EMBO/SourceData huggingface dataset:
Figure 3(A) Composite of bright field and Whi5-tdTomato (BF/Whi5) and CLN2-PP7 mRNA labelled with PCP-NLS-GFP (CLN2 mRNA) in mother (M) and daughter (D) cells of the indicated strains. Arrows indicate nuclear foci. Brighter GFP nucleoplasmic areas may correspond to the nucleolus. Insets show enhanced-contrast images to visualise cytoplasmic mRNA particles (arrowheads). Numbers indicate minutes relative to Whi5 nuclear import. Maximum projections of whole-cell Z-stacks are shown for Whi5 and CLN2 mRNA except in the inset and in selected esa1-ts images, where single Z-slices are shown for clarity. Scale bar: 4 µm (inset: 1 µm).(B) Fraction of cells of the indicated strains with nuclear CLN2-PP7 mRNA foci, aligned relative to Whi5 import. Symbols represent individual values; lines were generated by smoothing of the nearest 3 neighbouring values (0th order polynomial). (C) Fraction of cells of the indicated strains with nuclear CLN2-PP7 mRNA foci, aligned relative to Whi5 export.(D) Mean fluorescence intensity of nuclear CLN2 mRNA foci at the time of Whi5 export, normalised relative to the nuclear background. Data information boxes include 50% of data points, lines represent the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; **, p ≤ 0.01; n.s., p > 0.05, ordinary one-way ANOVA with Tukey multiple comparisons test. Adjusted p-values in (D): WT vs. esa1-ts p < 0.0001, WT vs. esa1-ts nup60-KN p < 0.0001, esa1-ts vs. esa1-ts nup60-KN p = 0.9845 Foci were scored in individual Z-slices spanning the entire cell volume. n = number of cells, pooled from two independent experiments with similar results.(E) Number of cytoplasmic CLN2-PP7 mRNA foci in early G1 (5 minutes after Whi5 import) and at the G1/S transition (5 min after Whi5 export). Data information: boxes include 50% of data points, lines represent the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; **, p ≤ 0.01; n.s., p > 0.05, ordinary one-way ANOVA with Tukey multiple comparisons test. Adjusted p-values in (E): WT vs. esa1-ts p < 0.0001, esa1-ts vs. esa1-ts nup60-KN p = 0.002. Foci were scored in individual Z-slices spanning the entire cell volume. n = number of cells, pooled from two independent experiments with similar results.(F) The position of nuclear CLN2-PP7 foci (marked with arrowheads in the examples) was scored relatively to the nuclear periphery (visualised with PCP-NLS-GFP) in the indicated strains. The fraction of non-perinuclear CLN2-PP7 foci is significantly increased in esa1-ts cells compared to the WT and rescued by nup60-KN mutation (two-sided Fisher's exact test p = 0.0209 and p = 0.0227). Scale bar, 1 µm.
Figure 4(B) Overexpression of MEX67 and MTR2 rescues the lethality of Hos3-NLS overexpression. 10-fold serial dilutions of the indicated strains transformed with the indicated plasmids spotted onto SC-Glu and SC-Gal medium and incubated at 25 °C for 3 days.(C) A high-copy plasmid containing SAC3 rescues the lethality of Hos3-NLS overexpression. Strains carrying a high copy plasmid containing the SAC3 ORF (nucleotides 467-3905), or the empty vector, were grown(D) Overexpression of Hos3-NLS promotes nuclear accumulation of mRNA. (Left) Cultures of the indicated strains were treated with galactose overnight to induce HOS3-NLS expression, cells were fixed, and FISH was performed using Cy3-Oligo(dT). (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions. Data information: , arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.(E) Inactivation of Esa1 impairs export of poly(A) RNA. (Left) Cultures of the indicated strains were incubated at 37 ºC. (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions Data information: , arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.(F) nup60-KN mutation partially rescues the mRNA export defects of esa1-ts. Cells of the indicated strains were processed Data information: arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.
