EMBO/soda-nel huggingface dataset:
PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex (2013)
figf5

(a) UVRAG knockdown impairs the interaction of the RINT-1–ZW10 complex with COPI in an autophagy-independent manner. Cells (HEK293T) were transfected with control shRNA, UVRAG shRNA or Atg16L1 shRNA for 72 h. Whole-cell lysates (WCLs) were used for immunoprecipitation with anti-RINT-1 (first and third panels), anti-ZW10 (second panel), or anti-UVRAG (fourth panel), followed by immunoblotting with the indicated antibodies. Input represents 10% whole-cell lysates. Note that
β′-COP interaction was suppressed by UVRAG knockdown but unaffected by Atg16L1knockdown. See Supplementary Fig. S9 for uncropped data. (b) Retrograde transport of tsO45-VSVG–KDELR–YFP from the Golgi to the ER is inhibited by UVRAG knockdown. HeLa cells serially transfected with UVRAG shRNA or control shRNA, then with VSVG–KDELR–YFP expression vector, were incubated at 32 °C (permissive condition) overnight. On shifting to 40 °C (non-permissive condition), the ER-like distribution pattern of VSVG–KDELR–YFP was registered. Representative confocal images of the distribution pattern of VSVG–KDELR at 40 °C and its co-localization with PDI are shown (upper panel). Data are mean±s.d.,
n = 100 cells obtained from three independent experiments;
**P0.01 (bottom panel). Scale bars, 10 μm. (c) UVRAG interaction with both PtdIns(3)P and RINT-1 is required for COPI-dependent retrograde transport, as shown by the redistribution of VSVG–KDELR from the Golgi to the ER. HeLa cells were transfected with control shRNA or UVRAG shRNA. The UVRAG-depleted cells were then complemented with empty vector, Flag–UVRAG, UVRAGΔ270−442, UVRAGK78A/R82A or UVRAGΔC2, along with the transfection of VSVG–KDELR–YFP. The ER pattern for the chimaeric KDELR was quantified (left panel). Data are the mean±s.d.;
n = 200 cells obtained from three independent experiments;
*P0.05;
**P0.01. Endogenous and reconstituted UVRAG expression was confirmed by immunoblotting (right panel). (d) PtdIns(3)P depletion inhibits Golgi-to-ER retrograde transport of VSVG–KDELR. HeLa cells were transfected with empty vector, wild-type MTMR3, the C413S mutant of MTMR3, or treated with wortmannin or rapamycin, and then the ER pattern of VSVG–KDELR was quantified. UT, untreated. Data are the mean±s.d.;
n = 100 cells for each group obtained from three independent experiments;
**P0.01. (e) Interaction between endogenous UVRAG and the ER tethering complex under wortmannin treatment. WCLs of HEK293T cells treated with wortmannin were used for immunoprecipitation with anti-UVRAG or anti-RINT-1 antibody, followed by immunoblotting with the indicated antibodies. The input panel shows endogenous protein expression. See Supplementary Fig. S9 for uncropped data.
PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex (2013)
figf6

(a) Golgi morphology on UVRAG depletion. HeLa cells were transfected with control or UVRAG-specific shRNA for 72 h and subjected to electron microscopy analysis (left panel). When compared with control cells (left), the Golgi structure was swollen and fragmented in cells lacking UVRAG. The level of Golgi fragmentation was quantified (right panel). Data represent mean±s.d.;
n = 50 cells obtained by gathering data from two independent experiments;
*P0.05. Scale bars, 500 nm. (b) Depletion of UVRAG leads to cis-Golgi dispersion. HeLa cells were transfected with control shRNA or UVRAG-specific shRNA expressing GFP as an expression marker and then stained for GM130 (left panel; red; highlighted by arrows). Nuclei were stained with DAPI (blue). The dispersed distribution of GM130 in shRNA-transfected cells was quantified (middle panel). Data are the mean±s.d.;
n = 500 cells obtained from five independent experiments. The expression of UVRAG and GM130 in treated cells is also shown (right panel). Scale bars, 10 μm. (c) The effect of UVRAG on membrane trafficking from the ER to the Golgi. HeLa cells serially transfected with UVRAG shRNA RINT-1 shRNA or control shRNA, then with VSVG–GFP expression vector, were incubated at 40 °C (non-permissive condition) overnight. The cells were then shifted to 32 °C (permissive condition) for 30 min to allow cargo transit from the ER to the Golgi. Before and after the temperature shift, the cells were fixed and labelled with anti-PDI and anti-GM130 antibodies to define the ER and Golgi region, respectively. Representative confocal microscopy images of the distribution pattern of the retrograde cargo at 40 °C and 32 °C are shown (left panel). The Golgi-like distribution pattern of VSVG–GFP was registered (right panel). Data are mean±s.d.;
n = 100 cells from three independent experiments;
**P0.01. Scale bars, 10 μm. (d,e) UVRAG interaction with PtdIns(3)P and RINT-1 is required or Golgi integrity. (d) HeLa cells were transfected with control shRNA (first row) or UVRAG shRNA (second to sixth rows). The UVRAG-depleted cells, as indicated by GFP expression, were then transfected with empty vector (second row), Flag–UVRAG (third row), UVRAGΔ270−442 mutant (fourth row), UVRAGK78A/R82A mutant (fifth row), or UVRAGΔCCD mutant (sixth row), followed by confocal microscopy using anti-GM130 (red) for
cis-Golgi staining, anti-Flag (purple) for the ectopically expressed UVRAG protein expression, and DAPI for nuclei (blue). Asterisks indicate shRNA-transfected cells and arrows denote the cells reconstituted with UVRAG (wild-type or mutant) expression. (e) The dispersed distribution of GM130 was quantified, and data represent the mean±s.d.;
n = 200 cells from four independent experiments;
*P0.05;
**P0.01.
PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex (2013)
figf7

