EMBO/soda-nel huggingface dataset:
Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKMH1 membrane cycling during autophagosome-lysosome fusion. (2019)
Figure 3

Figure 3. Increased production of PI(4,5)P2 in Rab7 positive endosomes causes Rab7 dissociation.

(A) Cartoon depicting the experimental design. A PIP5Kγ construct fused to FKBP12 is expressed together with an FRB module fused to Rab7 and the GFP-tagged PLCδ1PH reporter for PI(4,5)P2. Addition of rapamycin causes recruitment of PIP5Kγ to the endosomes where it makes more PI(4,5)P2 and attracts the PI(4,5)P2 reporter.

(B) Representative confocal pictures showing these changes in live cells. Cells were transfected with the indicated constructs and imaged before and after addition of rapamycin (100 nM) for 10 min. Note that the Rab7 recruiter itself is dissociating from the membranes after PI(4,5)P2 generation. Left panels show some cells enlarged. Scale bars: 20 µm.

(C) Quantification of the changes in BRET experiments. Here the BRET sensor contained the PLCδ1PH-fused to Sluc and the Venus targeted with Rab7 expressed from a single vector and it was co-transfected with the Rab7-targeted FRB (tagged with iRFP) and the FKBP12-fused PIP5Kγ in which the CFP was mutated to eliminate its fluorescence (CFP*). Recruitment of the PIP5Kγ but not its kinase-dead version caused an increase in the BRET signal indicating the increased PI(4,5)P2 in this compartment (means ± S.E.M., from three separate experiments performed in triplicates).

(D) PI(4,5)P2 increases were larger when the Venus part of the BRET sensor was targeted with the GTP-locked form of Rab7 (Q67L) and was negligible with the GDP-locked form of Rab7 (N125I) in the BRET construct. Also note that the Rab7 wild-type-based BRET signal slowly returned toward the green trace consistent with Rab7 falling off from the membrane both in the BRET and the recruiting constructs (means ± S.E.M., from three separate experiments performed in triplicates). (E) The response of PI4K2A K/O cells was significantly reduced in these latter experiments. Means ± S.E.M. are shown from three experiments performed in triplicates.

(F) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments shown in panel E (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P = 0.0478 and 0.0287 for #20 and #26 clones, respectively).
Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKMH1 membrane cycling during autophagosome-lysosome fusion. (2019)
Figure 4

Figure 4. Increased level of PI4P in Rab7 endosomes is associated with PI(4,5)P2 production. (A) Representative confocal images of HEK293-AT1 cells, which show that treatment with OSW1 (20 nM), a drug that inhibits the cholesterol-PI4P transport protein, OSBP, causes accumulation of PI4P in the Golgi and in the endosomes (upper panels). Note that the accumulation is much reduced in the endosome but less so in the Golgi in the PI4K2A K/O cells. Scale bars: 20 µm

(B) Quantification of these changes by BRET analysis. The PI4P-Rab7 BRET sensor construct was transfected into the respective cell lines and the cells treated with OSW1 (20 nM) for the indicated times. The BRET ratios were expressed relative to those of DMSO treated cells.

(C) Detection of endosomal PI(4,5)P2 in cells pre-treated with OSW1 for 1 h in serum-free medium using confocal microscopy and the PLCδ1PH-GFP PI(4,5)P2 reporter. Angiotensin II (AngII, 100 nM) (a Gq and PLC-activating agonist) was used to liberate the PLCδ1PH-GFP PI(4,5)P2 reporter from the PM, which was necessary to detect the endosomal PI(4,5)P2. The signal is transient as the AngII receptors show desensitization. Note the reduced signal in the two PI4K2A K/O clones. Scale bars: 20 µm.

(D) Quantification of the same changes by BRET analysis in wild-type cells using the PI(4,5)P2 sensor with Rab7-Q67L-targeting. Note the transient signal increase after AngII stimulation only in cells treated with OSW1.

(E) This increase is barely detectable in the PI4K2A K/O cells.

