EMBO/soda-nel huggingface dataset:
Intrinsic lipid-binding activity of ATG16L1 supports efficient membrane anchoring and autophagy (2019)
Figure 1

Figure 1 - ATG16L1 membrane targeting activity is retained in the absence of ATG5 or WIPI2.

A. Analyses of protein expression in lysates of various cell lines by western blotting against the indicated antibodies.

B. Fluorescence analyses of GFP-ATG5 or ATG16L1-GFP stably expressed in ATG5-/- or ATG16L1-/-. Cells were amino acid starved for 2 hr prior to fixation and imaging of the GFP fluorescence. Scale bar: 10 μm.

C. Assessment of ATG16L1 levels in the cytosolic (C) and membrane (M) fractions in lysates of the indicated cell lines using western blot analyses and antibodies against ATG16L1. ATG16L1-/- and ATG5-/- stably expressed ATG16L1-GFP while ATG3-/- stably expressed Flag-S-ATG16L1. Antibodies against α-tubulin and integrin β1 were used as controls for fractionation. Exogenous (exo) ATG16L1 was detected using antibodies against ATG16L1.

D. Lack of WIPI2 expression is confirmed by western blot analyses in wild type MEFs (WIPI2+/+) and WIPI2-/- cells.

E. Immunofluorescence analyses of ATG16L1-/- and WIPI2-/- stably expressing Flag-S-ATG16L1. Cells were amino acid starved (AA starve) for 2 hr in the presence or absence of 3'MA (and additional pretreatment for 30 min) followed by fixation and immuno-staining using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm. Right panel represents quantification of 3 independent experiments and error bars depicting SEM values. * P ≤ 0.05, ** P ≤ 0.01 (pairwise unpaired Student's t-test).
Intrinsic lipid-binding activity of ATG16L1 supports efficient membrane anchoring and autophagy (2019)
Figure 2

Figure 2 - PAS targeting activity of ATG16L1 lies within its CCD.

A. Schematic presentation of ATG16L1 fragments and mutants used in this study. The following fragments encompassed the indicated amino acids: Wild type (WT): 1-623; ∆1: 39-632; ∆2: 120-623; ∆3: 206-623 and ∆4: 336-623. Mutant ∆2∆182-205 consists of the ∆2 fragment with an additional deletion in amino acids 182-205. All constructs contained a Flag-S tag at the N-terminal end.

B. Fragments depicted in (A) were expressed in U2OS cells and amino acid starved for 5 hr followed by fixation and immunostaining against S tag to detect S-ATG16L1. Scale bar: 10 μm.

C. U2OS cells expressing Flag-ATG16L1∆2 or Flag-ATG16L1∆2∆182-205 were treated as in (B) and stained using antibodies against Flag tag (to detect ATG16L1, green) and FIP200 (red). Scale bar: 10 μm.

D. Protein-protein interaction assay in 293T cells transiently transfected with the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.

E. Homodimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.

F. Homodimerisation assay similar to (E).

G. Flag-ATG16L1WT or Flag-ATG16L1∆182-205 were stably expressed in ATG5-/- cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.
Intrinsic lipid-binding activity of ATG16L1 supports efficient membrane anchoring and autophagy (2019)
Figure 3

Figure 3 - ATG16L1 binds liposomes through CCD sequences.

A. Sequence alignment of ATG16L1 CCD segment from various species and ATG16L2 (Sc: Saccharomyces cerevisiae; Xt: Xenopus (Silurana) tropicalis; Hs: Homo sapiens; Mm: Mus musculus). Residues that mediate WIPI2b and FIP200 binding are highlighted in orange and magenta, respectively. Cyan-shaded residues (I171, K179 and R193) are exposed conserved residues predicted to not contribute to the dimer-dimer interface and are mutated in this study. Cherry-shaded residues are mutated in Fig 6 and 7.

B. Microscopy-based protein-liposome binding assay. ATG16L1-GFP immobilised on beads incubated in the presence of rhodamine-labelled liposome preparations containing the indicated phosphoinositides. Scale bar: 50 μm.

C. Quantification of relative liposome binding in (B).

D. Structural modelling of ATG16L1 residues 160-205 (magenta helix) in the presence of lipid bilayer (green lines). Highlighted residues include I171, K179 and R193 as sticks, which are mutated in this study. Shown in yellow stick, a PI3P molecule embedded in the lipid bilayer and interacting with some of the positively charged residues of ATG16L1.

E. Microscopy-based protein-liposome binding assay as in (B). ATG16L1WT- and ATG16L1LD-GFP immobilised on beads were incubated with rhodamine-labelled, PI3P-positive liposome preparations. Scale bar: 50 μm. Right panel shows quantification of liposome binding relative to ATG16L1WT from 3 independent experiments including SEM values.

