EMBO/sd-character-level-ner huggingface dataset:
FUNDC1 and ULK1 cooperatively regulate mitophagy A ULK1+/+ or ULK1-/- cells were cultured under hypoxic (1% O2) or normoxic conditions in the absence or presence of 50 nM bafilomycin A1 (BAF1).Figure 4A ULK1+/+ or ULK1−/− cells were cultured under hypoxic (1% O2) or normoxic conditions in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1, and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc, then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays. B ULK1+/+ or ULK1−/− cells were treated with or without FCCP (20 μM) in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1 and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc and then treated with FCCP (20 μM) for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays. C ULK1+/+, ULK1−/− FUNDC1‐KD, or ULK1−/− FUNDC1‐KD cells rescued by introduction of HA‐ULK1 and FUNDC1‐Myc were cultured under hypoxic (1% O2) conditions for 24 h or with FCCP (20 μM) for 24 h. Samples were analyzed by electron microscopy. Scale bar, 2 μm. The yellow asterisks mark mitochondria. The red arrows indicate double‐membraned autophagic structures. The yellow arrow denotes autolysosomes. D-E The mitochondrial coverage and mitochondrial−containing AVs shown in (C) were quantified. Recon, reconstitution. F Provisional working model of the interplay between ULK1 and FUNDC1 in mitophagy regulation.
Inhibition of autophagy blocks endogenous presentation of the NeoR‐derived peptide on MHC class II.Figure 6(A) RCC1.24‐NeoR (endogenous), and RCC1.24 cells to which NeoR protein had been added (exogenous), were treated with different concentrations of 3‐MA and cell surface presentation of the NeoR‐derived peptide on HLA‐DP3 monitored with the T cell clone 20-4/A4‐specific T cell clone 20-4/A4. Inhibition of autophagy by 3‐MA causes an almost complete inhibition of endogenous antigen presentation, while presentation of exogenous NeoR is not affected. (B) EBV‐B1.11 cells transfected with pINCO‐NeoR (endogenous), or incubated with recombinant NeoR protein (exogenous), were treated with the inhibitors of autophagy, wortmannin and 3‐MA, and HLA‐DP3‐restricted presentation of the NeoR‐derived peptide determined. Inhibition of antigen presentation by leupeptin served as control. (C) Presentation of the HLA‐A2‐restricted tyrosinase peptide in EBV‐B1.11 cells transfected with pINCO‐Tyrosinase is not affected by these inhibitors of autophagy. FACS analysis of HLA‐class I and HLA‐DP expression on RCC1.24 (D) and EBV‐B1.11 cells (E) As compared to untreated cells (thick‐lined histogram), treatment of cells with 7.5 mM 3‐MA (shaded histogram) does not cause down‐regulation of HLA‐class I or HLA‐DP. Isotype control is shown as thin‐lined histogram.
Figure 1A) Graphs showing the enzymatic activity of class I (left panel), class IIa (middle panel) and class I/IIb (right panel) HDACs performed in FAPs isolated from WY, WO, YC, YT, OC and OT mice. B) Cartoon illustrating the experimental strategy. Mdx mice at 1.5 months (young) and 12 months (old) of age were treated with TSA or its vehicle of control for 15 days. At the end of the treatment FAPs were isolated by FACS from hindlimb muscles to perform H3K9/14ac and H3K27ac ChIP-seq and RNA-seq. C) Heatmap for H3K9/14ac ChIP-seq signal in the experimental conditions described in B). D) NGS plot showing H3K9/14ac comparative patterns in the experimental conditions described in B). E) Heatmap for H3K27ac ChIP-seq signal in the experimental conditions described in B). F) NGS plot showing H3K27ac comparative patterns in the experimental conditions described in B). G) Upset graph showing the intersection size (in black) between the differentially acetylated loci for H3K9/14ac (in blue) in the experimental conditions described in B). The top 10 intersections are shown. H) Upset graph showing the intersection size (in black) between the differentially acetylated loci for H3K27ac (in blue) in the experimental conditions described in B). The top 10 intersections are shown. I) Heatmap showing 2 clusters of DE genes identified across all the experimental conditions described in B). Gene expression is represented as z-score calculated across the rows. J) Gene Ontology performed on cluster 1 (left panel) and cluster 2 (right panel) genes. K) Heatmap showing the differential expression levels by log2 Fold Change for representative genes of cluster 1 and cluster 2 in the RNA-seq comparisons of YT vs YC, OC vs YC and OT vs OC FAPs.
