EMBO/SourceData huggingface dataset:
Figure 5CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-DIO-Kir2.1-P2A-dTomato or control AAV-Flex-mCherry vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. Weekly body weight gain after viral delivery (A, *p<0.0001, unpaired Student's t test), at 15 weeks after viral delivery.CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-DIO-Kir2.1-P2A-dTomato or control AAV-Flex-mCherry vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. measurements of fat mass (B, **p=0.0006, unpaired Student's t test) and lean mass (C, p=0.3728, unpaired Student's t test) at 15 weeks after viral delivery.(D-F) Measurements of energy expenditure during the 7th week after viral delivery. Comparison of real time O2 consumption traces during the 4-day measurement period (D), of average O2 consumption between the 2 groups (E, *p=0.0007, unpaired Student's t test), and of the difference between day and night periods between the 2 groups (F, p=0.0791, unpaired Student's t test).(G-I) Measurements of feeding during the 7th week after viral delivery. Comparison of real time feeding c traces during the 4-day measurement period (G), of average food intake between the 2 groups (H, **p=0.0019, unpaired Student's t test), and of the difference in food intake between day and night periods between the 2 groups (I, *p=0.0363, unpaired Student's t test). n=6 each. All data presented as mean +/- SEM.
Figure 1B. Size-exclusion chromatography (SEC) experiments of Mdm12, tMmm1, and the Mdm12-tMmm1 complex comparing the molecular size of these proteins in solution. The proteins indicated were injected into a Superdex 200 column (GE Healthcare) with a buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM DTT. The standard molecular masses for the SEC experiments (top) are shown for relative molecular weight comparison (blue dextran, void; ferritin, 440 kDa; aldolase, 158 kDa; cobalbumin, 75 kDa; ovalbumin, 44 kDa; and carbonic anhydrase, 29 kDa).C. Graph indicating the molecular weights of Mdm12, His6-Mdm12, and the Mdm12-tMmm1 complex in solution as measured by analytical ultracentrifugation.
Figure 2A. Ribbon diagram of the yeastMdm12 dimer. The crystal structure of full-length Mdm12 was determined by SAD and refined with native data to 3.1 Å resolution. Lipids bound to Mdm12 are drawn with black stick models.B. Schematic diagram indicating the secondary structure elements and their organization in Mdm12. Three structural elements of Mdm12 are highlighted in different colored boxes.C, D. Surface representations of the Mdm12 dimer are shown in different orientations.
Figure 3A. Ribbon diagram showing the 2-fold dimerization interface of Mdm12. Oxygen and nitrogen atoms are shown in red and blue, respectively. The orange dotted lines indicate intermolecular hydrogen bonds between two protomers of Mdm12. The sequence alignment of yeastMdm12 orthologs is shown to highlight the sequence conservation in the N-terminus β1-strand. Ten orthologs are aligned from residues 1 to 11. Absolute and highly conserved residues are indicated in red and orange, respectively.B. The molecular weight of the Mdm12 (I5P) mutant was measured by size-exclusion chromatography (below) and ultracentrifugation (top) as in Figure 1B and C.C. Ribbon diagram showing the structures of the SMP domain in E-SYT2, CETP, and BPI for the comparison of dimeric interfaces among SMP domains. Note that CETP and BPI are not dimers but monomers containing two tandem SMP domains.
Figure 4A. Simulated annealing omit map (Fo-Fc, contoured at 1.5σ) showing the molecule bound to Mdm12 (left). The final model for the bound PE is shown as in stick representation. The electron density (2Fo-Fc) calculated in the final model is shown with the stick model of PE in the right (3.1 Å resolution, contoured at 0.8σ).B. Surface representation of the Mdm12 dimer. Hydrophobic amino acids lining the Mdm12 channel are indicated by a blue mesh. Lipids built in Mdm12 are in space-filling representation.C. Ribbon diagram showing lipid coordination by Mdm12. Mdm12 residues and lipid fatty acids are colored in green and yellow, respectively.D. Mdm12 binds NBD-PE. Wild-type and monomeric (I5P mutant) Mdm12 were incubated with NBD-PE and separated from free NBD-PE in native PAGE. Coomassie staining (left) and fluorescent (right) detection indicates that Mdm12 directly interacts with NBD-PEin vitro.E. Quantitative data showing binding affinities for NBD-PE by Mdm12. The binding affinities of Mdm12 (monomer/dimer shown in Native PAGE and I5P mutant) for NBD-PE was measured with a NBD-PE concentration-dependent manner. All experiments were carried out three times and the means ± SD are given.F. Mdm12 mutants (L256W, I262W, and L256W/I262W double mutant) were incubated with NBD-PE, and subjected to native PAGE. Because wild-type Mdm12 separates as both monomer and dimer on native PAGE, the purely monomeric form (I5P) of Mdm12 was used as the wild type for clarity. The graph in the right indicates the quantities measured in the experiments. The bar shows the relative amounts of the band ratio (fluorescence/Coomassie). Values represent the means and SD from three independent experiments.
