EMBO/SourceData huggingface dataset:
Figure 2S 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 3B. Plots showing the distribution and relationship between the fractions of deposited or free Ura3p, its magnitude of effect on fitness (α/β/γ) and the selection coefficient (S) in different environments.C. Average of the selection coefficient (left) and average of the cytosolic fluorescence (equivalent to the fractional abundance of the free protein in URA3agg, right).
Figure 4(B) Drug response curves upon treatment of organoid models with nutlin‑3a, 5‑FU, oxaliplatin, ibrutinib, panobinostat and entinostat (biological replicates n=3, data are shown as mean ±SD).
Figure 5Drug response curves upon treatment with inhibitors for (B) EGFR (afatinib, gefitinib), (biological replicates n=3, data are shown as mean ±SD).Drug response curves upon treatment with inhibitors for (C) B‑RAF (LY3009120, TAK‑632, vemurafenib, dabrafenib), (biological replicates n=3, data are shown as mean ±SD).Drug response curves upon treatment with inhibitors for (D) MEK1/2 (selumetinib, binimetinib) (biological replicates n=3, data are shown as mean ±SD).
Figure 6(A) Brigthfield images of human patient derived organoids (PDOs) from gastric cancer with and without RTK/MAPK alterations (scale bar 100µm, zoom in 40µm).(B) Drug response curves of PDOs upon treatment with the MEK1/2 inhibitor trametinib (biological replicates n=3, data are presented as mean, SD values are shown in Table EV2). (C) Drug response curves upon HDAC inhibition with panobinostat (PAN) and entinostat (ENT) (biological replicates n=3, data are presented as mean, SD values are shown in Table EV2). (D) Comparison of the relative area under the curve (AUCrel) upon trametinib treatment in RTK/MAPK altered (mean AUCrel: 0.5308; biological replicate n=7) vs. non‑altered (mean AUCrel: 0.7101; biological replicates n=6) human PDOs (two‑tailed Student's t‑Test; *p= 0.0356).
Figure 1A Fluorescence microscopy showing UBQLN2 (Alexa Fluor 647) in solutions contained 50 µM FL UBQLN2 with increasing amounts of K48- and K63-linked chains of different lengths in 20 mM NaPhosphate and 200 mM NaCl (pH 6.8) at 30 ºC. [Ub]:[UBQLN2] reflects the ratio between Ub monomers and UBQLN2 molecules. The same image is used for the 0:1 condition. Scale bar, 5 µm. Yellow cartoons illustrate one major solution conformation of each chain.B Representative results from spectrophotometric turbidity assay comparing the effects of varying amounts of K48- and K63-linked chains of different lengths on UBQLN2 LLPS at 30 μM UBQLN2.
Figure 2A CSPs for UBQLN2 450-624 residues at 1:1 ratio with Ub4. The highlighted regions in the domain map are important for the homotypic interactions that drive UBQLN2 LLPS (Dao et al, 2018). B Amide peak intensities between the unbound and Ub4-bound forms of UBQLN2 450-624 with the 555-570 region highlighted in gray. C CSPs for residues in UBQLN2 487-624 at 1:1 ratio with Ub4. D CSPs for residues in UBQLN2 450-624 lacking residues 551-569 (red/gray region) at 1:1 ratio with Ub4. E Normalized titration curves of 11 amide resonances for UBQLN2 450-624.F Comparison of binding affinity (Kd) measurements obtained from NMR and fluorescence anisotropy. Error bars for the FA numbers represent the standard deviation over three biological trials and for the TITAN numbers are from jackknife analysis. For visual comparison, gray dashed line is Kd of Ub binding to UBQLN2 450-624 as determined from NMR and FA experiments (green bars).G Fluorescence anisotropy values for fluorescently-labeled UBQLN2 450-624 in the presence of increasing K48- or K63-Ub4. Error bars represent the standard deviation over three biological trials.H Fluorescence anisotropy values for fluorescently-labeled K48- or K63-Ub4 in the presence of increasing UBQLN2 450-624. Error bars represent the standard deviation over three biological trials.