Figure 3A. Co-immunoprecipitation of RIPK3 with Aβ(1-42) in HT22, BV2 cell lines, and primary astrocytes in response to Aβ(1-42) aggregates. IP, immunoprecipitation; IB, immunoblot. B. A close-up view of the interactions between Nec-1 and surrounding residues in RIPK1. Nec-1 is shown in green. The hydrophobic residues around Nec-1 in RIPK1 kinase domain are shown in sticks. Hydrogen bonds in this and all other figures are represented by yellow dashed lines. Docking score of Nec-1 in RIPK1 was -9.550. C. Surface treated view of 12-mer Aβ fibrils (2LMO, VDW radius = 0.55 Å) having hydrophobic pockets plausible for Nec-1 binding. D. Docked structure of 12-mer Aβ fibrils (2LMO) with 4 molecules of Nec-1. Docking scores for four top-ranked Glide docking poses of Nec-1 in Aβ plaques; -8.338, -4.755, -8.493, and -6.754 in clockwise order starting from the top-left corner. E. Surface plasmon resonance sensorgrams of Nec-1 targeting Aβ(1-42) aggregates (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.
Loss of OMA1 does not restore ocular dysfunction and deteriorates axonal degeneration in the spinal cord of NYKO mice.Figure 8Immunoblot analysis of retinal and spinal cord lysates from 6-7 week old WT, NYKO, NOKO and NYOKO mice (n = 3) using the indicated antibodies. SDHA was used as a loading control. * was not reproducibly detectable in all experiments. B Frequency distribution of mitochondrial lengths in spinal cords of 6-7 week old NYOKO mice (176 mitochondria) compared to WT Kruskal-Wallis test, ns = not significant. Data were analyzed using one-way ANOVA. Representative images of eyes and lenses from 6-7 week old WT and mice lacking OMA1 (NOKO) or both YME1L and OMA1 (NYOKO) in the nervous system. Orange dashed lines mark eye morphology. Scale bars 2.5 mm. D Immunoblot analysis of retina lysates of 6-7 week old WT, NYKO, NOKO and NYOKO mice (n = 3). SDHA was used to control for gel loading. E Transverse semithin sections of spinal cords of 6-7 week old WT, NOKO and NYOKO mice. Sections were stained with toluidine blue. Orange arrows point to MDBs indicating degenerating neurons. Scale bars 25 µm. F MDBs in dorso-lateral tracts of 6-7 week old WT (n = 3), NOKO (n = 3), and NYOKO (n = 3) mice. Data were analyzed using one-way ANOVA. ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM. G Walking beam test of 6-7 week old WT (n = 6), NOKO (n = 5) and NYOKO (n = 7) mice. Data were analyzed using one-way ANOVA. ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.
