EMBO/SourceData huggingface dataset:
Figure 3A-C In vitro ubiquitylation assays were performed as described in Figure 2, but with the addition of immunoprecipitatedWNK1 in (A). In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation.A-C In vitro ubiquitylation assays were performed as described in Figure 2, but with the addition of immunoprecipitated over-expressed FLAG-WNK4 in (B, C) (see Materials and Methods). The WNK kinases are modified by CUL3WT-KLHL3, with the higher molecular weight smear observed in anti-WNK1 and anti-FLAG panels representative of multiple ubiquitin molecules being covalently attached to the WNK protein. CUL3Δ403-459 is unable to modify WNKs. Samples from the same assay reactions were divided to allow immunodetection of the different protein components modified within the same assay reaction. (C) CUL3WT, CUL3Δ403-459 and an equimolar solution CUL3WT:CUL3Δ403-459 (1:1 Mix) were incubated with KLHL3 and immunoprecipitatedFLAG-WNK4 in ubiquitylation reactions to determine the influence of CUL3Δ403-459 on the ubiquitylation activity of CUL3WT. Notably, the presence of CUL3Δ403-459 does not inhibit WNK ubiquitylation by CUL3WT.In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation.
Figure 4Western blot of whole kidney lysates from mice culled after a minimum 4-h fast. Following exsanguination after surgery, mouse tissues were rapidly harvested and stored, samples were then homogenised, clarified and quantified prior to SDS-PAGE. Immunodetection with the antibodies shown highlights elevated signalling through the WNK kinase pathway in CUL3WT/Δ403-459 versus CUL3WT mice. The lowest panel is an anti-CUL3 immunoprecipitation of the kidney lysate samples, whereby the CUL3 antibody was cross-linked to Protein G sepharose and used to affinity purify CUL3WT and CUL3Δ403-459 from kidney lysates. The samples were incubated together overnight to allow deneddylation of CUL3 proteins to occur and maximise CUL3 binding to the anti-CUL3 resin. Samples were thoroughly washed and eluted from the resin prior to SDS-PAGE and immunodetection for CUL3. The IP highlights that CUL3Δ403-459 is indeed present within the kidney lysate.Fresh frozen kidney tissues from transplant patient and donor cadavers were homogenised, clarified and quantified prior to SDS-PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human kidneys.Plasma aldosterone after a minimum 4-h fast was calculated by HTRF (homogeneous time-resolved fluorescence) aldosterone assay. The average aldosterone level per mouse was calculated from duplicate samples run in parallel on the assay. Blood was rapidly harvested in heparin-coated plasma extraction tubes following exsanguination after surgery, and samples were snap-frozen for storage. A 58% increase in aldosterone was detected in CUL3WT/Δ403-459 versus CUL3WT mice (*P = 0.0245). Two-tailed unpaired Student's t-test; data are mean ± SEM.Arterial blood biochemistries after a minimum 4-h fast. Under anaesthesia, the right carotid artery was cannulated to minimise atmospheric exposure of samples collected for iSTATbloodgas and electrolyte measurements. CUL3WT/Δ403-459mice present with abnormal electrolyte homoeostasis compared to CUL3WTmice, exhibiting hyperkalaemia (***P = 0.0004) and hyperchloraemia (***P = 9.5 × 10−5) with a compensated metabolic acidosis (P = 0.7766), marked by a decrease in bicarbonate () (***P = 3.4 × 10−5), base excess (BE) (***P = 9.1 × 10−5) and partial pressure of carbon dioxide (pCO2) (***P = 0.0038). Two-tailed unpaired Student's t-test; data are mean ± SEM.