Figure 3B Proximal Ub resonances in Ub4 were compared to monoUb, as noted in schematic. CSPs > 0.05 ppm (orange dashed line) are mapped onto the proximal Ub unit of Ub4 in panel A.C 15N-1H SOFAST-HMQC NMR spectra of proximal Ub of K48-Ub4 or K63-Ub4 at 50 µM in the absence and presence of UBQLN2 450-624 at a 2:1 UBQLN2:Ub4 ratio. Unbound monoUb spectrum (black) is overlaid for comparison. NMR spectra collected and processed under identical conditions.D CSPs of the same proximal Ub resonances in K63-Ub4 vs K48-Ub4. Gray bars indicate K48-Ub4 resonances that were too weak to be detected.
Figure 4B Normalized pair distance distribution function P(r) from SAXS data for four Ub4 chain types.C CSPs at 1:1 Ub4:UBQLN2 for all chain types, but clustered into two categories for clarity.F Normalized titration curves of UBA resonances showing more pronounced sigmoidal behaviors upon titration of K48-Ub4 into UBQLN2 450-624 than upon titrations of K11-Ub4, K63-Ub4, M1-Ub4 and HOTag6-Ub.G Fluorescence anisotropy values for fluorescently-labeled UBQLN2 450-624 in the presence of increasing amounts of different Ub4 chains. Error bars represent the standard deviation over three biological trials.H Comparison of Kd values obtained from NMR and fluorescence anisotropy. Error bars for the FA numbers represent the standard deviation over three biological trials and for the TITAN numbers are from jackknife analysis. For visual comparison, gray dashed line is Kd of Ub binding to UBQLN2 450-624 as determined from NMR and FA experiments (green bars).
Figure 5A Fluorescence microscopy showing enhancement or inhibition of UBQLN2 droplet formation at increasing amounts of chains at 50 µM UBQLN2 in 20 mM NaPhosphate and 200 mM NaCl (pH 6.8) at 30 ºC. [Ub]:[UBQLN2] reflects the ratio between Ub monomers and UBQLN2 molecules. Solution is spiked with Alexa Fluor 647-labeled UBQLN2. The same image is used for the 0:1 condition. For visual comparison, images for K48-Ub4 and K63-Ub4 are identical to those shown in Figure 1A. Scale bar, 5 µm.B Representative results from spectrophotometric turbidity assay as a function of temperature at 30 μM UBQLN2.E Fluorescence microscopy showing the distribution of Ub4 (spiked with Dylight 488-labeled chains) inside and outside droplets containing 50 µM UBQLN2 (spiked with Alexa Fluor 647-labeled UBQLN2). Scale bar, 5 µm.
Figure 6A SDS-PAGE gels monitoring the formation of K63-linked polyUb chains and possibly ubiquitinated GST-Ubc13 over time, as indicated by asterisks and curly brackets. K63-Ub3 migrates on the gel similarly as His-Mms2, right below 20 kDa. Experimental conditions: ± 50 µM Ub and UBQLN2, 30 nM Dylight 650-labeled UBQLN2, 1 µM mE1, 2 µM His-Mms2, 4 µM GST-Ubc13, 10 mM ATP and MgCl2, 3 mM TCEP, 50 mM Tris pH8, 37 ºC. The K63-linked polyUb chain reactions were done in the absence (top) and presence (bottom) of UBQLN2. Since UBQLN2 competes with E1 and E2 to bind to Ub, the presence of UBQLN2 slowed down chain formation (indicated by comparing disappearance rates of Ub). The band intensity for GST-Ubc13 decreased with time. The time-dependent appearance of species between 50-85 kDa were obstructed in the presence of UBQLN2. These species are most likely ubiquitinated GST-Ubc13, and not free polyUb chains, since these bands were observed with BioRad Stain-free gels (right) that detect proteins with tryptophan residues unlike Ub or polyUb chains.