Figure 2(A) Scheme of actin filaments growing on functionalized glass surfaces containing WAVE1 micropatterns in the presence of profilin-actin in solution. (B) TIRF microscopy of actin monomers (blue, 1uM total, 30% Cy5 labeled) in the presence of equimolar profilin recruited to a WAVE1 pattern (magenta). (C) TIRF microscopy of actin polymerization in- (blue arrows) and outside (red arrows) of WAVE1-micropatterns (magenta) in the presence of 1uM profilin-actin visualized by a filament-binding probe (5nM Alexa488-UTRNN). (D) Kymograph of a growing filament barbed end entering a WAVE1 micropattern. Conditions as in (C) (E) Distributions of barbed end growth velocities for filaments growing outside (green, N=115) or inside (magenta, N=102) of WAVE1-micropatterns compared to a negative control (non-NPF treated PEG surface, black, N=61) (F) Left: Barbed end growth velocities (dots) in 1uM profilin-actin as a function of measured mCherry-WAVE1 surface density. NPF surface density was varied using varying amounts of dark mCherry competitor (see Methods). The red line is a linear fit to the data. Error bars are SD. Right: Surface mCherry-WAVE1 fluorescence and corresponding kymographs of barbed end growth for indicated examples. (G) Surface mCherry-NPF fluorescence and corresponding kymographs of barbed end growth for indicated WAVE1, N-WASP or dark mCherry control constructs in 1uM profilin-actin. (H) Barbed end growth velocities for indicated WAVE1, N-WASP or dark mCherry control constructs in 1uM profilin-actin.
Figure 2(A) Immunocytochemistry analyses show that glutamate stimulation (Glut, 100 µM 5 min + 25 min washout) increased PRR7 in the nucleus. Cycloheximide (100 µM) added 30 min prior to glutamate stimulation did not affect this increase (Cont=100±12.5%, Glut=202.1±15.2%; +cycloheximide Cont=104.9±3.6, Glut=231.4±10.9) (n=~30-50 cells). (B) Decreased synaptic PRR7 immunostaining following glutamate stimulation in DIV 21 hippocampal neurons. Epoxomicin (50 nM) added 20 min prior to stimulation did not affect this decrease (Cont=100±3.6%, Glut=32.3±1.3%; +epoxomicin Cont=99.8±4.6, Glut=42.9±1.1). (n=14-17 dendrites, >1200 puncta for each group). Statistical analyses for (A) and (B) were performed using the Mann Whitney test. (C) Quantification of nuclear PRR7 immunostaining in cultured hippocampal neurons. Treatments (APV [50 µM, APV-(2R)-amino-5-phosphonovaleric acid], CNQX [40 µM, 6-cyano-7 nitroquinoxaline-2,3-dione], TTX [1 µM, tetrodotoxin], NMDA [50 µM, N-Methyl-D-Aspartate]) as indicated. Only APV treatment blocked the glutamate dependent increase in nuclear PRR7 (108.1 ± 19%). Statistical significance was determined using one-way ANOVA in conjugation with Sidak post-hoc test. (D) PRR7-GFP accumulates in the nucleus of hippocampal neurons upon NMDA treatment. Depicted are representative averaged confocal images (30x30 µm2) from optical sections (300 nm step size) of PRR7-GFP expressing neurons (DIV17) before and after bleaching. Prior to stimulation there was substantial PRR7-GFP in the nucleus due to basal neuronal activity (upper panel). This was reduced by incubation overnight with MK801 (10 µM) and TTX (1 µM) (lower panel). NMDA (50 µM) stimulation, but not MK-801, led to recovery of nuclear PRR7-GFP fluorescence following bleaching of nuclear ROIs at t0`(blue arrow in E).(E) Quantification of changes in nuclear GFP fluorescent intensities were normalized to time point t0` and corrected for bleaching. No significant differences in PRR7-GFPFRAP were found in control (MK801) conditions. GFP intensities are displayed via look-up-table (LUT). Data are represented as mean±SEM; n=9 cells (NMDA) and n=5 cells (MK801). Analysis was done using one-way ANOVA using Bonferroni post-hoc correction. (F) PRR7-mEos translocates from distal dendrites to the nucleus upon NMDA treatment. (Left panels) Confocal maximal intensity projection images of hippocampal neurons expressing PRR7-mEos before and after photoconversion of distal dendrites (ROIs) through the image z-stack from from green (488nm) to red (568nm) using a UV laser (405 nm). (Right panels) Averaged intensity projection images from nuclear planes showing accumulation of photoconverted PRR7-mEos (568nm) in the nucleus following NMDA stimulation (50 µM- 30min).(G) Paired quantification of changes in background corrected fluorescent intensities of nuclear PRR7-mEos at 0 min and 30 min following NMDA stimulation or MK801 treatment. (Average increase; NMDA=617.8±47.7%, n=11 cells, p<0.0005; MK801=206.1±13.6%, n=5 cells, n.s.). Statistical significance was determined by two-way ANOVA using Sidak's correction for multiple comparisons.