Figure 5Representative pseudocoloured maximum-intensity z projections of immunofluorescence stained kidney sections showing the distribution of the WNK/SPAK pathway components between CUL3WT/Δ403-459 and CUL3WTmice at a minimum 4-h fasting baseline (n = 4 per genotype). CUL3 and KLHL3 are comparable between genotypes, with significantly higher levels of CUL3 in the proximal convoluted tubule (PCT) compared to the weak staining in distal convoluted tubule (DCT) and thick ascending limb of the loop of Henle (TAL), while KLHL3 shows higher expression in the DCT/TAL cytosol with staining of the PCT confined to the apical membrane. Total (t) and phospho (p) NCC Thr44 and SPAK Thr243 show increased apical membrane expression in the parvalbumin (PVALB)-positive early and late (PVALB-negative) DCT of CUL3WT/Δ403-459mice. Unexpectedly, the increased levels of WNK4 and SPAK resulted in the formation of discrete puncta specifically in the DCT of CUL3WT/Δ403-459mice. It is possible that the autophagy-lysosomal system may attempt to compensate and degrade these excess proteins although the WNK4 puncta do not colocalise with the lysosomal marker LAMP1. Scale bar, 20 μm.Representative immunohistochemistry staining of KLHL3 and CUL3 in humankidney sections (n = 6). KLHL3 shows preferential DCT/TAL cytosolic staining similar to the mouse despite no evidence of PCT apical staining as seen in (A). CUL3 exhibits preferential basolateral cytosolic staining of the PCT with similarly low levels of diffuse staining in the DCT/TAL to that of the mouse. Scale bar, 100 μm.
Figure 6Continuous blood pressure measurements were taken in mice under general anaesthesia with thermostatically controlled body temperature, right carotid artery pressure transducer catheterisation and ECG Lead II heart rate monitoring. CUL3WT/Δ403-459 mice have elevated systolic (SBP) (***P = 1 × 10−12) and diastolic (DBP) blood pressure (***P = 8.5 × 10−8), although they also present with 7% lower heart rates (*P = 0.0485) when compared to CUL3WT mice. Two-tailed unpaired Student's t-test; data are mean ± SEM.Pulse waveform analysis of blood pressure traces obtained in (A) reveals that CUL3WT/Δ403-459 mice have an increased pulse pressure (PP) [SBP - DBP] (***P = 2.1 × 10−10), augmentation pressure (AP) [SBP - anacrotic notch (AN) pressure] (***P = 3.5 × 10−6), dicrotic notch (DN) pressure (***P = 4.7 × 10−13) and mean arterial pressure (MAP) [1/3 × SBP + 2/3 × DBP] (***P = 1.3 × 10−9). This hypertensive phenotype is in part due to changes in vascular contractility in CUL3WT/Δ403-459 mice as evidenced by their higher augmentation index (AIx) [AP/PP] (**P = 0.0096), a marker of arterial stiffness, and is further supported by an increase in their diastolic pressure decay time constant (τbourgeois) [1/slope of diastolic pressure decay; measured 30 ms after DN and 20 ms before end DBP] (**P = 0.0083), a surrogate marker of increased vascular resistance. Two-tailed unpaired Student's t-test; data are mean ± SEMIn vivo dose-responses to phenylephrine and angiotensin II. After baseline measurements were obtained from mice in (A), the right external jugular vein was cannulated for administration of bolus doses of vasopressors in increasing half-log steps. Dose-response curves were generated and data analysed using a logistical function to determine the estimated dose producing half-maximal response (ED50) and maximum response (Emax). The fitted Emax for phenylephrine was increased in CUL3WT/Δ403-459 versus CUL3WT (183.9 ± 2.5 versus 164.9 ± 1.4 mmHg) (***P = 1 × 10−6) indicating an increased vasoconstrictor response to adrenergic stimulation elevating systolic blood pressure substantially above the CUL3WT maximum. Similarly, the fitted Emax for angiotensin II stimulation was higher in CUL3WT/Δ403-459 versus CUL3WT (155.5 ± 1.8 versus 138.3 ± 2.2 mmHg) (***P = 1.7 × 10−6). However, there was no change between CUL3WT/Δ403-459 versus CUL3WT sensitivity (as measured by ED50) to phenylephrine (36.6 ± 5.1 versus 31.4 ± 2.8 μg/kg bw) (P = 0.3778) or angiotensin II (0.77 ± 0.16 versus 0.93 ± 0.13 μg/kg bw) (P = 0.4401). Two-tailed unpaired Student's t-test; data are mean ± SEM.
