
Figure 4

Figure 4 Slc7a5-null neural tube exhibits slightly reduced cell proliferation and aberrant mTORC1 activity, and qPCR validation of significantly changed genes in Slc7a5-null embryos

(A-D) Proliferation was assessed in the spinal cord (forelimb level) (A, B) using a phospho-Histone3 (phospho-H3) antibody (green) to identify mitotic cells and DAPI (blue) to label nuclei; cells were counted in 3 wildtype (18 sections) and 3 Slc7a5-null embryos (18 sections) and mitotic index calculated; (C, D) Proliferation was also assessed in the forebrain in 3 wildtype embryos (17 sections) and 3 Slc7a5-null embryos (16 sections). White dashed lines indicate midline.

(E, F) Comparison of mitotic index in wildtype and Slc7a5-null (E) spinal cord, p = 0.872 ,and (F) forebrain *p = 0.049, each dot represents the average for one embryo, unpaired t-test (see original source data).

(G, H) mTORC1 activity in the spinal cord (hindlimb level) was assessed by immunofluorescence using an antibody against the phospho-ribosomal protein S6 (phosho-S6 Ser 235/236).

(I) Labelling intensity was measured and plotted relative to DAPI intensity; 6 wildtype embryos (18 sections) and 5 Slc7a5-null embryos (19 sections). Each dot represents the average for one embryo, (*p = 0.0457) unpaired t-test with Welch correction (see original source data).

(J) Western blot of individual E9.5 wildtype (n=3) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-S6K (Thr389) and total P70S6K for loading control. Band intensities were measured with FIJI and an unpaired t-test was performed for statistical analysis (see original source data).

(K-L) (K) Validation by qPCR of the targets identified by RNAseq; qPCR was performed on individual E8.5 embryos of each genotype (wildtype n=5, heterozygous n=9 and Slc7a5-null n=7). (L) qPCR was performed on individual E9.5 embryos (wildtype n=5, heterozygous n=3 and Slc7a5-null n=4) using primers specific for genes associated with the integrated stress response. A one-way Anova test with a Turkey post-test was performed.

Data information: *p<0.05, **p<0.01, ***p<0.001, for actual p values see original source data file. Error bars indicate SEM. All scale bars 50 µm.
Wnt regulates amino acid transporter Slc7a5 constraining integrated stress response in mouse embryos (2019)
Figure 5

Figure 5 Slc7a5-null embryos exhibit localised integrated stress response

(A-L) mRNA in situ hybridization was performed at E9.5 to detect Chac1 in (A-a6) wildtype and (B-b6) Slc7a5-null littermate embryos (n=3 for each condition); Chac1 expression at E10 in emerging limb buds assessed in (C) wildtype and (D) Slc7a5-null littermate embryos (n=2 for each condition); Trib3 mRNA detected in (E-e6) wildtype and (F-f6) Slc7a5-null littermate embryos (n=3 for each condition) and in emerging limb buds at E10.5 assessed in (G) wildtype and (H) Slc7a5-null littermate embryos (n=2 for each condition). Chac1 and Trib3 transcripts were also detected in wildtype E9.5 embryos after >3 days in situ hybridisation reaction (n=3 and n=6 respectively). Lateral view (I and K), optic vesicles (white arrowheads), branchial arches (ba) and otic vesicles (ov). Dorsal view (J and L), neural tube (nt) and limb buds (white arrowheads). Scale bars 200 µm for whole embryo images and 100 µm for sections.

(M) Schematic indicating regions (purple) in which high levels of Slc7a5 (see Figures 1C-E) and ISR genes Trib3 and Chac1 are detected in wildtype embryos.
Wnt regulates amino acid transporter Slc7a5 constraining integrated stress response in mouse embryos (2019)
Figure 6

Figure 6 Cell death is increased in Slc7a5-null embryos and exogenous ER Stress induction in wildtype embryos upregulates Chac1 and Trib3

(A, B) Western blot of individual E9.5 wildtype (n=4) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-GCN2 (Thr899) and total GCN2 (loading control), ***p = 0.0001 ; or against phospho-eIF2α (Ser51) and total eIFα (loading control), *p = 0.0482, band intensities measured with FIJI and analysed with unpaired t-test (see original source data).