Arginine-rich DPR causes functional impairment of EXOSC10/the RNA exosome complex A.Figure 8A. Schematic diagrams of codon optimized (lacking extensive G4C2 repeat) plasmids expressing N-terminally EGFP fused 177 repeats of poly-GR or 166 repeats of poly-PR under the control of CMV promoter. B, C. Increased expression of 3'extended snoRA48 precursor upon EXOSC10 knockdown (B) or GFP-poly-GR177/ GFP-poly-PR166 expression (C) on RT-qPCR. D, E. Increased expression of 3'extended snoRA68 precursor upon EXOSC10 knockdown (D) or GFP-poly-GR177 /GFP-poly-PR166 expressions (E) on RT-qPCR. F. RT-qPCR analysis showed increased cellular G4C2 repeat RNA expression from co-transfected EF1 (G4C2)80 repeat construct in the presence of GFP, GFP-poly-GR177 or GFP-poly-PR166.
Schematic model for Spns1 function under the control of the network module of autophagy-senescence signaling cascades differentially regulated through Beclin 1 and p53.Figure 6(A) Beclin 1 is essential for the early stage of autophagy and its depletion suppresses the Spns1 defect by blocking the 'autophagic process' and its progression. BafA can decelerate 'lysosomal biogenesis', which subsequently presumably prevents autophagosome-lysosome fusion, through the inhibition of the v-ATPase, and contributes to amelioration of the Spns1 defect at least temporarily. Basal p53 activity may suppress the intersection between the 'autophagic progress' and 'lysosomal biogenesis' where the Beclin 1 depletion was not sufficient, but the v-ATPase inhibition was still effective enough, to compete with the p53 loss to suppress the Spns1 deficiency. By switching the basal p53 state to the activated version with UV irradiation, p53 can promote autophagy. Spns1 might be a gatekeeper of autolysosomal maturation followed by lysosomal biogenesis. It remains unknown how p53 can mechanistically be linked to the lysosomal 'efflux' function of Spns1 as well as the lysosomal 'influx' function of v-ATPase, and further investigations will be required to explore this connection. (B) Roles of Spns1, p53 and Beclin 1 in senescence equilibrium. Loss of Spns1 leads to an imbalance in homeostasis and increased senescence. This effect can be ameliorated by concurrent knockdown of Beclin 1. p53 has a comparatively less dramatic impact on Spns1-loss-induced embryonic senescence. When in the "basal" state, p53 helps retain equilibrium. When p53 is "activated" by UV irradiation, a modest increase in senescence is observed. The higher level of senescence is seen during loss of Spns1 in the absence of basal p53 or in the presence of activated p53. During loss/knockdown of all three genes (spns1, p53 and beclin 1), a state of moderate senescence is observed. An increase in senescence is accompanied by a p53-dependent decrease in cellular proliferation.
GA aggregates significantly impair synaptic unloading Cortical neurons were loaded with FM4-64 dye 48 hours after transfection.Figure 4Cortical neurons were loaded with FM4-64 dye 48 hours after transfection. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. A) Representative dye loading showing defined puncta regions along neurites 40x magnification, scale bar indicates 10μm. B) Representative intensities of FM4-64 puncta regions pre- and post- stimulation demonstrate loss of synaptic puncta fluorescence intensity following stimulation in GFP but not GA50 containing neurons. These images visually reflect the process performed in an automated manner through our imaging software to determine puncta fluorescence intensity. An unbiased constant threshold is placed on each image per experiment, then fluorescence intensity is measured over time. The threshold applied isolates puncta from background, giving distinct regions. 8-bit images were thresholded and pseudo-color (red) was applied to enlarged puncta regions of interest, scale bar indicates 10μm. C) Graphical representation is the change in fluorescence (ΔF) normalized to basal fluorescence (F), ΔF/F. The final 10 seconds of baseline recording and 10 seconds of the depolarization phase are shown, arrow indicates start of depolarization. Non-linear regression was performed in GraphPad Prism7 using the plateau followed by one phase decay model. The decay constant (k) for GFP was 0.4971 s-1, while for GA50 it was 219.5 s-1. Similarly the tau (τ) values were 2.012 seconds for GFP and 0.00455 seconds for GA50, respectively. Data presented as mean ± SEM for each imaged timepoint. Statistical significance was determined using the Sidak-Bonferroni method, ****p < 0.0001, n=20 puncta regions from 4 biological replicates.