Figure 7Western blot of tunica media-intimathoracic aorta lysates from mice culled after a minimum 4-h fast. Following exsanguination after surgery, mouse tissues were rapidly harvested and the tunica adventitia removed before storage, and later, the samples were homogenised, clarified and quantified prior to SDS-PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3. Similar to the kidney, the aorta of CUL3WT/Δ403-459 showed slightly lower levels of CUL3 compared to CUL3WT without any change in KLHL3 levels.Fresh frozen thoracic aorta tissues from donor cadavers were homogenised, clarified and quantified prior to SDS-PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human aorta.Representative maximum-intensity z projections of immunofluorescence stained thoracic aorta sections showing the distribution of CUL3 and KLHL3 between CUL3WT/Δ403-459 and CUL3WT mice at a minimum 4-h fasting baseline (n = 4 per genotype). CUL3 and KLHL3 localisation is comparable between genotypes. The highest levels were detected in the vascular smooth muscle cells and endothelium, with a minimal expression in the perivascular adipose tissue of the adventitia. Scale bar, 50 μm.Representative immunohistochemistry staining of KLHL3 and CUL3 in humanthoracic aorta sections (n = 6). Similar to the mouse staining seen in (C), vascular smooth muscle cells in the tunica media of the aortic wall are positive for KLHL3 and CUL3. Scale bar, 50 μm.Morphometric analysis of thoracic aortae reveals vascular remodelling in CUL3WT/Δ403-459 mice. There is an increase of ˜21% in the vessel wall intima-media thickness (demarcated by arrows) of CUL3WT/Δ403-459 compared to CUL3WT mice (***P = 0.0003). However, there is no change in the number of elastin laminae (P = 0.1458) and therefore no increase in the number of medial muscle layers between genotypes. Two-tailed unpaired Student's t-test; data are mean ± SEM.
Figure 1A. Retinal whole mount immunostaining for Isolectin-B4 (red) and Desmin (green) in WT (top panel) and Glut1iECKO (bottom panel) mice showing pericyte coverage in the vasculature. Left and right panels show representative images with 20x or 63x magnification, respectively. B. Quantification of the pericytes coverage in the retinal vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=9 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)C. Immunostaining for CD31 (green), CD13 (white; pericyte marker) and αSMA (cyan) using paraffin section from WT (top panel) and Glut1iECKO (bottom panel) mice.D. Quantification of the pericytes coverage using CD13 immunostaining in the brain vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=7 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) E. Quantification of the smooth muscle cell coverage using αSMA immunostaining in the brain vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=13 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)J. Representative confocal images of fibrin (green), CD31 (red) and DAPI (blue) immunostaining in the brain vasculature of WT (top panel) and Glut1iECKO (bottom panel) mice K. Quantification of the number of cadaverine 555 positive brain parenchyma cells in WT (blue) and Glut1iECKO (orange) mice. (n=13 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) L. Quantification of extravascular fibrin deposits in WT (blue) and Glut1iECKO (orange) mice. (n=9 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) M. Quantification of lactate in the cerebrospinal fluid of WT (blue) and Glut1iECKO (orange) mice. (n=3 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)
Figure 2A. Quantification of the mTFP/Venus fluorescence ratio of laconic (lactate sensor) showing concentration-dependent lactate uptake in HBVPs. (n=8 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM)) B. Representative images showing mTFP (blue) and Venus (yellow) fluorescence in control and lactate (20mM) treated HBVPs.F. Representative images (high magnification; 60x) showing mTFP (cyan), Venus (yellow) and VECAD (purple) fluorescence from BMECs and laconic-infected HBVPs on a 2 well chamber. The upper panel shows HBVPs with endothelial contact and the lower panel shows HBVPs without endothelial contact. G. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs with endothelial contact (EC-PC contact) or HBVPs alone (PC only). (n=16 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)H. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or GLUT1 (lower panel) siRNA treated BMECs. VECAD immunostaining (purple) was used for endothelial staining. I. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or GLUT1 (siGLUT1) siRNA-treated BMECs. (n=12 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)
Figure 6E. Western blot analysis for siRNA efficiency of MCT1.G. Western blot analysis for siRNA efficiency of MCT12.I. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or MCT1 (lower panel) siRNA-treated BMECs. VECAD immunostaining was used for endothelial staining. J. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or MCT1 (siMCT1) siRNA-treated BMECs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)K. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or MCT5 (lower panel) siRNA-treated BMECs. VECAD immunostaining was used for endothelial staining. L. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or MCT5 (siMCT5) siRNA-treated BMECs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)
Figure 7D. Western blot analysis showing expression level of MCT12 and β-actin in HBVPs and BMECs. E. Western blot analysis for siRNA efficiency of MCT12.G. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in control (upper panel) or MCT12 (lower panel) siRNA treated HBVPs in a 2 well chamber assay system with BMECs. VECAD (purple) immunostaining was used for endothelial staining. H. Quantification of the mTFP/Venus fluorescence ratio of Laconic in control (blue) or MCT12 (orange) siRNA treated HBVPs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.