Data information: (B, D, E) Means ± S.E.M. are shown from three experiments performed in triplicates. The values in (D) and (E) were measured in the same 96-well plate at the same time.
Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKMH1 membrane cycling during autophagosome-lysosome fusion. (2019)
Figure 5

Figure 5. PIP5Kγ is the primary source of PI(4,5)P2 in late endosomes and LC3 accumulation detected in PI4K2A K/O cells.

(A) BRET experiment in parental HEK293-AT1 cells in OSW1-pretreated cells after RNAi mediated knock-down of PIP5Kβ or PIP5Kγ. The BRET experiment was performed the same way as described in the legend to Fig.4D (means ± S.E.M. from three experiments, each performed in triplicates). Note the reduced response in the PIP5Kγ knock-down cells and the slight reduction in the case of PIP5Kβ knock-down.

(B) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments shown in panel A (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P=0.0241; **P=0.0088).

(C) These reductions are proportional to the reduction in PIP5Kγ levels as assessed by Western blot analysis of cell lysates obtained from cells treated with siRNAs at the same time.

(D) Increased appearance of GFP-LC3 positive vesicles in PI4K2A K/O cells and reduced acidification of the LC3-positive vesicles using the GFP-mCherry-LC3 reporter. Cells were transfected with the indicated constructs for one day and observed by confocal microscopy. Scale bars: 20 µm.

(E) Quantification of the LC3 positive vesicles. (n = 67, 34, 26, 13, 30, 27 for the various groups, respectively from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P = 0.0004 and 0.0012 for #20 and #26 clones relative to control, respectively.

(F) Co-localization analysis using the Pearson coefficient (n = 31, 21 and 14 cells analyzed for the different groups from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P value < 0.0001 for both groups.
Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKMH1 membrane cycling during autophagosome-lysosome fusion. (2019)
Figure 6

Figure 6. PIP5Kγ knock-down causes defects in autophagosome-lysosome fusion and tubulation of Rab7 compartments.

(A, B) Distribution of mCherry-GFP-LC3 in HEK293-AT1 cells (without starvation) after treatment with control siRNA (A) or siRNA for PIP5Kγ (PIP5K1C). Scale bars: 20 µm. Note the massive tubulation of the LC3 compartment in some of the knocked-down cells (B, lower images, scale bar: 10 µm).

(C) Comparison of Pearson coefficients from cells treated with control or PIP5Kγ RNAi. For statistical analysis the unpaired t-test was used (n = 50 and 45 cells for control a PIP5Kγ knock down groups, respectively; *** P< 0.0001).

(D) Representative confocal images of live HEK293-AT1 cells expressing GFP-Rab7 treated with control or PIP5Kγ RNAi. Note the tubulation in the PIP5Kγ RNAi-treated cells. Scale bars: 10 µm.
Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKMH1 membrane cycling during autophagosome-lysosome fusion. (2019)
Figure 7

Figure 7. PI(4,5)P2 production releases PLEKHM1 from the Rab7 compartment.

(A) HEK293-AT1 cells were transfected with PLEKHM1-GFP-Rab7 along with CFP-FKBP-PIP5Kγ and iRFP-FRB-Rab7 constructs for one day and examined live by confocal microscopy. Representative cells show reduced PLEKHM1 localization when PIP5Kγ was recruited to the Rab7 endosomes (lower row images).

(B) This response is reduced in PI4K2A K/O cells.

(C) Quantification of these changes by BRET analysis where the full-length PLEKHM1 was fused to Sluc and Venus was fused to Rab7 in the BRET construct. For PIP5Kγ recruitment a mutant dark CFP(W66A)-FKBP-PIP5Kγ was used with an iRFP-FRB-Rab7. BRET values were normalized to those of DMSO treated cells. Comparisons were made for parental (blue) and PI4K2A K/O (green and orange) cells.

(D) Similar BRET analysis where the PLEKHM1 was replaced by either RILP or Vps35 in the BRET construct.