Figure 5(A) Cells were immunolabeled for Mic60 and DNA and visualized by confocal fluorescence microscopy before and after induction of Mic60 expression for 48 h. (B) Protein levels of MICOS proteins after Mic60 induction. Cell lysates were analyzed by western blotting at the indicated time points. (C) Recovery of mitochondrial networks upon Mic60 re-expression. Cells were induced with doxycycline hyclate for 72 h, immunolabeled for TOM20 and visualized by confocal microscopy. (D-G) TEM of Mic60-TO cells before and after induction. (D) TEM recordings of Mic60-TO cells at the indicated time points. (E) Quantification of the cristae morphology. (F) CJ frequency. The number of CJs on TEM recordings was normalized to the length of the OM. At least 90 mitochondrial sections were analyzed for each sample. (G) Quantification of the cristae morphology of Mic60-TO cells. Only mitochondria exhibiting at least one CJ were analyzed. Data information: n: Number of mitochondrial sections. Scale bars: 20 µm (A), 10 µm (C), 500 nm (D).
Figure 6Growth phenotype observed on CSY medium supplemented with different carbohydrates (1% final concentration) compared to iJL208 predictions. Growth observations and predictions were normalized by growth on sucrose. Discrepancies between experimental data and predictions are highlighted (red rectangle). The metabolic network reconstruction allowed the identification of potential candidates carrying promiscuous reactions (pink) responsible for maltose and mannose catabolism. 3D structures of candidates (Mfl499, Mfl120, and Mfl254; red) are superimposed to available structures in the Protein Data Bank (light blue) for which the suspected enzymatic activity is annotated. FATCAT p-values are shown. The 173 genes where the transcriptomic and proteomic expression data (Exp) are consistent (see Appendix Figure S8) are compared with flux states predictions generated by parsimonious flux balance analysis (Flux). True Positives (TP) and True Negatives (TN) correspond to genes where both predictions and observations are consistent, while False Positives (FP) and False Negatives (TN) where they are inconsistent. Genes considered expressed or with an active flux are represented in dark blue, and silent in light blue. Revised gene essentiality data (see Figure EV3) compared to essentiality predictions from iJL208. Essential and non-essential protein-coding genes are represented in green and red, respectively. TP, TN, FP, and TN are as in c. The pentose phosphate pathway (PPP) of M. florum along with ribose import. The transaldolase (TALA) reaction (pink) was missing in the network. Structural similarity between two 2-deoxy-ribose-phosphate aldolases (Mfl121, red in left box; Mfl639, red in right box) to a known transaldolase from the PDB (light blue) served as basis for the addition of this gene-associated reaction in the model. The observed essentiality (Ess.) and expression (Exp.) of genes (see panels c and d) associated with each reaction of the PPP are indicated by green (essential or expressed) or red (non-essential or non-expressed) colored squares.