Figure 8A. Permeability assay using vibratome section showing the cadaverine 555 dye (red), CD31 (green) and CD13 (purple) in the brain vasculature of Glut1iECKO mice with (bottom) or without (top) lactate administration. Note the extravascular accumulation of cadaverine dye in control Glut1iECKO mice, but not in lactate supplemented Glut1iECKO mice.B. Quantification of the CD13-positive pericytes coverage in the brain vasculature of Glut1iECKO mice with (orange) or without (blue) lactate administration. (n=16 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.) C. Quantification of the number of cadaverine 555 positive brain parenchyma cells in the brain vasculature of Glut1iECKO mice with (orange) or without (blue) lactate administration. (n=24 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.) D. Quantification of CSF lactate level in WT and Glut1iECKO mice with/without lactate supplementation. (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.)
Figure 1(a-b) eIF4A knockdown in either S2 (a) or Kc167 (b) cells blunts the inactivation of TORC1 upon amino acid removal. Cells were treated with dsRNA targeting GFP as a negative control, or three independent, non-overlapping dsRNAs targeting eIF4A for 5 days and then incubated with complete medium or medium lacking only amino acids for 30 minutes. Representative of 3 biological replicates.(d) Impaired inactivation of TORC1 in response to eIF4A knockdown is most apparent upon partial depletion of amino acids, caused by removal of amino acid subsets. After 5 days of knockdown, S2 cells were treated for 30 min with either complete Schneider's medium (+aa), Schneider's medium lacking all amino acids (-aa) or various subsets of amino acids, as indicated (where EAA "essential amino acids" = H,I,L,K,M,T,W,V). Error bars: std dev. n=3 biological replicates.(e) Time course of amino-acid removal reveals that eIF4A knockdown Kc167 cells maintain elevated S6K phosphorylation up to the maximum possible timepoint of 60 minutes when the cells start dying (see drop in S6K and tubulin levels). Representative of two biological replicates.(f) eIF4A mutant larvae have impaired TORC1 inactivation upon shifting to food lacking amino acids. Control (w1118) or eIF4A1006/1013 1st instar larvae were transferred from standard food to plates containing either standard fly food or PBS/1% agarose+2%sucrose for 1h prior to lysis and immunoblot analysis. Two biological replicates are shown. Representative of 3 biological replicates.