Data information: (C, D) Means ± S.E.M. are shown from three experiments performed in triplicates. (A, B) Scale bars: 20 µm.

Legend to Extended View Figures:
Nonsense-mediated mRNA Decay Involves Two Distinct Upf1-bound Complexes (2018)
Figure 1

Fig. 1: Enrichment analysis accurately describes Upf1-associated proteins.

A-C. Positioning of Upf1, Upf2 and Upf3 in the abundance distribution of all yeast proteins, based on the data compiled by Ho et al., 2018 (A). The distribution of abundance for the proteins identified by mass spectrometry in input (B), and purified samples (C), show the expected bias for proteins present in high copy numbers. Light grey colour highlights the positions of proteins that were only identified in the Upf1-TAP samples (6 replicates) and not in the total extract (3 replicates).

D. Correlation of LTOP2-based protein level estimates in a total yeast protein extract with published protein abundance data (Ho et al, 2018).

E. Distribution of the intensity for the proteins quantified in association with Upf1 (LTOP2 score, log2 scale). Positioning of Upf1 is highlighted as a white rectangle while Upf2 and Upf3 are highlighted in red. The dark grey line indicates the region of intensities corresponding to ribosomal proteins.

F. Distribution of the protein enrichment values for Upf1 associated proteins based on LTOP2 scores and known protein abundance in a total yeast extract. Upf1, Upf2 and Upf3 positions are also highlighted as white rectangles.

G. A combination of the data presented in (E) and (F) as a scatter plot, to see both the amount and enrichment of proteins in Upf1-TAP purified samples. The horizontal axis represents the LTOP2 score, with the vertical axis showing enrichment over input values. Both axes use log2 transformed values.
Nonsense-mediated mRNA Decay Involves Two Distinct Upf1-bound Complexes (2018)
Figure 2

Fig. 2: Purification of NMD factors reveals two distinct complexes containing Upf1.

A-F. Enrichment values for proteins identified in tagged Upf1 (A), Upf2 (B), Upf3 (C), Nmd4 (D), Ebs1 (E), and Dcp1 (F) purifications. Tagged proteins are indicated in bold for each experiment. Black bars correspond to average enrichment values obtained in purifications done without RNase and orange bars with an RNase A and RNase T1 treatment. Error bars represent SD. Dots represent individual enrichment values for each protein in the 6 replicates for Upf1-TAP, 3 for Upf1-TAP and RNase, 6 for Upf2-TAP, 3 for Upf2-TAP and RNase, 5 for Upf3-TAP, 3 for Upf3-TAP and RNase, 4 for Nmd4-TAP, 3 for Nmd4-TAP and RNase, 3 for Ebs1-TAP, with and without RNase, and 3 for Dcp1-TAP with or without RNase. Only proteins enriched by a factor of 16 or more in one of the purifications presented here are shown. For clarity, groups of related proteins were combined (the group of Lsm1 to Lsm7 is marked as "Lsm1-7" and the values correspond to the mean of all the values found in purifications; CK2 group corresponds to Cka1, Cka2, Ckb1 and Ckb2).

G. Immunoblot validation of Upf1-TAP interactions with HA tagged Nmd4 and Ebs1. Control strains did not express Upf1-TAP.

H. Immunoblot validation of Upf1-TAP interaction with Pat1-HA, Lsm1-HA and Edc3-HA. The purification was performed with a mix of three strains, expressing Upf1-TAP or not (control).

I. Representation of binary interactions identified by our experiments: dashed lines correspond to interactions that are sensitive to RNase treatment; the arrows start at the tagged protein and indicate the enriched factor.
Nonsense-mediated mRNA Decay Involves Two Distinct Upf1-bound Complexes (2018)
Figure 3

Fig. 3: Nmd4 and Ebs1 are the only Upf1-decapping components that interact with Upf1 independent of the N-terminal CH domain.