Figure 1A Generation of myeloid cells genetically inactivated for Elp3. Elp3 mRNA and protein levels were assessed by Real-Time PCR with extracts isolated from peritoneal macrophages of Elp3Control and Elp3∆Mye mice. mRNA levels in Elp3Control mice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 3 mice; mean +/- SD, Student T test, ***= p< 0.001). An anti-Elp3 western blot (WB) is also illustrated. The anti-Gapdh blot is shown for normalization purposes.C and D Defective mTORC2 signaling upon Elp3 deficiency in myeloid cells. Peritoneal macrophages from Elp3Control and Elp3∆Mye mice were isolated and cultured ex-vivo. They were untreated or stimulated with a combination of LPS (100 ng/ml) and IFNγ (50 ng/ml) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses.F mTORC2 activation by M1 polarization signals relies on Ctu2. Control and Ctu2-depleted bone marrow-derived macrophages (BMDMs) (SiRNA-mediated depletions) were treated or not with IFNγ (50 ng/ml) and LPS (100 ng/ml) and the resulting cell extracts were subjected to WB analyses.
Figure 2C Elp3 deficiency in myeloid cells exacerbates the decrease of colon length upon DSS administration (n = 7 mice; mean +/- SD, **= p< 0.01).E More severe histological score in mice lacking Elp3 in myeloid cells upon DSS administration. Representative intestinal crypts from the indicated genotypes are illustrated. The histological score was established as described in methods (n = 5 mice per genotype; mean +/- SD, Student T test, *= p< 0.05).F Increased infiltration of iNOS+/CD68+ cells upon DSS administration in Elp3∆Mye mice. The percentage of iNOS+ in CD68+ cells was established in both genotypes treated with DSS (n = 7 mice per genotype; mean +/- SD, Student T test, **= p< 0.01). Anti-iNOS and -CD68 immunofluorescence analyses carried out in intestinal crypts from both genotypes are illustrated.G Patients suffering from ulcerative colitis (UC) show reduced levels of both Elp1 and Elp3. The central band represents the mean value of relative expression in the investigated cohort. Boxes represent 75th and 25th percentile. Whiskers represent maximum and minimum values before the upper and lower fence, respectively (**= p< 0.01, ***= p<0.001) (GSE9452-GEO DataSets, Definition of an ulcerative colitis pre-inflammatory state).
Figure 3A IL-4 and IL-13 induce the expression of tRNA-modifying enzymes in macrophages. Peritoneal macrophages were untreated (« Control ») or stimulated with IL-4 (10 ng/ml), IL-13 (10 ng/ml) or with both cytokines for 24 hours and cell extracts from the resulting cells were subjected to WB analyses.B The pool of thiolated tRNAs in macrophages is enhanced by IL-4/IL-13 but decreased by LPS/IFNγ. On the left, Northern blot analysis assessing t:Q(UUG) tRNA thiolation in Thioglycollate-elicited peritoneal macrophages subjected to the indicated treatments for 24 hours. On the right, quantification of t:Q(UUG) tRNA thiolation, calculated as the ration of thiolated over nonthiolated t:Q(UUG), in the indicated experimental conditions.C Elp3 induction by IL-4/IL-13 occurs through a PI3K-dependent pathway. Peritoneal macrophages were pretreated with DMSO (control vehicle) or with Wortmannin (200 nM) for one hour and subsequently left untreated or stimulated with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for the indicated periods of time. Cell extracts from the resulting cells were subjected to WB analyses.E Rictor but not Raptor deficiency impairs Elp3 expression. BMDMs were transfected with the indicated siRNAs and the resulting cells were treated or not with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for 24 hours. Cell extracts were subjected to WB analyses.F IL-4/IL-13 rely on Stat6 to induce Elp3 expression. Peritoneal macrophages were pretreated with the control vehicle (DMSO) or with the Stat6 pharmacological inhibitor AS1517499 (5 μM) for one hour and subsequently left unstimulated or treated with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for the indicated periods of time. Cell extracts from the resulting cells were subjected to WB analyses.Elp3 deficiency in myeloid cells impairs multiple signaling pathways triggered by IL-4 (H, , IL-13 (H) or by both cytokines (H). Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4 (10 ng/ml) (H, IL-13 (10 ng/ml) (H) or with both cytokines (H) for 8 hours (H) and the resulting cell extracts were subjected to WB analyses.J Elp3 deficiency in myeloid cells impairs multiple signaling pathways triggered by IL-4 I and J) Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4 (10 ng/ml) , I and J), or for the indicated periods of time (I and J) and the resulting cell extracts were subjected to WB analyses.L Elp3 promotes M2 macrophage polarization. Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4/IL-13 (10 ng/ml) for 24 hours and the resulting cell extracts were subjected to WB analyses.