Figure 2(a-a') Although knockdown of eIF4A or eIF3-S2 equally blunt translation of EGFP from an inducible plasmid (a), only knockdown of eIF4A but not eIF3-S2 impairs TORC1 inactivation upon amino acid removal (a'). (a) S2 cells, treated with indicated dsRNAs for 5 days, on day 3 transfected with an inducible EGFP plasmid (pMT-EGFP) and on day 4 induced for 18h. Non-targeting LacZ dsRNA used as a negative control. (a') S2 cells treated with indicated dsRNAs for 5 days, then incubated with medium lacking amino acids for indicated time prior to lysis. Representative of two biological replicates.(b) Knockdown of eIF4A, but not other translation initiation factors, blunts TORC1 inactivation upon amino acid withdrawal. Kc167 cells treated with indicated dsRNAs for 4 days and then incubated with complete Schneider's medium or Schneider's medium lacking the indicated amino acids for 30 minutes prior to lysis.(c) Inhibition of translation with cycloheximide does not block inactivation of TORC1 upon amino acid withdrawal in Kc167 cells. Kc167 cells treated with either eIF4A dsRNA for 4 days, or cycloheximide (50 µg/mL, "CHX") for 5 min prior to, as well as during removal of amino acids (-LIVASTQP). CHX treated cells still inactivate TORC1 (compare lanes 8 to 7) whereas eIF4A knockdown cells do not (lanes 14 vs 13). Quantifications of two biological replicates are shown. CHX and eIF4A samples were normalized to their respective control conditions.(d) Knockdown of eIF4A in Kc167 cells does not prevent a drop in intracellular amino acids levels when amino acids are removed from the medium for 30 min. Quantification of total intracellular amino acids, analyzed in a blinded fashion. Levels of individual amino acids shown in Figure S2D. For CHX samples, cycloheximide (50µg/mL) was added 5 minutes prior to, and during treatment with medium containing or lacking amino acids. Statistical significance tested by ANOVA2 using Scheffé's multiple comparisons method (p<0.05). Error bars: std. dev. n=5.
Figure 3(a) Knockdown of eIF4F components (eIF-4E and eIF-4G) partially phenocopy the eIF4A knockdown, leading to elevated TORC1 activity upon amino acid removal (30 min). Knockdown of all other tested translation initiation factors do not cause elevated TORC1 upon amino acid removal (main Figures 1c, 2a' and 2b). Error bars: std. dev. n=3 biological replicates.(b) Quantification of de novo protein synthesis rates by OPP incorporation reveals that eIF4E and eIF4G knockdowns deplete eIF4F function less efficiently than the eIF4A knockdown, explaining why the effects of eIF4E and eIF4G knockdowns on TORC1 activity (panel a) are a bit milder than the eIF4A knockdown. Kc167 cells treated with dsRNA for 4 days then incubated with 20μM Click-it OPP reagent 30min before fixation and fluorescent labeling. Quantification of OPP fluorescence per cell (nuclear count) for two independent experiments is displayed (3 independent images per condition), normalized to the no dsRNA condition. Scale bars: 25µm.
Figure 4(a) Binding of eIF4G to the Rag GTPases detected by co-immunoprecipitation from Kc167 cells in absence or presence of the chemical cross-linker DSP. Representative of >3 biological replicates.(b) Binding between eIF4G and the Rag GTPase complex is mainly mediated by RagC, detected by expressing and immunoprecipitating either RagA or RagC alone. Representative of two biological replicates.(c) Interaction between eIF4A and Raptor detected via co-immunoprecipitation of epitope-tagged proteins. Proteins were cross-linked with DSP prior to lysis and immunoprecipitation. Representative of 3 biological replicates.(d-d'') eIF4A and Raptor form a complex, detected by proximity ligation assay (PLA). Specificity of the PLA signal was controlled by knocking down either eIF4A or Raptor in Kc167. (d) Representative images. Cells outlined in white. (d') Quantification of number of PLA spots per cell. (n>200. ***p<0.0001, ANOVA1, Dunnett's multiple comparisons test, was performed on log-transformed data, box = +/- 1 quartile, whiskers = max/min). (d'') Immunoblotting to detect efficiency of eIF4A and Raptor knockdowns.(e-f) The eIF4A-Raptor interaction does not take place on lysosomes (e) or the endoplasmic reticulum (ER) (f). Kc167 cells were either incubated with 100 µg/mL Dextran for 1 hour, washed and incubated for 14 hours in normal growth medium to label late endosomes and lysosomes (e), or transfected to express an ER-resident GFP (f). Amino acid removal was for 45 min. Pearson's correlation coefficients for eIF4A-Raptor PLA and Dextran in panel (e) are 0.09 and 0.08 for +aa and -LIVASTQP respectively (calculated for all cells in the acquired fields, which were chosen randomly, n=40). Scale bars: 5µm.