Figure 5(A) Depletion of Hos3 or expression of acetyl-mimic Nup60 (nup60-KN) increases the nuclear localisation of Sac3 in G1. Cells of the indicated strains were imaged by time-lapse microscopy and the fluorescence levels of the indicated proteins were determined in G1 (after cytokinesis). The NPC component Nup49 was used as a control for Nuclear Pore Complex protein levels. Fluorescence intensity was measured in sum projections of whole-cell Z-stacks, by segmentation of either the nuclear area in the mCherry channel, or of the whole cell in the brightfield channel. The ratio of Sac3/Nup49 intensities was then normalised relative to the mean intensity of wild-type mothers. Data information arrowheads point to daughter cells boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.(B) Inactivation of Esa1 decreases Sac3 nuclear levels. Wild-type (WT) and esa1-ts cells were arrested in mitosis by treatment with nocodazole at 25 ºC, shifted to 37 ºC, released from the mitosis block in fresh medium at 37 ºC, and imaged by time-lapse microscopy. Fluorescence levels were quantified in G1 Data information: arrowheads point to daughter cells boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.(C) Rapamycin-dependent dimerisation abolishes Sac3 mother/daughter asymmetries. NUP60-mCherry-FKBP SAC3-GFP-FRB cells were incubated with rapamycin (RAPA) to trigger FRB-FKBP heterodimerization, or with DMSO as control. Fluorescence levels were quantified in G1 cells 15 to 30 minutes after addition of the drug. Data information arrowheads point to daughter cells, boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.(D) Sac3 anchoring to the nuclear basket advances Start in esa1-ts daughter cells. Composite of bright field and Whi5-mGFP (top) and quantification of Whi5 nuclear export timing (bottom) in wild-type (WT) and esa1-ts mother (M) and daughter (D) cells treated with either rapamycin (RAPA) or DMSO and expressing Nup60-FRB and Sac3-mCherry-FKBP. Sac3 anchoring to Nup60 does not alter Whi5 export timing in WT mother or daughter cells (DMSO vs RAPA, p > 0.05, Log-rank Mantel-Cox test), but it advances Whi5 export in esa1-ts daughters (p = 0.0001). Whi5 export efficiency was slightly improved also in mother cells (p = 0.0374). 8 z-confocal slices spaced 0.4 µm were acquired every 3 min; maximum projections are shown. Time is indicated in minutes; t=0 marks Whi5 nuclear import. Scale bar, 5 µm. Data information: arrowheads point to Whi5 export.
Figure 6(A) Time Lapse microscopy of WT and nup60-KN cells expressing GAL1pr:sfGFP and Nup60-mCherry at the indicated times of galactose induction. Scale bar, 4 µm.(B) Depletion of Hos3, and expression of acetyl-mimic Nup60 (nup60-KN) enhances GAL1 expression. WT, hos3Δ, nup60-KN and nup60-KN hos3Δ cells were shifted to galactose and imaged by time-lapse microscopy to monitor GAL1pr:sfGFP expression during 7 hours. Nuclear fluorescence was scored by segmentation of the nuclear area in the mCherry channel and mean fluorescence of nuclear GFP and Nup60-mCherry was quantified from sum projections of whole-cell Z-stacks at the indicated times. At least 200 cells were scored for each strain and time point. Shaded areas indicate the SEM.(C) mRNA levels of GAL1 were determined for wild type (WT) and nup60-KN cells at the indicated times after galactose addition. One of two independent experiments with similar results is shown (mean and SEM from 3 technical replicates).(D) The GFP intensity of mother/daughter pairs for cells in B, at 5-minute intervals after galactose addition (left) and 425 min after galactose addition (right). Boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.001; ns, p > 0.05, two-tailed paired t-test. n = number of cells. One of two independent experiments with similar results is shown.(E) Expression of GAL1pr:sfGFP induced with galactose (left) or ß-estradiol (right), in the presence of the ß-estradiol-dependent transactivator Gal4-ER-VP16. Smooth lines show GFP fluorescence intensity (left x axis); lines with circles show the difference in GFP intensity between the indicated strains (right x axis). The difference between wild type and hos3∆, and between wild type and nup60-KN, increases continuously over time in response to galactose, but not to ß-estradiol.
Figure 7(A) Cells of the indicated strains were incubated with galactose in the presence of either rapamycin (RAPA) to induce FRB-FKBP heterodimerization, or DMSO as control. GAL1pr:sfGFP expression was monitored over time(B) (Top) Representative images of the indicated cells in rapamycin or DMSO, 250 min after galactose addition. Scale bar 4 µm. (Bottom) Mother/daughter pairs were quantified as in Figure 6D at 250 min after galactose addition (pink shaded area in A). Boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. **, p ≤ 0.01; *, p ≤ 0.05; ns, p > 0.05, two-tailed paired t-test for M-D comparisons, unpaired for comparisons between strains. n = number of cells. One of two independent experiments with similar results is shown.