Figure 4B Defective induction of Pparγ by IL-4/IL-13 upon Elp3 deficiency. Cell extracts from peritoneal macrophages of Elp3Control and Elp3∆Mye mice treated or not with IL-4/IL-13 were subjected to WB analyses.
Figure 7A The induction of Ric8b expression by IL-4 relies on Elp3. BMDMs from the indicated genotypes were treated or not with IL-4 (10 ng/ml) up to 24 hours and the resulting cell extracts were subjected to WB analyses.B Elp3 promotes Ric8B mRNA translation. BMDMs from Elp3Control and Elp3∆Mye mice were treated by IL-4 for 24 hours, followed by a treatment with 10 μg/ml puromycin for 5 minutes. To detect newly synthesized Ric8b proteins, a Puro-PLA assay was conducted. Representative images detecting Ric8b (Red) and α-Tubulin+ microtubules (green) in BMDMs are illustrated. On the right, the graph shows a quantification of signals for Ric8b in α-Tubulin+ areas. A random of six different areas were counted (6 technical replicates, mean +/- SD, Student's t-test, *= p< 0.05).C Ric8b is a direct target of Elp3. BMDMs from Elp3Control and Elp3∆Mye mice were infected with a lentiviral GFP construct (negative control), Flag-Ric8b WT or with Flag-Ricb MUT, as indicated and the resulting protein extracts were subjected to WB analyses to assess Ric8b and Elp3 protein levels. Gapdh was used as a loading control.D and E Ric8b promotes IL-4-dependent mTORC2 activation. Control or Ric8b-depleted BMDMs were treated or not with IL-4 (10 ng/ml) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses.
Figure 8C Elp3 promotes mitochondrial translation. BMDMs from the indicated genotypes were treated or not with IL-4/IL-13 (10 ng/ml) for the indicated hours and the resulting cell extracts were subjected to WB analyses.
Figure 9D Elp3 expression in myeloid cells maintains the pool of tumor-associated macrophages (TAMs). The number of TAMs, defined as CD301+/F4/80+ cells among F4/80+ cells was quantified in intestinal crypts from both Apc+/Min Elp3Control and Apc+/Min Elp3∆Mye mice (n = 5 mice per genotype; mean +/- SD, Student T test, *= p< 0.05). Representative immunofluorescence analyses are illustrated.
Figure 1D. Northern blot analysis of V. cholerae wild-type strain examined for the expression of VcdRP monitored over bacterial growth in the indicated media. The solid triangle represents the band corresponding to the full length primary vcdRP transcript, whereas the open triangles correspond to the different processed isoforms. Probing with 5S rRNA confirmed equal loading.
Figure 2A. Northern blot analysis of the expression of VcdRP and Spot42 sRNA monitored at low cell density (OD600 of 0.1) and high cell density (OD600 of 2.0), in the absence (-) or presence (+) of cAMP (5mM f.c.) supplemented externally in LB medium. The solid triangle indicates the band corresponding to the full-length primary transcript. Probing with 5S rRNA confirmed equal loading.
Figure 3B. Western blot analysis (top) of CTX levels detected in secreted protein fractions of V. cholerae ΔhapR cells grown under AKI conditions and carrying the indicated plasmids. Coomassie-stained SDS gel (bottom) confirmed equal loading of the protein fractions. The normalized band intensities are indicated for each expression plasmid relative to pCtrl, which was set to 100%.