Figure 5A. Structural views suggesting that Mdm12 might have a preference for certain phospholipids. The surface representation of Mdm12 positioned around the binding site for the head group of PE is shown with charge distribution (left) and the sequence conservation (right) in the same orientation. Surface electrostatics and sequence conservation were calculated using an APBS program [31] with the nonlinear Poisson-Boltzmann equation and contoured at ±3kT/e, and ConSurf website (consurf.tau.ac.il) [32] with 34 different yeast orthologs, respectively. The ribbon diagram shown in the middle indicates the overall orientation of Mdm12.B. Lipid displacement experiments to identity the natural ligands of Mdm12in vitro (see material and methods section for details). NBD-PE-preloaded Mdm12-GST was mixed with a series of glycerophospholipids at different concentrations, and displacement of NBD-PE by non-labeled ligand was estimated from the decrease in fluorescence. Means ± SD are shown (n=3 independently performed experiments).
Figure 6A. Overall structure (left) and electron density (right) of Mdm12 in the asymmetric unit. Four molecules (two Mdm12 dimers) are organized with 2-fold rotation symmetry. The 2-fold axes are indicated with a black dotted line.B. Overall structure (left) and electron density (right) of ΔMdm12 in the asymmetric unit. Six ΔMdm12 molecules (three Mdm12 dimers) are arranged with 2-fold rotation symmetry as shown above.C. Ribbon diagram showing a 2-fold interface (tail-to-tail) in two crystal structures (See text for details).D. Size-exclusion chromatography revealing that the N-terminus-truncated version of Mdm12 (residues 11-271) retains the ability to interact with Mmm1. The experiment was performed as in Figure 1B. Eluted fractions indicated by shading were subjected to SDS-PAGE followed by Coomassie Blue staining.
Figure 1(C) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2. C-terminally 6xHA tagged Rx and C-terminally 4xMyc-tagged NRC2EEE were co-expressed with either free GFP or C-terminally GFP-tagged CP. Free mCherry-4xMyc and mCherry-6xHA fusions were used as controls in the treatments without NRC2 and Rx, respectively. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisks indicate bands corresponding to the activated NRC2 complex. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.
Figure 2(B) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2 using different heavy and light sensor-helper combinations. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC2 complex. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.
Figure 3(B) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2 using different "heavy" and "light" helper combinations. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. A non-specific band was observed at ~110 kDa for the anti-FLAG antibody. Red asterisks indicate bands corresponding to the activated "heavy" NRC2 complexes. Blue asterisks indicate bands corresponding to "light" NRC2 complexes. Purple bands indicate intermediate molecular weight complexes combining "heavy" and "light" NRC2. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.
Figure 4(B) BN-PAGE and SDS-PAGE assays with inactive and activated NRC-dependent sensors and NRC2. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC2 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.
Figure 5BN-PAGE and SDS-PAGE assays with inactive and activated NRC-dependent sensors and NRC4AAA. Total protein was extracted with a HEPES-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC4 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.
Figure 6C-terminally GFP-tagged NRC2EEE and C-terminally RFP-tagged Rx were co-expressed either with an EV-4xMyc construct or a CP-4xMyc construct in leaves of nrc2/3/4 N. benthamiana CRISPR mutant lines. (A-B) Single-plane confocal micrographs show the localization of both components of the inactive and active Rx-NRC2 system, together with PM marker RPW8.2-BFP. Scale bars represent 10 µm. (A) NRC2EEE-GFP and Rx-RFP co-localize in the cytoplasm prior to activation. (B) Upon co-expression of CP and activation of the system, NRC2EEE forms puncta associated with the PM while Rx remains in the cytoplasm.(C) Membrane enrichment assays are consistent with microscopy, showing that inactive NRC2EEE-GFP is mostly present in the soluble (cytoplasmic) fraction, whereas activated NRC2EEE-GFP exhibits equal distribution across soluble and membrane fractions. Rx is mostly present in the soluble fraction and exhibits no change upon activation of the system with CP. T = total, S = soluble, M = membrane. ATPase was used as a membrane marker. Rubisco was used as a marker for total and soluble fractions and visualized by Ponceau staining (PS). Red asterisks indicate bands matching the expected MW for each protein. The experiment was repeated two times.