(a,b) Differential interaction of UVRAG with the RINT-1–ZW10–NAG tethering complex and the beclin-1–Bif-1–PI(3)KC3 autophagy complex after treatment with rapamycin (a) and HBSS (b). Cells (HEK293T) treated with rapamycin (50 nM) in a or with HBSS in b were subjected to immunoprecipitation with anti-UVRAG (first panel), anti-RINT-1 (second panel) or anti-PI(3)KC3 (third panel), followed by immunoblotting with the indicated antibodies. Input (10%) shows endogenous protein expression. See Supplementary Fig. S9 for uncropped data. (c) Gel filtration analysis of UVRAG complex formation under normal conditions (untreated, UT) and after rapamycin-induced autophagy (50 nM). Affinity-purified UVRAG complexes from HCT116 cells stably expressing Flag–UVRAG were fractionated by Superose-6 gel filtration column and the eluates were analysed by western blotting for UVRAG, RINT-1, ZW10, beclin 1, PI(3)KC3 and Bif-1 proteins. Whole-cell lysates (2.5%) were used as the input (lane-1). The elution profile of each protein was quantified by densitometry analysis and normalized. Relative densitometry units were plotted against fraction number. Black arrows indicate the positions of the molecular weight size markers. Red arrows indicate the peak shift of the UVRAG eluates.
PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex (2013)
figf8

(a–c) UVRAG interaction with beclin 1 and PtdIns(3)P, but not with RINT-1, is required for autophagy-induced Atg9 dispersal. (a) HeLa cells were stably transfected with control shRNA (first row) or
UVRAG shRNA (second–sixth rows). The UVRAG-depleted cells were then transfected with empty vector (second row), Flag–UVRAG (third row), UVRAGΔ270−442 mutant (fourth row), UVRAGK78A/R82A mutant (fifth row) or UVRAGΔCCD mutant (sixth row), and incubated in complete medium (untreated, UT, first panel), starvation medium (HBSS 2 h, third panel), or treated with rapamycin (50 nM 2 h, second panel) or SMER28 (50 μM, 2 h, fourth panel). The distribution pattern of endogenous Atg9 (green) in the UVRAG (wild-type or mutant)-transduced cells (red) was analysed by confocal microscopy (a). Asterisks denote condensed (non-dispersed) Atg9 in UVRAG-depleted HeLa cells and arrows denote the cells complemented with wild-type or mutant UVRAG. (b,c) The percentage of cells with Atg9 translocation was quantified (c) and the expression of UVRAG and actin was determined by immunoblotting (b). Data represent the mean±s.d.;
n = 300 cells from three independent experiments; NS, not significant;
**P0.01;
***P0.001. Scale bars, 10 μm. (d) Effect of knockdown of UVRAG, beclin 1 andRINT-1 on the translocation of Atg9 to the LC3-labelled autophagsomes during autophagy. Control-, UVRAG-, BECN1- or
RINT-1-knockdown HeLa cells were incubated in complete medium (normal condition), starvation medium (HBSS, 2 h), or treated with rapamycin (100 nM, 2 h) or SMER28 (50 μM, 2 h). The cells were then stained for endogenous Atg9 (green) and LC3 (red) followed by confocal microscopy analysis (top and bottom left panels). The percentage of Atg9 vesicles co-localized with LC3+-autophagic puncta was quantified (bottom middle panel). Endogenous UVRAG, beclin 1 and RINT-1 protein expression are shown (bottom right). Data represent mean±s.d.;
n = 200 cells obtained from three independent experiments. NS, not significant;
*P0.05;
**P0.01;
***P0.001. Scale bars, 10 μm.
FAM111 protease activity undermines cellular fitness and is amplified by gain-of-function mutations in human disease (2020)
Figure 1

Figure 1. Human FAM111A and FAM111B are active proteases.