F. Protein-protein interaction assay in 293T cells transiently transfected with the indicated S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.

G. Dimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated S-tagged ATG16L1 constructs and analysed as in (F).

Data information: Quantifications depict means and error bars (SEM) from at least three independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (pairwise unpaired Student's t-test).
Intrinsic lipid-binding activity of ATG16L1 supports efficient membrane anchoring and autophagy (2019)
Figure 4

Figure 4 - Binding of ATG16L1 to lipids is required for PAS recruitment.

A. ATG16L1-/- stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and ATG5 (red). Scale bar: 9 μm. Right panels show quantifications of average number of Flag- and ATG5-positive dots per cell.

B. Cells, as in (A), were immunostained using antibodies against S tag (to detect ATG16L1, red) and WIPI2 (green). Scale bar: 9 μm. Lower panel shows quantification of average number of WIPI2 positive dots per cell.

C. Average intensities of individual Flag-ATG16L1 dots in (A). Underlying grey circles represent individual data points. Dots were quantified (n=1225 for ATG16L1WT; n=334 for ATG16L1LD).

D. WIPI2-/- cells reconstituted with the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and FIP200 (red). Scale bar: 9 μm. Lower panel shows quantification of average number of Flag positive dots per puncta-positive cell.

Data information: Quantifications depict means and error bars (SEM) from at least three independent experiments. * P ≤ 0.05, *** P ≤ 0.001, **** P ≤ 0.0001 (pairwise unpaired Student's t-test).
Intrinsic lipid-binding activity of ATG16L1 supports efficient membrane anchoring and autophagy (2019)
Figure 5

Figure 5 - Binding of ATG16L1 to lipids is required for autophagy­.

ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies.

A. Cells were amino acid starved (AA starved) to induce mTORC1-dependent autophagy in the presence or absence of BafA1 for 2 hr prior to lysis.

B. Mitophagy was induced by CCCP (10 μM) treatment for 6 hr prior to lysis.

C. Glucose starvation (Glu starved) for 20 hr was used to induce mTORC1-independent autophagy. BafA1 was added to all conditions 2 hr prior to lysis.

D. LC3-associated phagocytosis (LAP)-like LC3 lipidation was induced by treating cells with CCCP (100 μM) for 2 hr prior to lysis. Right panel shows quantification of LC3-II/LC3-I ratio. Means and error bars (SEM) are shown (n=4). ** P ≤ 0.01 (pairwise unpaired Student's t-test).

E. LAP-like LC3 lipidation induced by monensin or ammonium chloride (NH4Cl) for 2 hr prior to lysis.
Intrinsic lipid-binding activity of ATG16L1 supports efficient membrane anchoring and autophagy (2019)
Figure 6

Figure 6 - Negatively charged residues within the CCD of ATG16L1 weakens its interaction to lipids.

A. ATG16L1 CCD region 164-198 depicted on a heptad repeat with the relevant residues highlighted in red (acidic; D164 and E165), purple (hydrophobic; I171), and blue (basic; K179 and R193).

B. Coomassie gel of recombinant ATG16L172-307 wild type and mutants. * indicates bacterial protein

C. Liposome co-sedimentation assay using recombinant ATG16L1 protein and sonicated liposomes. Supernatant (S) and pellet (P) were analysed by immunoblotting against T7 tag to detect ATG16L1. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel). Error bars (SEM). ** P ≤ 0.01 (unpaired Student's t-test).

D. Liposome co-sedimentation assay, as in (C), using ATG16L172-307LE and sonicated liposomes that contain or lack PI3P. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel) with error bars depicting SEM values. ns P > 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (pairwise unpaired Student's t-test).
Intrinsic lipid-binding activity of ATG16L1 supports efficient membrane anchoring and autophagy (2019)
Figure 7

Figure 7 - Persistent localisation of ATG16L1 to PAS inhibits autophagy.

ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LE-expression constructs and autophagy assessed during amino acid starvation (AA starve) followed by immunoblotting or immunofluorescence analyses.

A. Immunofluorescence analyses of amino acid starved cells (2 hr) using antibodies against WIPI2 (red) or ATG16L1 (green). Scale bar: 9 μm.

B. Cells as in (A) with the addition of 3'MA (and pretreatment with the drug for 30 min). Scale bar: 9 μm.

C. Quantification of percentage of cells positive for ATG16L1 puncta in (A) and (B).

D. Cells were left untreated, amino acid starved for 2 hr, or amino acid starved for 2 hr followed by refeeding with full growth media for 1 hr. Puncta formation was assessed by immunofluorescence staining using antibodies against S tag to detect ATG16L1.­­ Scale bar: 10 μm.