Figure 4I-L. Relative fluorescence intensities (y-axis) of E. coli strains harboring the gene-specific translational reporters as in (A-D) or the corresponding M2* variant for each gene, combined with an empty control plasmid or vcdRP expression plasmids (pVcdRP and pVcdRP M2) measured using plate reader (I-K) or by Western blot analysis (L). The fluorescence of pCtrl (for each reporter fusion) was set to 1.
Figure 5A. Immunoprecipitation of chromosomally tagged GltA::HA combined with either an empty vector control (pCtrl) or SPA-tagged vcdP over-expression plasmid, grown in LB medium to exponential phase (OD600 of 0.5). The SPA epitope contains the 3×FLAG and the calmodulin binding peptide sequences separated by a TEV protease cleavage site (Zeghouf et al, 2004). Therefore, protein samples corresponding to the total input and cell lysates before and after subjecting to immunoprecipitation with anti-Flag antibody were loaded on a SDS-PAGE gel. Western blotting with anti-HA and anti-Flag antibodies confirmed the interaction of GltA with VcdP in vivo. RNAP served as loading control. The solid triangles indicate the corresponding protein sizes.B. Citrate synthase enzyme activity measurements performed on cellular lysates. An empty vector control (pCtrl) or vcdP expression plasmids (pVcdP and pVcdP*) were conjugated into V. cholerae wild-type, ΔvcdRP, ΔgltA or F383A GltA backgrounds. Cells were grown in LB medium to stationary phase and subsequently lysed. The protein concentrations of these cellular extracts were measured, and served as input for the assay. The resulting colorimetric product at 412nm was proportional to the enzymatic activity of citrate synthase present. The enzyme kinetics were assayed at 15 second intervals for at least 30 minutes and the activity per milligram of lysate was determined from the initial velocities.
Figure 1(a) Widefield immunofluorescence images of PFA fixed HeLa cells showing endogenous FLCN and LAMP1 staining in (top) normal growth or (bottom) starvation conditions. Orange arrows highlight association of FLCN with LAMP1 positive membranes in the peri-nuclear region. White dotted line shows cell periphery in starved condition. Scale bar = 10μm.(b) (left) Quantification of FLCN/LAMP1 association in growth and starvation conditions. A cell is scored as positive if 5 or more discrete FLCN/LAMP1 puncta were observed at 100x magnification using a widefield microscope. Data represents 60 cells from 3 independent experiments, error bars show S.E.M, *** = p<0.001. (right) Western blot shows levels of phosphorylated of S6K and 4EBP in whole cell extracts under the same conditions.(c) Western blot showing typical efficiency of FLCN knockdown in HeLa cells using a pool consisting of 4 oligonucleotides and 2 independent oligonucleotides from the pool. Graph shows relative FLCN expression (n=3, error bars show S.E.M.)(d) Representative confocal fluorescence microscopy images showing LAMP1 (magenta) distribution in HeLa cells under normal growth or starvation conditions in cells depleted of FLCN or control cells transfected with a non-targeting siRNA. The location of the Golgi (green) is highlighted by giantin staining.(e) Schematic illustrating application of the cumulative intensity distribution method for quantification of lysosome distribution.(f) Graph showing cumulative distribution of LAMP1 intensity in normal growth and starvation conditions in HeLa cells. P-value is determined by the extra sum of F-squares test following non-linear regression and curve fitting. Error bars show S.E.M. from 30 cells in 3 replicates.(g) Graph showing cumulative distribution of LAMP1 intensity in starvation conditions for control cells transfected with non-targeting siRNA or cells depleted of FLCN. Error bars show S.E.M. from 30 cells in 3 replicates.