A. Domain organization of human FAM111A and FAM111B proteins.

B. Alignment of the serine protease domains in human FAM111A, human FAM111B and E. coli DegS. Red boxes denote fully conserved residues; yellow boxes indicate conservative amino acid substitutions. Purple stars indicate catalytic triad residues.

C. Crystal structure of monomeric E. coli DegS protease (PDB ID: 2R3U). Zoomed-in view shows position of catalytic triad residues (yellow).

D. Homology-based protease domain model of human FAM111A (residues 371-555; teal). Zoomed-in view shows catalytic triad residues (yellow). Residues mutated in human disease (red) are indicated.

E. Homology-based protease domain model of human FAM111B (residues 471-664; blue). Zoomed-in view shows catalytic triad residues (yellow). Residues mutated in human disease (red) are indicated.

F. Overlay of the DegS, FAM111A and FAM111B protease domains in (C-E).

G. Purified recombinant FLAG-FAM111A proteins were incubated at indicated temperatures for 4 h, and FAM111A auto-proteolytic activity was analyzed by immunoblotting with FLAG antibody.

H. As in (G), using recombinant human FLAG-FAM111B proteins.

I. U2OS cell lines conditionally expressing indicated GFP-FAM111A alleles were fixed at the indicated times after treatment with Doxycycline (DOX) to induce expression of the transgenes and stained with crystal violet. Levels of stably expressed GFP-FAM111A proteins are shown in Figure 2A.

Data information: Data are representative of at least three (G-I) independent experiments with similar outcomes.
FAM111 protease activity undermines cellular fitness and is amplified by gain-of-function mutations in human disease (2020)
Figure 2

Figure 2. FAM111A proteolytic activity displaces RFC from chromatin and inhibits DNA replication.

A. Immunoblot analysis of stable U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of WT or mutant forms of GFP-FAM111A.

B. DNA replication rates in U2OS/GFP-FAM111A cells treated with DOX for the indicated times, pulse-labeled with EdU and stained with DAPI were analyzed by quantifying EdU signal intensity in S phase cells using quantitative image-based cytometry (QIBC) (red bars, mean (A.U., arbitrary units); n>2000 cells per condition). See also Appendix Figure S6A.

C. Cells treated as in (B) were pre-extracted, fixed and stained with PCNA or MCM2 antibody, and subsequently analyzed by QIBC (n>2000 cells per condition).

D-F. Quantification of data in (C) (red bars, mean). Cells in S phase were identified based on EdU positivity. See also Appendix Figure S6B-D.

G. Analysis of FAM111A interactors. U2OS/GFP-FAM111A WT cells were treated or not with DOX for 4 h, subjected to GFP immunoprecipitation (IP) and analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (+DOX/-DOX ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR<0.05; s0=1).

H. U2OS or U2OS/∆FAM111A cells were subjected to IP with IgG (control) or RFC1 antibody followed by immunoblotting with indicated antibodies.

I. U2OS cells transfected with empty vector (EV) or indicated RFC subunit expression plasmids were subjected to FLAG IP and immunoblotted with indicated antibodies.

J. As in (C), except that cells were stained with RFC1 antibody (n>2000 cells per condition).

K. Quantification of data in (J) for S phase (EdU-positive) cells (red bars, mean).

Data information: Data are representative of at least three (A-F,H-K) and two (G) independent experiments with similar outcomes.
FAM111 protease activity undermines cellular fitness and is amplified by gain-of-function mutations in human disease (2020)
Figure 3

Figure 3. FAM111A protease activity suppresses transcription and triggers Caspase-dependent apoptosis.

A. Immunoblot analysis of U2OS/GFP-FAM111A WT cells treated with DOX for the indicated times.

B. Representative images of U2OS/GFP-FAM111A cell lines that were treated or not with DOX, fixed and stained with γ-H2AX antibody and DAPI.

C. Cells in (B) were subjected to QIBC analysis of γ-H2AX signal intensity (red bars, mean (A.U., arbitrary units); n>2000 cells per condition). See also Appendix Figure S6E.

D. Immunoblot analysis of U2OS/GFP-FAM111A WT cells treated or not with DOX and the pan-Caspase inhibitor Z-VAD-FMK as indicated.