(C-F) phosphorylated eIFα was assessed by immunofluorescence in E9.5 embryos in the forebrain, wildtype (C,c1) (n=2 embryos, 0/14 sections) and Slc7a5-null (D, d1) (n=2 embryos 16/16 sections), and in hindbrain/anterior spinal cord, wildtype (E-e2) (n= 4 embryos, 0/62 sections) and Slc7a5-null (F-f2) (n= 4 embryos, 54/54 sections) including in the otic vesicle (compare e2, f2). Scale bars in (C-e1, D-f1) 100 µm and in (e2 and f2) 25µm. Arrowheads indicate ventral midline.

(G) An increase in Trib3 protein was detected by Western blot in Slc7a5-null embryos, each lane represents an E9.5 embryo lysate (wildtype n=3, Slc7a5-null, n=4) with α-Tubulin loading control, unpaired t-test *p = 0.0153 (see original source data).

(H-L) TUNEL assay to detect apoptotic cells in wildtype (n=3 embryos, 32 sections) or Slc7a5-null (n=5 embryos, 47 sections); (H, I) transverse sections through the head at level of the forebrain and (J, K) the spinal cord, (H-K) scale bars 50 µm, arrowheads indicate ventral midline. (L) Quantification of TUNEL positive cells within spinal cord sections was performed as stated in Materials and Methods. Each dot represents the average apoptosis cell count for a single embryo, Welch's correction unpaired t-test, p = 0.250, F-test to compare variances, p = 0.0003 (see original source data).

Data information: All error bars indicate SEM.
Wnt regulates amino acid transporter Slc7a5 constraining integrated stress response in mouse embryos (2019)
Figure 7

Figure 7 Wnt β-catenin signalling is unaffected in Slc7a5-null embryos, but is required for Slc7a5 transcription and β-catenin loss induces ISR gene Trib3

(A-B) (A,a1) Wnt β-catenin target Axin2 strongly expressed in littermate, (a2, a3) in TS, and (B, b1) in Scl7a5-null embryos (b2, b3) in TS (n=2 littermates, n=6/6 Slc7a5-null embryos).

(C, d1) Axin2 transcripts in embryo trunk explant exposed to DMSO (C, c1, n=12/12) were reduced in Wnt-c59 exposed explants (D, d1, n=14/14). Images in (c1, d1) show TS through explants (data from 3 independent embryo explant experiments).

(E, F) Slc7a5 transcripts in embryo trunk explant exposed to DMSO (E, e1, n=9/10) or Wnt-c59 (F, f1, n=15/15). Images in (e1, f1) show TS through explants (data from 3 independent embryo explant experiments).

(G, H) Slc7a5 expression in control T-Cre;Ctnnb1flLOF/Δ heterozygous littermate embryo (G, g1) and in TS (g2, g3), and in T-Cre;Ctnnb1flLOF/Δ mutant embryo where Slc7a5 transcripts are absent in posterior neural tube (the region of T-cre recombination, indicated with white arrowhead in H) and shown in TS in (h2, h3) (n= 0/6 littermate and n=6/6 mutant embryos).

(I, J) Trib3 is lacking in T-Cre;Ctnnb1flLOF/Δ heterozygous control embryos at E7.75 (I), caudal region dissected from the same embryo (white dashed box in I) (i1) and viewed in TS (i2); Trib3 detected in homozygous T-Cre;Ctnnb1flLOF/Δ embryo (J), in caudal region dissected from the same embryo (white dashed box in J) (j1) and shown in TS in caudal epiblast and remnant primitive streak (ps) (j2) (n= 3/3 T-Cre;Ctnnb1flLOF/Δ embryos and 14 littermate controls). Trib3 was also detected in the more rostral forming neural tube at this early stage (seen in J) and so in cells not directly experiencing β-catenin loss; this may reflect the failure to form paraxial mesoderm, which provides signals that promote and support neurogenesis.

(K-M) At E9.5 Trib3 was not detected in T-Cre;Ctnnb1flLOF/Δ heterozygous littermate embryos (K, k1) and in TS ( k2, k3), but was detected in homozygous T-Cre;Ctnnb1flLOF/Δ mutant embryos (L-m3) in patches of cells in neural tube (nt) (L - l2), gut (g) (l3) and somites (s) (m2, arrowheads) (n=9/9 T-Cre;Ctnnb1flLOF/Δ embryos and 12 littermate controls).

Data information: White dashed lines indicate position of sections. Scale bars, in (A, a1, B, b1, G, g1, H, h1) = 200 µm, in (C, D, E, F) = 100 µm, all sections 50 µm.

Expanded View figure legends
Cryo-EM structure of native human uromodulin, a zona pellucida module polymer (2020)
Figure 1

Figure 1. Overall structure of human UMOD filaments.