Figure 2A. Experimental design to measure the selection coefficient (S) upon growing URA3agg in competition with URA3sol. B. The selection coefficient, S, is proportional to the difference in growth rates between URA3agg and URA3sol ω is the growth rate, which corresponds to the inverse of doubling time, τ. The S values measured at standard conditions (30°C) The S values measured at standard conditions (30°C) indicate that, depending on the media composition (+Ura, -Ura, +5FOA), the formation of protein deposits can be neutral, deleterious or even advantageous for the cell as the function becomes non-essential, essential or toxic, respectively. S values measured in different environments. In all cases the standard error is below 5%. Color scale: purple in absence of uracil (-Ura), white in the presence of uracil (+Ura) and orange in the presence of 5FOA (+5FOA). At 37°C, the heat stress together with the inability to fold Ura3p have such a strong fitness effect that the strains barely grew in absence of uracil, impeding the measurement of S. A bar of purple-white diagonal lines indicates a presumed value of S at 37°C without uracil.
Figure 6A Whole mount immunofluorescence at 12w of the organ of Corti of a Syne4-/- mouse injected with AAV.Syne4 mouse injected with AAV.Syne4. B Whole mount immunofluorescence at 12w of the 12 kHz region of WT, Syne4-/- mouse, and Syne4-/- mouse injected with AAV.Syne4. C IHC and OHC counts, n=3 for WT, n=4 for Mut, n=6 for Mut + AAV.Syne4. D Correlation between HC count and ABR threshold at 12kHz. E 3D surface projection of an OHC at 12w from the 12kHz region of WT and Syne4-/- mice injected with AAV.Syne4. Open arrows denote basal end, white arrows denote cuticular plate. F Image analysis of nucleus position of OHC. G Quantification of nuclear distance from the cuticular plate. A total of 30 OHC from 3 WT mice and 38 OHC from 4 Syne4-/- mice injected with AAV.Syne4 were measured.
Figure 1A A schematic representation of dendrite pruning in ddaC neurons. B-H Live confocal images of ddaC neurons expressing mCD8-GFP by ppk-Gal4 at WP stage or 16 h APF. Neurons expressing efa6 RNAi #1-#3 (C-E), efa612 mutant MARCM clones (F), and efa612/GX6w- transheterozygous mutant neurons (G) showed consistent dendrite pruning defects at 16 h APF, as compared to the wild-type neurons (B). The dendrite pruning defects were fully rescued when overexpressing full-length Efa6 in the efa612 mutant clones (H). Red arrowheads point to the ddaC somas. I-J Quantification of dendrite severing defects and unpruned dendrite lengths at 16 h APF.
Figure 4(A) PARP2 and DDL association in Arabidopsis protoplasts. PARP2-HA and DDL-FLAG were transiently expressed in Col-0protoplasts, immunoprecipitated with α-FLAG antibody (IP: -FLAG), and immunoblotted with α-HA (IB: α-HA) or α-FLAG antibody (IB: -FLAG) (top two panels). The protein inputs are shown with immunoblotting before immunoprecipitation (bottom two panels). Protoplasts were treated without or with 100 nM flg22 for 30 min. (B) PARP2 and DDL association in N. benthamiana. PARP2-FLAG and DDL-HA were transiently co-expressed in leaves of N. benthamiana. The samples were collected at 2 dpi for Co-IP assay with 100nM flg22 treatment for 1 hr. (C) PARP2 and DDL association with BiFC assay. Different combinations of constructs were transiently expressed in Arabidopsis Col-0 protoplasts and the fluorescence signals were observed with a confocal microscope. An NLS-RFP construct was co-transfected to show the nuclear signals. Bar=10 µm. (D) PARP2 and DDL interaction in vitro. GST-PARP2 protein immobilized on glutathione beads was used to pull-downHIS-SUMO-DDL-HA proteins (left panels). A PARylation reaction was performed prior to the pull-down assay (the third lane of left panels). The PARylated PARP2 and DDL were detected with α-PAR antibody (right panels). (E) In vitro PARylation of DDL by PARP2. Immunoprecipitated DDL-FLAG proteins from Arabidopsis protoplasts were incubated with GST-PARP2 in a PARylation reaction containing Biotin-NAD+. The reactions in lane 4 and 5 contained 1 mM 3-AB or GST-PARG1 respectively. PARylated proteins were detected by streptavidin-HRP antibody (Strep), which recognizes Biotin-labelled PAR.