E-G. Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX for the indicated times, pulse-labeled with EU and stained with DAPI were analyzed by QIBC (red bars, mean; n>2000 cells per condition). See also Figure EV3I for the impact of the FAM111A *PIP mutant and Appendix Figure S6H.

H. U2OS/GFP-FAM111A cell lines treated or not with DOX were pre-extracted, fixed and stained with RPB1 antibody, and analyzed by QIBC (n>2000 cells per condition).

I. Quantification of data in (H) (red bars, mean). See also Appendix Figure S6I.

J. Representative images from the experiment in (H). Scale bars, 10 μm.

Data information: Data (A-J) are representative of at least three independent experiments with similar outcomes.
FAM111 protease activity undermines cellular fitness and is amplified by gain-of-function mutations in human disease (2020)
Figure 4

Figure 4. Patient-associated mutations hyperactivate FAM111A protease activity to exacerbate its adverse impact on cellular fitness.

A. Overview of heterozygous FAM111A mutations found in patients with gracile bone dysplasia or Kenny-Caffey syndrome.

B. Immunoblot analysis of U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of the indicated GFP-FAM111A alleles. U2OS/GFP-FAM111A WT (low) cells express the transgene at a lower level than U2OS/GFP-FAM111A WT cells used in Figure 1 and 2 (see Figure EV4A).

C. U2OS/GFP-FAM111A cell lines treated or not with DOX, pulse-labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC.

D. Quantification of data in (C) for S phase (EdU-positive) cells (red bars, mean (A.U., arbitrary units); n>2000 cells per condition). See also Appendix Figure S6J.

E. As in (C), except that cells were pulse-labeled with EU.

F. Quantification of EU incorporation in cells in (E) (red bars, mean; n>2000 cells per condition). See also Appendix Figure S6L.

G. U2OS/GFP-FAM111A cell lines that were treated or not with DOX were stained with RFC1 antibody, pre-extracted and fixed, and stained with DAPI. RFC1 and DAPI signal intensities were analyzed by QIBC.

H. As in (G), except that cells were stained with RPB1 antibody.

I. Immunoblot analysis of U2OS/GFP-FAM111A cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK.

J. As in (I), using U2OS cell lines conditionally expressing ectopic untagged FAM111A alleles.

K. Purified recombinant FLAG-FAM111A proteins were incubated at indicated temperatures for 4 h, and FAM111A auto-proteolytic activity was analyzed by immunoblotting.

L. GFP IPs from U2OS cell lines expressing GFP-FAM111A WT or D528G mutant were analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (WT/D528G ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR<0.05; s0=1).

Data information: Data are representative of at least three (B-K) and two (L) independent experiments with similar outcomes.
FAM111 protease activity undermines cellular fitness and is amplified by gain-of-function mutations in human disease (2020)
Figure 5

Figure 5. Disease-associated FAM111B mutants have amplified proteolytic activity and phenocopy FAM111A-induced cellular phenotypes.

A. Domain organization of human FAM111B and overview of disease-associated heterozygous missense mutations.

B. U2OS/GFP-FAM111A WT cells treated or not with DOX were subjected to GFP IP followed by immunoblotting with indicated antibodies.

C. As in (B), using U2OS/GFP-FAM111B WT cells.

D. Purified recombinant FLAG-FAM111A and FLAG-FAM111B proteins were incubated separately or mixed, subjected to IP with FAM111A antibody and immunoblotted with FLAG antibody.

E. Immunoblot analysis of U2OS/GFP-FAM111B cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK.

F. Cells in (E) were pulse-labeled with EdU, stained with DAPI and analyzed for DAPI and EdU signal intensity using QIBC.

G. Quantification of data in (F) for S phase (EdU-positive) cells (red bars, mean (A.U., arbitrary units); n>2000 cells per condition). See also Appendix Figure S6N.

H. As in (F), except that cells were pulse-labeled with EU.

I. Quantification of EU incorporation in cells in (H) (red bars, mean; n>2000 cells per condition).

J. U2OS/GFP-FAM111B cell lines (Figure 5E) were fixed at the indicated times after DOX treatment and stained with crystal violet.

K. Cells in (E) were fixed, immunostained with γ-H2AX antibody and subjected to QIBC analysis of γ-H2AX signal intensity (red bars, mean; n>2000 cells per condition). See also Appendix Figure S6O.

L. Immunoblot analysis of purified recombinant FLAG-FAM111B proteins and their auto-cleavage products.

Data information: Data (B-L) are representative of at least three independent experiments with similar outcomes.