A Electron micrograph of unstained UMOD filament sheets in human urine. The inset highlights a double helix-like structure resulting from juxtaposition of two individual filaments. Scale bars: 50 nm and 10 nm (inset).

B, C "Tree" front view and "zig-zag" side view of purified native UMOD filaments, imaged using a Volta phase plate. Scale bars: 10 nm.

D, E Front and side views of UMODe filaments, showing absence of branches. Scale bars: 10 nm.

F Orthogonal views of the sharpened cryo-EM map of UMODfl (3.8 Å resolution), oriented as in panels B and C, respectively. The map is fitted with an atomic model that consists of a complete EGF IV+ZP module (chain A; blue), the ZP-C domain of a second molecule (chain B; teal) and the EGF IV+ZP-N domain of a third one (chain C; magenta).

G-J Details of the UMODfl map shown in panel F: ZP-N N396 glycan (G); interdomain linker α1β (H) and β1 (I); ZP-C αEFβ/ZP-N βF' intermolecular interface (J).

K Sharpened cryo-EM map of UMODe (4.0 Å resolution) in two orthogonal views oriented as in panels D, E. Comparison of this map with that of UMODfl identifies density belonging to the N-terminal half of UMOD (salmon contour in the front view of panel F), which is lost upon site-specific cleavage by elastase (orange arrow).

L Goodsell-style depiction of a complete UMODfl filament model, with protein subunits shown in different colors.
Cryo-EM structure of native human uromodulin, a zona pellucida module polymer (2020)
Figure 4

Figure 4. Effect of polymerization interface mutations on UMOD filament assembly.

A Immunofluorescence of unpermeabilized stably transfected MDCK cells shows that, unlike wt full-length UMOD, mutants ZP-N (R415A) or ZP-C (ΔFA) do not assemble into filaments. Scale bar: 50 µm.

B-G Immunofluorescence of unpermeabilized cells co-expressing FLAG-tagged wt UMOD (green) and the indicated HA-tagged isoforms (red). UMOD R415A does not incorporate into polymers that only contain wt protein (C). UMOD ΔFA has a dominant negative effect on wt protein polymerization (D) that is rescued in a double mutant carrying both ZP-N and ZP-C mutations (F). Similarly, the dominant negative effect of a CCS mutation (4A) that prevents EHP dissociation (E) is suppressed by the introducing the R415A mutation in the 4A isoform (G). Scale bar: 50 µm.

H Immunofluorescence of permeabilized cells expressing soluble isoforms of UMOD truncated before the EHP (UMOD-CCS) shows that wt forms intracellular polymers whereas polymerization interface mutants do not. The intracellular polymers are localized in the endoplasmic reticulum (ER), as shown by co-staining with the KDEL sequence used as ER marker. Scale bar: 10 µm.
Cryo-EM structure of native human uromodulin, a zona pellucida module polymer (2020)
Figure 6

Figure 6. Modelling of UMOD sheet architecture and ZP module filament binding sites for UPEC and sperm.

A Negative stain micrograph of a sheet formed by purified UMOD filaments in 100 mM NaCl (top). Analysis of this material by correlation averaging reveals a periodic pattern (bottom) with a two-fold symmetry axis perpendicular to the plane of the sheet (white symbol). Scale bars: 250 Å (top) and 125 Å (bottom).

B UMOD sheet model, generated by antiparallel juxtaposition of multiple copies of the UMODfl composite map, according to the 2D information shown in panel A. Due to lack of information in the direction perpendicular to the plane of the sheets, the exact position of adjacent filaments relative to this plane is unknown; for simplicity, the depicted model has been generated by assuming that the cores of the filaments making up the sheet lie on the same plane. Individual filaments are alternately colored light and dark grey, with N-terminal protein regions highlighted in pink and magenta, respectively. The zig-zag view of two adjacent filaments in the sheet resembles the projection of a double helix (see inset of Fig 1A) and the regular presentation of UMOD branches generates a molecular Velcro surface for the capture of UPEC.

C Comparison of the UMOD sheet model with the bottom part of panel A suggests that the diffuse density protruding into the space between adjacent filaments (cyan arrow) corresponds to the glycans attached to N396 and N513 (light and dark blue arrows, respectively).