A. Upf1 fragments tested: FL is for the full-length protein; HD-Cter for the region containing the helicase domain and the C-terminal part of Upf1 (208-971); CH for the N-terminal domain (2-208) that contains the N-terminal unstructured region and the Cysteine-Histidine rich domain; CH-HD for a version a full-length Upf1 lacking the C-terminal region 854-971; and HD for the helicase domain of Upf1 alone (not to scale). N-terminal TAP-tagged Upf1 versions were expressed from a single copy vector under an inducible tetO7 promoter.

B-D. Results of purifications using Upf1-FL (B), Upf1-CH (C) and Upf1-Cter (D) as bait with the x-axis showing the average enrichment value (log2 scale) for Upf1-decapping and Upf1-23 components. The colour of the bars illustrates the treatment used during the purification, black without RNase, and orange with an RNase treatment. Error bars represent SD. White dots correspond to enrichment values obtained in individual experiments.

E. Comparison of the enrichment of Upf1, Nmd4 and Ebs1 in the purification of the Upf1 fragments. The Upf1 C-terminal region (854-971) affected the association with Nmd4 or Ebs1 (Student t-test with a one-sided alternative hypothesis). We compared the enrichment of each of Ebs1 and Nmd4 between purifications of Upf1 fragments having this region (Upf1-FL and Upf1-HD-Cter, 6 experiments) and purifications of Upf1 fragments lacking the extension (Upf1-CH-HD and Upf1-HD, 4 experiments). The C-terminal region had no effect on Nmd4 enrichment (p-value ≈ 0.98), whereas there was significantly less associated Ebs1 on this small region (p-value ≈ 0.0013).

F. Western blot of input and eluates of Upf1 domains purification in a Nmd4-HA strain. The band with the # might corresponds to a dimer of Upf1-CH, bands marked with a star correspond to residual signal with the anti-HA antibodies (Nmd4). Fragments in the eluate have a smaller size because the protein A part of the tag was removed by digestion with the TEV protease. G6PDH served as a loading control in the input samples.
Nonsense-mediated mRNA Decay Involves Two Distinct Upf1-bound Complexes (2018)
Figure 4

Fig. 4: Nmd4 interaction with the Upf1-decapping complex is direct and mediated by Upf1.

A. Average enrichment of Upf1-23 and Upf1-decapping components in purified Upf1-TAP in the presence (black bars) and absence (blue bars) of NMD4. Pink and dark blue vertical lines highlight proteins of the Upf1-23 and Upf1-decapping complexes respectively. White dots correspond to individual enrichment values obtained in replicate experiments, 6 for Upf1-TAP and 3 for the nmd4Δ condition. Error bars correspond to SD.

B. Average enrichment of Upf1-decapping components in Nmd4-TAP in the presence (black bars) and absence (blue bars) of UPF1. White dots correspond to individual enrichment values obtained in replicate experiments, 4 for Nmd4-TAP and 2 for the upf1Δ condition. Error bars correspond to SD.

C. Schematics of Upf1 fragments used for the in vitro interaction assay; yUpf1-HD is the helicase domain (220-851) of the yeast Upf1 protein, hUpf1-HD is the helicase domain (295-914) of human Upf1 (not to scale).

D. CBP-Nmd4 was mixed with hUPF1-HD or yUPF1-HD (all the proteins overexpressed in E. coli and purified). Protein mixtures before (input, 20% of total) or after purification on calmodulin affinity beads were separated on 10% SDS-PAGE (w/v) acrylamide gels.

E. Upf1 interaction with a 30-mer 3' biotinylated RNA fragment was tested by mixing purified Upf1 helicase domain with Nmd4 and testing the fraction of recovered protein on streptavidin beads after washes with 150 mM and 300 mM NaCl containing buffer solution.
Nonsense-mediated mRNA Decay Involves Two Distinct Upf1-bound Complexes (2018)
Figure 5

Fig. 5: Nmd4 and Ebs1 are essential for NMD elicited by the overexpression of the helicase domain of Upf1.