figf1(a) Induction of GFP+ dots in differentiated THP-1 cells stably expressing GFP-LC3, left untransfected or transfected for 6 h with poly(dA:dT) (1.5 μg/ml), then exposed to control antibody (Control Ab), antibody to neutralize IL-1β (Anti-IL-1β), dimethyl sulfoxide (DMSO) or 3-MA (5 mM); results are presented as quantification of GFP-LC3+ dots (top) and images of individual cells (middle). Scale bars, 10 μm.(b) Expression of AIM2 and beclin-1 in cells transfected with control siRNA or siRNA targeting AIM2 or beclin-1; numbers below lanes indicate band intensity (ratio of cells transfected with targeting siRNA to those transfected with control siRNA).(c) Induction of GFP+ dots in differentiated THP-1 cells stably expressing GFP-LC3 and treated with various siRNAs (below graph), quantified 6 h after transfection with poly(dA:dT) (+) or no transfection (-).(d) Immunoblot analysis of the expression of LC3-I and LC3-II (above) in wild-type (WT) and Aim2−/− BMDMs left untransfected or transfected for 6 h with poly(dA:dT); numbers below lanes indicate division of the ratio of induced LC3-I to induced LC3-II by the ratio of basal LC3-I to basal LC3-II (band intensities). Below, immunoblot analysis of IL-1β in supernatants after overnight culture.(e) Immunoblot analysis of LC3-I and LC3-II in or wild-type, ASC-deficient (Pycard-/-; called 'Asc-/-' here) or caspase-1-deficient (Casp1-/-) BMDMs primed with LPS (20 ng/ml) and then left untransfected or transfected with poly(dA:dT) or left untreated or treated with nigericin for 4 h; numbers below lanes as in d. Actin serves as a loading control throughout. NS, not significant; *P 0.05 and **P 0.01 (unpaired t-test). Data are from three individual experiments
figf2(a) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells transfected for 3 h with Cy3-labeled poly(dA:dT) (1.5 μg/ml). Yellow dots (Merge) indicate colocalization of green (LC-3) and red (Cy3) signals. Scale bars, 10 μm.(b) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells transfected to express Cy3-poly(dA:dT), imaged every 15 min starting 1 h after transfection (numbers in bottom left corners indicate time in min (m)). Arrow indicates coalescence of the Cy3 and GFP signals (also in bottom right image).(c) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells sham-transfected with no poly(dA:dT) (Control) or transfected with poly(dA:dT), then fixed and immunostained for ASC (red). Insets (bottom right), 5× enlargement of areas indicated by arrowheads at left. Scale bars, 10 μm (top) or 20 μm (bottom).(d) Confocal microscopy of differentiated THP-1 cells transfected with poly(dA:dT) and immunostained for LAMP-1 and ASC; below, z-axis projections. Scale bars, 10 μm.(e) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells treated for 2 h with LPS (500 ng/ml) and ATP (3 mM), then immunostained for ASC (red): left image in each pair, three-dimensional volume renderings reconstructed from z-stack images; right image in each pair, surface-segmentation models (2× enlargement). Scale bars, 10 μm.(f) Electron microscopy of differentiated THP-1 cells stimulated with LPS and ATP (left) or transfected with poly(dA:dT) (right) for 2 h and immunostained for ASC, visualized with 15-nm gold-conjugated protein A. Red arrows indicate ASC immunostaining. Right image in each pair, 4.5× enlargement of outlined area at left. Scale bars, 500 nm. Data are representative of three experiments (a,c) or two experiments (b,d-f).
figf3(a) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells left untransfected (Control) or transfected for 2 h with poly(dA:dT) (1.5 μg/ml), then immunostained for endogenous AIM2(b) Confocal microscopy of differentiated THP-1 cells transfected for 3 h with poly(dA:dT), then immunostained for LAMP-1 and AIM2.(c) Confocal microscopy of differentiated THP-1 cells left untreated or treated for 2 h with LPS (500 ng/ml) and ATP (3 mM), then immunostained for endogenous LC3 and NLRP3. Scale bars, 10 μm. Data are representative of at least three experiments.