D Detail of a porcine ZP filament model, generated by superposition of homology models of adjacent ZP3 and ZP4 subunits (green and purple, respectively) onto the structure of UMODfl (semi-transparent grey), and projection of the residues of the interdomain linker of another ZP3 subunit onto the UMOD ZP-N/ZP-C linker (red). A cluster of experimentally verified glycosylation sites is shown, with side chain atoms depicted as spheres and boxes highlighting the proximity of ZP3 N271 and ZP4 N220, whose N-glycans mediate sperm binding in pig (Yonezawa, 2014). Note how these residues are also close to the conserved N- (dashed underline) and O- (site 1, single underline; site 2, double underline) glycosylation sites in the ZP3 interdomain linker. The invariant ZP3 Trp that follows site 1 is shown in stick representation at the interface with ZP4.

E Detail of the ZP3/ZP1 interface of an avian egg coat filament model, assembled and represented as described for panel D. The box highlights the location of O-glycosylation site 1, important for in vitro binding of chicken ZP3 to sperm (Han et al, 2010).
Paraspeckles are constructed as block copolymer micelles (2021)
Figure 1

Figure 1. Deletion of the 3′ terminal domain of NEAT1_2 causes core localization of the 3′ ends of NEAT1_2 within the paraspeckle.

A The schematics of human NEAT1_2 (WT) and the mutants with deletions in the 3′ terminal regions. The NEAT1 transcripts are shown above with a scale. The gray dashed lines represent the deleted regions. The positions of NEAT1 probes used in SRM (blue) and EM (orange) are shown.

B (left) SRM images of paraspeckles in HAP1 NEAT1 ∆3′ mutant cells (∆3′) detected by NEAT1_5′ (green) and NEAT1_15k (magenta) FISH probes in the presence of MG132 (5 μM for 6 h). Scale bar, 500 nm. (right) Graph showing the proportion of paraspeckles with localization of the NEAT1 3′ ends in the core or in the core and shell (n = 44).

C (upper) EM observation of the paraspeckles in ∆3′ cells using NEAT1_D2 probes in the presence of MG132 (5 μM for 17 h). Scale bar, 100 nm. (lower, left) Graph showing the proportion of localization of the NEAT1 region detected by NEAT1_D2 probe (248 gold particles) within the paraspeckles in ∆3′ cells. (lower, right) Graph showing the proportion of localization of NEAT1_D2 probes in each paraspeckle in ∆3′ cells (n = 17). The box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top).

D SRM images of the paraspeckles in ∆16.6-20.2 kb and ∆20.2-22.6 kb cells detected by NEAT1_5′ (green) and NEAT1_3′ or 19k (magenta) FISH probes in the presence of MG132 (5 μM for 6 h). Scale bar, 500 nm.

E Graph showing the proportion of paraspeckles with localization of the NEAT1 3′ ends to the core and shell or the core in ∆16.6-20.2 kb and ∆20.2-22.6 kb cells treated with MG132 (5 μM for 6 h). (∆16.6-20.2 kb: n = 230, ∆20.2-22.6 kb: n = 99)

F Schematics of the NEAT1_2 configuration in WT and deletion mutants.
Paraspeckles are constructed as block copolymer micelles (2021)
Figure 2

Figure 2. Deletion of 5′ terminal domain of NEAT1_2 causes random distribution of the NEAT1_2 5′ ends within the paraspeckle.

A The schematics of WT NEAT1_2 and mutants with deletions in the 5′ terminal regions are shown in Figure 2A. The positions of NEAT1 probes used in SRM (blue) and EM (orange) are shown.

B SRM images of the paraspeckles in ∆0-0.8kb and ∆0-1.9kb (∆5′) cells treated with MG132 (5 μM for 6 h) detected by NEAT1_5′, 1k, and 2k (green) and NEAT1_3′ (magenta) FISH probes. Scale bar, 500 nm.

C Graph showing the proportion of paraspeckles with localization of the NEAT1 5′ ends to the core and shell or the shell in WT, ∆0-0.8 kb, and ∆5′ cells treated with MG132 (5 μM for 6 h). (WT: n = 159, ∆0-0.8 kb: n = 54, ∆5′: n = 48)

D (left) EM observation of the paraspeckles in ∆5′ cells treated with MG132 (5 μM for 17 h) using NEAT1_5′ probes. Scale bar, 100 nm. (middle) Graph showing the proportion of localization of NEAT1_5′ probes (347 gold particles) within the paraspeckles in ∆5′ cells. (right) Graph showing the proportion of the localization of NEAT1_5′ probes in each paraspeckle in ∆5′ cells (n = 20). The box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top).

E Schematics of the NEAT1_2 configuration in the WT and ∆5′ mutant.