A. Schematic representation of the domain structure of Nmd4 and Ebs1 from S. cerevisiae compared with human (h) Smg6, Smg5 and Smg7. 14-3-3, HHR and PIN domains were defined based on literature data (Fukuhara et al, 2005; Luke et al, 2007). Boundaries of the PIN domains were chosen based on a Mafft alignment of Smg5/6 of several species, while for Nmd4 the entire protein sequence was used. Drawing is not to scale. Identity percentages among the different domains of Smg proteins, Nmd4 and Ebs1 are indicated. Values represent percent of identical residues as a fraction of all the aligned residues.

B. Scatter plot of the mean fold change of transcripts in nmd4∆ RNAseq experiment against transcript mean fold change in ebs1∆ (Pearson correlation, r = 0.66, p-value < 2.10-16). Black dots correspond to NMD substrate examples for which individual traces are shown in Fig. EV3. Dashed lines represent boundaries of five bins of equal numbers of transcripts (2046 transcripts or notable features per bin), with bin 5 containing the transcripts that were most increased in the mutant condition compared with wild type.

C. Percentage of transcripts affected by UPF1 deletion (increase by at least 1.4 fold) among nmd4∆ bins, as defined in B. Differences between percentages of upf1∆ affected transcripts in bin 4 and 5 and bin 3 and 4 were significant (binomial test, p<10-24).

D. Same as in C, for ebs1∆. Differences between distribution of transcripts in bin n and bin n+1 were significant (binomial test, p<10-9).

E. NMD efficiency of WT, upf1∆ or double mutant strains complemented with Upf1-FL, Upf1-HD-Cter or an empty plasmid. The NMD efficiency for each strain is based on reverse-transcription followed by quantitative PCR for RPL28 pre-mRNA. A wild-type strain has 100% NMD efficiency and a upf1∆ strain has 0% NMD efficiency, by definition. Each value is an average of replicate experiments, 3 for the WT condition, 5 for upf1Δ, 3 for ufp1Δ/upf2Δ, upf1Δ/ufp3Δ and upf1Δ/ebs1Δ and 4 for the upf1Δ/nmd4Δ condition. Error bars represent SD. Dots represent values obtained in the various replicate experiments.

F. Schematic drawing of an NMD reporter whose transcription can be repressed by doxycycline addition to the culture.

G. Changes in the steady-state level of the reporter encoded HA-tagged protein in upf1Δ, nmd4Δ, ebs1Δ and the nmd4Δ/ebs1Δ strains were estimated by immunoblot from three independent experiments, with one example shown. H. RNA decay in a wild-type and nmd4Δ/ebs1Δ strains was tested by reverse-transcription quantitative PCR specific to the 5' end of the reporter RNA at different times after transcription shut-off. The quantifications represent mean RNA amounts and standard deviation of the average (three independent experiments).
Nonsense-mediated mRNA Decay Involves Two Distinct Upf1-bound Complexes (2018)
Figure 6

Fig. 6: Upf2 and Upf3 function as a heterodimer in the formation of Upf1-23.

A-B. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf1-TAP purified samples in the presence (grey bars) or absence (blue bars) of UPF2 (A) or UPF3 (B). Error bars represent SD. White dots represent enrichment values for individual replicates, 6 for Upf1-TAP, 3 for the upf2Δ and upf3Δ conditions.

C-D. Evaluation of the effects of UPF2 deletion on Upf3-TAP levels (C) and of UPF3 deletion on Upf2-TAP levels (D) in total extracts, in comparison with a loading control (G6PDH) was done by immunoblot.

E. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf2-TAP purified samples in the presence (grey bars, 6 experiments) or absence (blue bars, 2 experiments) of UPF1. Error bars represent SD. The levels of expression of Upf2-TAP and Upf3-TAP proteins have been verified in this condition (Appendix Fig. S2).
Nonsense-mediated mRNA Decay Involves Two Distinct Upf1-bound Complexes (2018)
Figure 7

Fig. 7: Association of Nmd4 to NMD substrates depends on Upf1-23.