Expanded View Figure legends
ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants (2014)
f1

Confocal images of the GFP fluorescent patterns in seedlings expressing ATG5-GFP or GFP-ATG8. (a,b) Control conditions: cytosolic signal associated with rare fluorescent puncta. (c,d) High increase of fluorescent puncta after 2 h of nutrient starvation. Note ring-like ATG5 structures in c. (e,f) Wortmannin treatment blocks the occurrence of fluorescent ATG5 or ATG8 structures. (g,h) Maximum intensity projections of vacuolar focal planes, allowing visualization of autophagic bodies after 4 h of nutrient starvation in the presence of concanamycin A. Vacuolar ATG5-GFP fluorescence is not detected (g), while GFP-ATG8 signals are abundant (h). An inset in g, corresponding to a cortical focal plane, shows that formation of cytosolic ATG5 structures in the cytosol is not inhibited by concanamycin A treatment. Maximum intensity projections of 10-μm-deep deconvolved stacks, of cortical (a–f, and inset in g) or vacuolar focal planes (g,h). Scale bar, 10 μm.
ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants (2014)
f2

(a) Differential interference contrast micrographs of root epidermal cells incubated for 8 h with concanamycin A, in nutrient starvation conditions. Autophagic bodies accumulation in the vacuole is clearly visible in wild-type seedlings, absent in the atg5-1 mutant and restored in atg5-1 seedlings expressing ATG5-GFP. (b) Confocal microscopy. The ATG5-GFP fluorescence pattern in the atg5-1 background is identical to that observed in wild-type seedling, including ring-like structures. Scale bar, 15 μm.
ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants (2014)
f3

(a) Dual observation of ATG5-GFP and mCh-ATG8 under nutrient starvation reveals a partial colocalization of the two reporters. Merge of the GFP (green) and ATG8 (magenta) channels of maximum intensity projections of 20-μm-deep deconvolved stacks. Scale bar, 3 μm. (b) Real-time imaging of an individual autophagosomal structure, showing one deconvolved image per 20 s. The ATG5 ring formation phase is defined as the steps up to establishment of the maximum diameter of the ring structure. The following steps, defined as the decay phase, being more dynamic, are depicted also at higher time resolution. Single-plane merged images of ATG5-GFP (green) and mCh-ATG8 (magenta) channels, deconvolved. Scale bar, 1 μm. (c) Representative FRAP experiment on mCh-ATG8 during the ring formation phase. Deconvolved images. Scale bar, 1 μm. The corresponding fluorescence recovery kinetic, measured on non-deconvolved images, is shown on the right. (d) Representative FRAP experiment of mCh-ATG8 during the ring decay phase, presented as in c. (e) Box and whiskers representations of fluorescence recoveries during ATG5 ring formation (n=11) and decay (n=17). Whiskers comprise the 10–90 percentile, the box the two central quartiles. The mean recovery values are indicated by an horizontal line. Recoveries during ATG5 ring formation and ring decay were significantly different according to the Mann–Whitney statistical test (***P0.001).
ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants (2014)
f6

(a) Correlative light-electron microscopy of GFP-ATG8-labelled autophagosomal structures. Correlation of a TEM micrograph of a root transverse section (1) with fluorescent imaging of GFP-ATG8 (arrowheads, 2) confirms the association of ATG8 signal with autophagosome (3). A closer view shows close contacts with ER-like membranes (empty arrowheads, 4). (b) Immunogold labelling of GFP-ATG8 on cryosections, employing an anti-GFP antibody (arrowheads). Similar interactions between ER membranes and autophagosomal structures can be detected either on cryofixed (samples c) or after chemical fixation (d). Empty arrowheads: connections with ER-like membranes. Scale bars, 5 μm in a(1–3); 0.5 μm in a(4) and b(1–3).
Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis (2016)
Figure 1

Figure 1. TLR2 inhibitor peptide co-localized with its corresponding TLR.

(A-C) Representative images of Cellular localization of TLR2-p and scrTLR2-p peptides in BM-derived macrophages were observed using confocal microscopy. (A) Cells were probed with anti TLR2 antibody following by staining with APC-labeled secondary antibody or (B) with TLR1 antibody following by staining with FITC-labeled secondary antibody (left panels, red). For membrane staining, cells were labelled with lipid dye, DiD (C). Then rhodamine-labeled fluorescent peptide was added to the cells (middle panels, green). Merged images are shown in the right panel. Scale bars 10µm. The mean Pearson correlation coefficient observed of TLR2-p or scrTLR2-p peptides with TLR2 TLR1 and DiD presents as mean of 2 independent experiments ± S.EM (n=19-61).