figf4(a) Immunoblot analysis of AIM2, ASC and LC3-II in lysates of differentiated THP-1 cells left untransfected or transfected with poly(dA:dT) (1.5 μg/ml) and left untreated or treated with 3-MA, rapamycin or amino-acid starvation, then separated into an inflammasome-enriched fraction (top) or an autophagosome-enriched fraction (middle). Bottom, immunoblot analysis of total cell lysates.(b) Immunoblot analysis of unprocessed and processed caspase-1 and IL-1β in lysates and supernatants of differentiated THP-1 cells treated as in a.(c) Immunoblot analysis of LC3-I and LC3-II (top) in lysates of mouse BMDMs primed with LPS (20 ng/ml) and left untreated or treated with alum, then left unexposed or exposed for 5 h to live (Live TB) or dead (Dead TB) M. tuberculosis (numbers below lanes as in Fig. 1d). Below, immunoblot analysis of IL-1β in supernatants of LPS-primed mouse BMDMs infected overnight with live M. tuberculosis in the presence or absence of 3-MA (2 mM); treatment with LPS and ATP serves as a positive control. Data are representative of at least two experiments.
figf5(a) Immunoblot analysis of ASC immunoprecipitates (IP; left) and total lysates (right) of differentiated THP-1 cells left untransfected or transfected for 2 h with poly(dA:dT) (1.5 μg/ml), probed for beclin-1 and ASC (below), then stripped and reprobed for p62 (above).(b) Confocal microscopy of differentiated THP-1 cells treated as in a, immunostained for p62 (green) and ASC (red); arrows indicate colocalized proteins.(c) Confocal microscopy of differentiated THP-1 cells treated for 2 h with LPS (500 ng/ml) and ATP (3mM), immunostained for p62 (green) and ASC (red): left two images, merged channel volume and surface rendering of a three-dimensional reconstruction; right four images, enlargement of images at left (yellow arrow), with arrows indicating removal of part of the signal to reveal the red signal (ASC) inside the green-stained area (p62).(d) Immunoblot analysis of lysates of cells transfected twice with siRNA with scrambled sequence (−) or siRNA specific for beclin-1 or p62; numbers below lanes indicate band intensity (as in Fig. 1b).(e) Immunoblot analysis of the inflammasome and autophagosome fractions and total lysates of differentiated THP-1 cells transfected as in d, with poly(dA:dT) included in the second transfection; lysates were prepared 4 h after the final transfection.(f) Immunoblot analysis of supernatants and lysates of differentiated THP-1 cells treated as in e; supernatants were collected after culture for 6 h in serum-free medium. Scale bars (b,c), 10 μm. Data are representative of at least two experiments.
figf6(a) Immunoblot analysis of ASC immunoprecipitates of differentiated THP-1 cells transfected for 2h with poly(dA:dT) (1.5 μg/ml), probed for ubiquitin (Ub), then stripped and reprobed for K63-linked ubiquitin (Ub-K63), then stripped and reprobed for ASC.(b) Immunoassay of differentiated THP-1 cells treated as in a with protein-dissociation buffer, followed by immunoprecipitation of ASC (24 kilodaltons (kDa)) and immunoblot analysis.(c) Confocal microscopy of differentiated THP-1 cells transfected for 2 h with poly(dA:dT), immunostained for ASC (red) and polyubiquitin (green). PolyUb, polyubiquitination. Scale bars, 10 μm. Brackets (right margins in a,b) indicate smear of ubiquitinated proteins. Data are representative of three (a,c) or two (b) experiments.
figf7(a) Immunoblot analysis of lysates of human monocytes-macrophages primed with LPS (10 ng) and then transfected with poly(dA:dT) (1.5 μg/ml) or treated with nigericin (4 μM) for 6 h (numbers below lanes as in Fig. 1d).(b) Immunoblot analysis of processed caspase-1 and IL-1β (top) and enzyme-linked immunosorbent assay of IL-1β (middle) in supernatants of monocytes-macrophages treated for 6 h with various combinations (bottom) of poly(dA:dT) (b) or nigericin (c), plus 3-MA and amino-acid starvation. *P 0.01 (unpaired t test).(d) Confocal microscopy of LPS-primed human monocytes-macrophages transfected for 3 h with poly(dA:dT) (top), treated for 3 h with nigericin (middle) or left unstimulated (bottom), immunostained for p62 (green) and ASC (red); arrows indicate colocalized proteins. Second row, three-dimensional volume rendering reconstructed from confocal z-stack images. Scale bars, 10 μm. Data are representative of at least two experiments.