A. Unspliced RPL28, an NMD substrate, was enriched in Nmd4-TAP purification in comparison with a control untagged strain, as measured by reverse transcription followed by quantitative PCR. Bars correspond to the mean of pre-RPL28 enrichment for 3 independent experiments, as compared with RIM1, a non-NMD mRNA. Error bars correspond to SD. The indicated p-value was computed using the Welch t-test, single-sided comparison.

B-D. Distribution of Nmd4-TAP, Nog1 (marker of free 60S subunits) and Rps8 (marker of 40S, 80S and polysome fractions) in wild type (B), upf1∆ (C) and upf2∆ (D) strains, tested by immunoblot. Fractions 1, 2, 3 are light fractions, fractions 4, 5 and 6 correspond respectively to ribosomal 40S, 60S and 80S positions, fractions 7 to 12 correspond to polysomes. The percent of total signal for Nmd4-TAP in three independent replicates along with standard deviation of the values are indicated for each fraction.

E. Quantified relative changes in Nmd4-TAP average levels in light and monosome/polysome fractions in upf1∆ and upf2∆ strains (100% correspond to levels in the wild type strain). Indicated p-values correspond to the Welch t-test, single-sided comparison of 4 replicate experiments (individual values are indicated as dots). Error bars correspond to SD.

F. Summary of the Upf1 domains interaction with Upf1-23 and Upf1-decapping components, based on the data presented in this work.

G. Proposed Upf1-23/Upf1-decapping sequence of events for yeast NMD.

Expanded view Figure legends
A lysosome‐to‐nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB (2012)
Figure 1

Lysosomal stress induces TFEB nuclear translocation. (A) Immunofluorescence of HEK‐293T cells that express TFEB–3 × FLAG, subjected to the indicated treatments and stained with antibodies against FLAG and the lysosomal marker LAMP2. The FLAG and LAMP2 channels are in green and red, respectively, in the merge. DAPI (blue) is included in the merge. Scale bars represent 10 μm. (B) Quantification of the number of cells with nuclear TFEB–3 × FLAG in the four conditions in (A). Each value represents mean±s.d. from three independent fields with N=300. (C) Immunofluorescence of HEK‐293T cells treated as indicated and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μm. (D) Immunoblotting of proteins extracted from HeLa cells that express TFEB–3 × FLAG treated with DMSO, chloroquine (CQ) or SalA, subjected to nuclear/cytosolic fractionation and blotted with antibody against FLAG to detect TFEB. H3 and tubulin were used as nuclear and cytosolic markers, respectively. Blots are representative of triplicate experiments.
A lysosome‐to‐nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB (2012)
Figure 3

mTORC1 phosphorylates TFEB at serine 142 (S142). (A) Torin 1 induces S142 dephosphorylation. HeLa cells were treated as indicated and total and nuclear extracts were probed with a TFEB p‐S142 phosphoantibody and with anti‐FLAG antibody. Disappearance of TFEB S142 phosphorylation upon starvation or Torin 1 treatment correlates with accumulation of TFEB in the nuclear fraction. (B) mTORC1 in‐vitro kinase assays. Highly purified FLAG–S6K1, TFEB–3 × FLAG, or TFEBS142A–3 × FLAG were incubated with radiolabelled ATP without kinase, with purified mTORC1 or with mTORC1+Torin 1, and analysed by autoradiography. The lower panel shows a FLAG immunoblot of the substrates. (C) Schematic representation of TFEB protein structure with the predicted mTORC1 phosphorylation sites and their conservation among vertebrates (for mTORC1 phophosite prediction see Material and methods). Numbering is according to human isoform 1. (D) Sequence conservation scores of the phosphosites and quantitative agreement between mTOR consensus motif and the sequence around the phosphosites of TFEB. (E) S142 and S211 regulate TFEB localization. FLAG immunostaining (red) of HeLa cells expressing serine‐to‐alanine mutated versions of TFEB–3 × FLAG. Nuclei were stained with DAPI (blue). Values are means of five fields containing at least 50 transfected cells. Student's t‐test (unpaired) ***P0.001. Scale bars represent 30 μm.