Figure 7(B) BN-PAGE and SDS-PAGE assays with infected and uninfected leaves expressing Rx and NRC2. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisks indicate bands corresponding to the activated NRC2 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.
Figure 8(A-B) Single-plane confocal micrographs show the localization of NRC2 together with Rx or free RFP during PVX infection. C-terminally GFP-tagged NRC2EEE was co-infiltrated with either EV-RFP or Rx-RFP in leaves of nrc2/3/4 N. benthamiana CRISPR mutant lines and infected with PVX by agroinfection. Scale bars represent 10 µm. (A) In the absence of Rx, NRC2EEE-GFP is localized to the cytoplasm during PVX infection. (B) When Rx is present, NRC2EEE forms puncta associated with the PM during PVX infection.
figf5a, GFP-MAP1LC3B foci after RNAi and/or rapamycin treatment in U2OS cells. Scale bar, 20 µm.
Figure 1B. Violin plots of the 3'READS signal on all the TNFα induced genes (n=307) or the set of TNFα induced genes longer than 40 kb (n=111). **** p < 0.0001. Statistical test: Friedman test with Dunn's multiple test correction. Violin plot: minimal to maximal value, dashed horizontal line: median, dotted horizontal lines: interquartile (25% and 75%).C. Western blot of total pol II, Ser2P, Xrn2, CPSF2, CPSF73, and histone H3 as a loading control, on the chromatin fraction. D. Western blot of Xrn2, CPSF2, and Nucleolin as a loading control, on the nucleoplasm fraction (the CPSF73 antibody does not provide reliable results on the nucleoplasm fraction). E. Quantification of the western blots shown in C and D. n=2 biological replicates, mean.F. Ratio of the different CPA and termination factors to total pol II. n=3 biological replicates, mean ± SEM, p-value: * p < 0.05, ** p < 0.01. Statistical test: two-tailed unpaired t test.
Figure 3A. Western blot of total pol II, Ser2P (Abcam or Cell Signaling), Ser5P (Abcam or Cell Signaling), and histone H3 as a loading control, on the chromatin fraction of CDK9as or wild-type HEK293 cells treated with DMSO or 15 µM 1-NA-PP1 for 15 or 30 minutes. B. Quantification of the western blots shown in A. n=2 biological replicates, except for Ser2P / pol II (Abcam) CDK9as NA 30 min, Ser5P / pol II (Abcam) CDK9as NA 15 min and NA 30 min, and Ser5P / pol II (Cell Signaling) CDK9as NA 30 min, where n=3 biological replicates.C. ChIP-qPCR of total pol II and of Ser2P (Abcam or Cell Signaling), and Ser5P (Abcam or Cell Signaling) ratioed to total pol II on KPNB1. n=3 biological replicates, mean ± SEM, p-value: * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical test: two-tailed unpaired t test.
Figure 4B. Western blot of total SF3B1, SF3B1 T142P, and histone H3 as a loading control, on the chromatin fraction of CDK9as cells treated for 30 minutes with DMSO, NA, tautomycetin (TT), calyculin A (CA), NA+TT, or NA+CA. The histone H3 loading control (CA samples) is the same as the loading control shown in Appendix Figure S3B as the same western blot experiment is shown in two different figures.C. Co-immunoprecipitation of CPSF2 in the CDK9as cell treated for 30 minutes with DMSO or NA followed by western blot with total pol II, CPSF2, CPSF3, SF3B1, SF3B3 and β-actin (negative control) antibodies.D. Co-immunoprecipitation of SF3B1 in the CDK9as cell treated for 30 minutes with DMSO, NA, or DRB followed by western blot with total pol II, SF3B1, SF3B2, and GAPDH (negative control) antibodies.
Figure 7B. qRT-PCR of nuclear polyadenylated mRNAs of several TNFα induced or non-induced genes with a 30 minutes DMSO, DRB, CA, or DRB+CA treatment.C. qRT-PCR of cytoplasmic polyadenylated mRNAs of several TNFα induced or non-induced genes with a 30 minutes DMSO, DRB, CA, or DRB+CA treatment.D. ChIP-qPCR of Ser2P, CPSF73, or CPSF2 ratioed to total pol II after 30 minutes treatment with DMSO, DRB, CA, or DRB+CA on KPNB1.E. Co-immunoprecipitation of total pol II from HeLa cells treated for 30 minutes with DMSO, DRB, CA, or DRB+CA followed by western blot with total pol II, Ser2P, SF3B1, CPSF2, Xrn2, and GAPDH antibodies.
Figure 1 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.
Figure 4 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.