Figure 2(a) Representative spinning disk confocal image showing FLCN-GFP and lysosomes (Lysotracker-Red) in a live FLCN-GFP/HA-FNIP2 transfected HeLa cell (from Movie EV1). Top panels show the first frame of a 180 frame (1fps) image series. Bottom panels show a maximum intensity projection of the image series to highlight mobile and immobile components. Boxed and magnified regions show typical peri-nuclear (i) and peripheral (ii) regions in the cell as well as a FLCN-GFP tubule (iii) Scale bar = 5μm.(b) Spinning disk confocal image series of a region live FLCN-GFP/HA-FNIP2 transfected HeLa cell highlighting dynamic FLCN-GFP tubules (Movie EV2). First merge panel shows FLCN-GFP and lysotracker-Red at T=0 Subsequent panels show only FLCN-GFP at 30 second intervals.c) Confocal image of a region of a methanol fixed FLCN-GFP/HA-FNIP2 transfected HeLa cell showing Rab7 (red) and LAMP1 (blue) association with FLCN-GFP tubules. Scale bar = 1μm.
Figure 3(a) Graphs showing cumulative distribution of LAMP1 intensity in control GFP (black), GFP-Rab34 (green) or GFP-Rab7 (blue) transfected cells. Note black line and symbols partially obscure blue.(b) GST-RILP pull-down analysis from HeLa cells in normal and starved conditions showing an enhanced capacity of endogenous Rab34 to bind RILP in starvation conditions. Quantification is mean of 3 independent experiments. Error is S.E.M.(c) Representative confocal fluorescence microscopy images of FLCN depleted or control cells, transfected with GFP-Rab34. Scale bar = 10μm.(d) (left) Graphs showing mean GFP-Rab34 expression (grey values) and cumulative distribution of LAMP1 intensity (right) in FLCN depleted or control siRNA cells transfected HeLa cells expressing GFP-Rab34. Error bars show S.E.M.. from 30 cells in 3 replicates. Scale bar = 10μm.(e) Western blot and graph showing typical efficiency of Rab34 siRNA knockdown after 48 hours in HeLa cells. Error bars show S.E.M from 3 experiments, *** = p<0.001.(f) Representative confocal fluorescence microscopy images showing LAMP1 (magenta) distribution in HeLa cells under normal growth or starvation conditions in cells depleted of Rab34 or control cells transfected with a non-targeting siRNA. Giantin is shown in green. Scale bar = 10μm.(g) Graphs showing cumulative distribution of LAMP1 intensity in normal growth or starvation conditions for control cells or cells depleted of Rab34 using siRNA.(h) Graph showing percentage of LAMP1 in cis-medial Golgi-region (giantin overlap) in the indicated conditions. Error bars show S.E.M from 30 cells in 3 replicates, *** = p<0.001.
Figure 4(a) (left) Representative confocal fluorescence microscopy image showing methanol fixed HeLa cell transfected with GFP-Rab34 and stained for LAMP1. Linescan analysis shows relative intensity of GFP and LAMP1 signals. Scale bar = 10μm.(b) Maximum intensity projection images from a confocal Z-stack showing LAMP1 and endogenous Rab34 in P-M fixed HeLa cells from normal and starved conditions. Whole cells and fields from which these panels are derived are shown in Figure EV4A. Boxes highlight regions shown in Figure 4C. Scale bar = 2μm.(c) Single confocal sections from the Z-stack in figure 4B. Orange arrows show close LAMP1/Rab34 association.(d) Analysis of Lysotracker-Red dynamics using PCC decay in HeLa cells transfected with either GFP or GFP-Rab34. Data is from 10 cells per condition. Error bars show S.E.M.(e) Live-cell spinning disk confocal microscopy image series at 10 sec intervals (from Movie EV3) showing GFP-Rab34 positive compartment making dynamic contacts with lysosomes. Scale bar = 1μm(f) Live-cell spinning disk confocal microscopy image series at 10 sec intervals (from Movie EV4) of FLCN-GFP and mCherry-Rab34 in the peri-nuclear region of a FLCN-GFP/HA-FNIP2/mCherry-Rab34 transfected HeLa cell. Blue arrow highlights FLCN-Rab34 association moving on a linear trajectory. Yellow arrow highlights shorter saltatory movements. White arrow highlights dynamic associations between distinct FLCN-GFP and Rab34 positive structures. Scale bar = 1μm(g) Confocal image showing localisation of endogenous FLCN and Rab34 in starved HeLa cells.