figf8(a) Immunoblot analysis of lysates of LPS (20 ng/ml)-primed mouse BMDMs exposed to LPS (500 ng/ml) and ATP (3 μM), poly(dA:dT) (1.5 μg/ml) or uric acid crystals (50 μg/ml), detecting GTP-bound RalB (collected by RaBP1-agarose affinity purification) and RalB in total lysates.(b) Immunoblot analysis of lysates and supernatants of mouse BMDMs transfected with control or RalB-specific siRNA on 2 sequential days, then primed with LPS and exposed for 6 h to poly(dA:dT) (1.5 μg/ml), probed for RalB, LC3 and IL-1β; supernatants were collected after overnight culture in serum-free medium (numbers below lanes as in Fig. 1d). Nonsp, nonspecific band.(c) Immunoblot analysis of lysates of wild-type and Aim2−/− mouse BMDMs transfected with control or AIM2-specific siRNA on 2 sequential days, primed for 2 h with LPS and then exposed for 1 h to poly(dA:dT) (numbers below lanes as in Fig. 1d).(d) Immunoblot analysis of ASC immunoprecipitates from LPS-primed mouse BMDMs left untreated or transfected for 1 h with poly(dA:dT) and treated for 30 min with LPS and ATP. Mock, immunoprecipitation with control antibody.(e) Confocal microscopy of LPS-primed mouse BMDMs left untransfected or transfected for 30 min with poly(dA:dT), immunostained for RalB and AIM2 (left three columns); RalB and ASC (middle columns); and beclin-1 and AIM2 (right two columns). Bottom row, 3× electronic enlargement of areas outlined above. Scale bars, 10 μm. Data are representative of at least two experiments.
Figure 5(a) Bipartite network of species (left) and metabolites (right) for metabolites that decreased by at least two-fold compared to the abundance of each metabolite at the beginning of the experiment. Colors represent modules containing many overlapping interactions in the network. Network partitioning into modules was performed using BiMat(Flores Metabolites in bold were depleted and secreted by distinct organisms. (b) Bipartite network of species (left) and metabolites (right) for metabolites that increased in abundance by at least two-fold compared to the beginning of the experiment. Metabolites highlighted in bold were depleted or secreted by different species. Colors represent modules containing many overlapping interactions in the network. Network partitioning into modules was performed using BiMat(Flores(c) Scatter plot of the number of consumed metabolites that decreased by at least two-fold compared to the beginning of the experiment vs. the OD600 value of the monospecies culture at the corresponding time point. Error bars represent 1 s.d. from the mean of three biological replicates.
Figure 1Example time trace when no binding event occurs (green laser excitation). EmCCD image shows signal from green and red channels before the addition of Cy3-labelled DNA when illuminated with green laser. Size of EmCCD image is 25µm x 50 µm.   Example time trace showing a binding event (green laser excitation). EmCCD image shows signal from green and red channels after the addition of Cy3-labeled DNA when illuminated with green laser. Size of EmCCD image is 25µm x 50 µm.
Figure 2Example time traces when DNA containing 3 PAM sequences is added (top) and when DNA containing a single PAM is added showing both short and long binding events (bottom).FRET histograms of all binding events for each construct. Y axis represents total number of counts, divided by the number of immobilized molecules in the field of view.Bar plot of all dwelltime values for all constructs showing short binding events for all DNA and long events only for the constructs containing PAM sequences. The times shown are mean values of dwelltimes obtained during four different experiments on four different days. Error bars represent standard error of the mean.Example dwelltime histograms from negative control and a construct containing a single PAM. Negative control dwelltime distribution is characterized by a single exponential decay (top) and dwelltime distribution for PAM-containing DNA is fitted by a double-exponential decay (blue line) (bottom). Equation for the double exponential decay fit: y=A1e-t/τ1 + A2e-t/τ2; ΔA1= 796.4 ΔA2= 28.3. Red line shows what a single-exponential decay fit for such distribution would be. Errors represent standard error of the mean.Scatter plot showing the average dwelltime (Δτav) values for the cases of 2 (d2) and 4 (d4) nucleotide separation between the PAM sites. The values are averages of four measurements over four different days for each case. Error bars represent the standard error of the mean. The average dwelltime was obtained using the equation τav=(A1Δτ12+A2τ22)/(A1Δτ1+A2Δτ2)Example trace showing direct FRET transitions for a 5-PAM containing DNA construct with separation between PAM sites being 4 nucleotides.Histogram showing the percentage of events that are longer than τ1 showing FRET fluctuations for multiple PAM constructs with PAM separation of 4 nucleotides. The values are averages of four measurements over four different days. The error bars represent standard error of the mean.