Figure 5 A Analytical SEC of UFE HCE/LCE-digests produces two protein peaks, F1 and F2. Reducing SDS-PAGE of SEC purified UFE digests with indicated molecular weight markers. The different SEC elution volumes of F1 and F2 (A) reflect different levels of egg coat filament digestion by HCE/LCE. native-PAGE of SEC purified UFE digests with indicated molecular weight markers. The different SEC elution volumes of F1 and F2 (A) reflect different levels of egg coat filament digestion by HCE/LCE. D, E TOF-MS spectrograms of purified F2 (D) and F1 (E) products. Round-shaped symbols near the peaks indicate the two moieties of the intact ZP module of ZI-3 (green) or the separated ZP-N and ZP-C domains of LCE-cleaved ZI-1,2 (purple).F, G TOF-MS spectrograms of cross-linked products of F2 (F) and F1 (G), with domain symbols indicating the deduced subunit composition of cross-linked products. I Electron micrograph of negatively-stained fish egg coat filaments. On the right are representative 2D classes, indicating a separation of ~65 Å between repeating units within the polymers (white arrows). Scale bars: 100 nm (left panel) and 10 nm (bottom right panel).
Figure 6 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).
Figure 2Aci2 and Pa were both grown in monoculture in the two-resource environment. The same data is shown in both plots. The left plot shows the average of eight technical replicates on a log scale, while the right plot shows each of the replicates on a linear scale. Overlain on the left plot are growth rate fits of gAci2 = 0.88hr-1 and gPa = 0.67hr-1. Data Information: Data in A were collected at 400nm.Aci2 was grown with alanine and glutamate supplied at 25 different ratios from 1:16 to 16:1 with the total supply kept constant at 0.1%w/ Data Information: Data were collected at 600nm.The population size at which Aci2's diauxic shift occurred is linearly correlated to the alanine supply, indicating that Aci2 initially consumes almost entirely alanine. Data Information: Data were collected at 600nm.Instantaneous growth rates were extracted from the Aci2 monoculture data A variation in Aci2's lag time is clearly visible in this representation. Data Information: Data were collected at 600nm.The same monoculture experiments were performed for Pa. The appearance of Pa having a long initial lag is primarily due to the growth that it needs to accomplish before its population size becomes significant on a linear scale as well as a small optical density artifact Data Information: Data were collected at 600nm.Pa's population size at the onset of its diauxic shift correlates to the supply concentration of whichever resource is supplied in a more limiting amount, indicating coutilization. Data Information: Data were collected at 600nm.Instantaneous growth rates extracted from the Pa monoculture data. Data Information: Data were collected at 600nm.Diauxic lag times were fit from the monoculture data and are plotted as circles. Lag times could not be fit for conditions with too little growth on the remaining glutamate. The lag times used in the modeling are shown as lines through the data.
Figure 6Monoculture experiments were performed to determine species' growth rates and lag times for each two-resource environment. For each pair of species, coculture competition experiments were performed in each two-resource environment, and qualitative competitive outcomes were determined There were 44 competitions in which both species could be assigned growth rates and lag times Each point represents one competition. Pie charts summarize the competitive outcomes for cases in which the slow-grower was the fast-switcher (above the Δtlag = 0 line) and in which the slow-grower was not the slow-switcher (on and below Δtlag = 0).
Figure 1Cross-sectional and longitudinal analysis of the association between amyloid-PET (in centiloid), CSF p-tau181, and CSF sTREM2 in early Aβ-accumulators (i.e. Aβ CSF+/PET−; n=70) and late Aβ-accumulators (i.e. Aβ CSF+/PET+; n=201). Cross-sectional linear regressions between A) centiloid and p-tau181 and B) centiloid and sTREM2. Standardized beta-estimates (β), T-values, and p-values were derived from linear regressions controlling for age, sex, education, and clinical status.Cross-sectional and longitudinal analysis of the association between amyloid-PET (in centiloid), CSF p-tau181, and CSF sTREM2 in early Aβ-accumulators (i.e. Aβ CSF+/PET−; n=70) and late Aβ-accumulators (i.e. Aβ CSF+/PET+; n=201). Longitudinal linear regressions between C) centiloid and change in p-tau181 and D) centiloid and change in sTREM2. Standardized beta-estimates (β), T-values, and p-values were derived from linear regressions controlling for age, sex, education, and clinical status.
Figure 2B) T-value projection of the association between sTREM2 on FDG-PET, stratified by group. FDG-PET z-scores were derived by referencing FDG-PET SUVRs to cognitively normal controls (i.e. n=131; Aβ CSF−/PET−).