E. Immunoblot analyses of amino acid starved cells in the presence of BafA1 for 2 hr. Cell lysates were analysed using the indicated antibodies.

F. WIPI2-/- cells reconstituted with the indicated ATG16L1 constructs were amino acid starved for 2 hr. BafA1 treatment was included in all conditions for 2 hr prior to lysis. Cell lysates were analysed by immunoblotting using the indicated antibodies. Quantification of LC3-II/LC3-I ratio of three independent experiments is shown on the right panel.

G. Ferroptosis assay in ATG16L1-/- cells stably expressing ATG16L1WT, ATG16L1LD or ATG16L1LE. Cells were cultured in amino acid free media (AA starve) in the presence of 10% FBS or 10% dialysed FBS (diFBS) for 24 hr. Quantification of percentage of PI-positive cells from at least three independent experiments is shown.

Data information:_Quantifications depict means and error bars (SEM) from at least three independent experiments. ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01 (pairwise unpaired Student's t-test).

Expanded View Figure Legends
Genetic deficiency of the mitochondrial protein PGAM5 causes a Parkinson’s-like movement disorder (2014)
f1

(a) WT and Pgam5 KO MEFs evaluated by TEM. The black arrows indicate mitochondria; (b) TEM analysis after 12 h CCCP treatment; black arrowheads indicate DMS. Inset shows a DMS with an encapsulated mitochondrion. The black arrows indicate mitochondria. Scale bar=0.5 micron; (c) WT and KO MEFs were treated with CCCP for 0, 3 or 6 h and cytosolic fractions were immunoblotted with anti-LC-3, anti-p62 or anti-actin as indicated. (d) Similar to c. Treated with CCCP for 6 and 12 h and immunoblotted for anti-COXIV, anti-Tomm20 or anti-actin as indicated. For c and d, the numbers below each lane indicate the fractional band density compared with the 0 h time point, which has been set at 1.0; (e) Confocal photomicrographs of mt-Keima-transduced WT and KO MEFs treated with CCCP or DMSO vehicle for 12 h (six individual cells from each group were scanned and representative cells are shown); Intracellular mt-Keima excited at 450 nm was shown in green colour, while red colour indicated the excitation by 550 nm in the same cell. Scale bar=20 μm. (f) Mitochondrial extracts of WT and Pgam5 KO MEFs treated with DMSO (−) or CCCP (+) for 3 h and analysed by immunoblot as above. (Note that mouse PINK1 antibody can only detect the full-length PINK1). Arrow indicates the 63 kDa form of PINK1. (g) Hela cells transduced with either nonspecific (NS) or PGAM5 shRNA viruses were treated with CCCP for 3 h, and mitochondrial fractions were immunoblotted for the proteins indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are indicated. For f and g, the numbers below indicate the full-length PINK1 band density compared with the first lane that has been set at 1.0 (h) Confocal micrographs of Hela cells as in g were transfected with PARKIN-YFP (green) plasmid before CCCP treatment, then (i) treated with CCCP for 12 h. Mitochondria were stained with anti-Tomm20 (red) 4',6-diamidino-2-phenylindole stained nucleus was shown in blue. Co-localization was indicated by white colour. Scale bar=10 μm. (j) Quantification of the co-localization percentage of Tomm20 (red) with parkin (green) per cell by Imaris software (n=7 for NS and PGAM5 shRNA group separately. Results are presented as mean and s.d. values and were analysed by the student t-test). All the blots and images are representative of three independent experiments. DMSO, dimethylsulphoxide.
Genetic deficiency of the mitochondrial protein PGAM5 causes a Parkinson’s-like movement disorder (2014)
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(a) Mitochondria from MEFs from Parl WT (+/+) and KO (−/−) mice were treated with the indicated doses of proteinase K (Prot.K) for 30 min on ice and then immunoblotted for the indicated proteins. Full-length and cleaved PGAM5 bands are indicated with arrows. (b) Pgam5 WT and KO MEFs were transduced with either nonspecific (NS) or Parl shRNA and either treated (+) or not (−) with CCCP for 3 h. Mitochondria were then purified and analysed by immunoblot for the indicated proteins. (c) WT PINK1 as well as kinase dead (KD) or Parkinson’s disease-associated PINK1 mutants (C92F, A168P, H271Q, G309D, L347P, G386S, R464H) were co-transfected into HEK293T cells with the same amount PGAM5 (lane 1–9) or GFP (lane 10–18) expression plasmids and total cell lysates were immunoblotting for PINK1, PGAM5 and actin. The ratio of WT full-length (63 kDa) versus cleaved (54 kDa) PINK1 (*/#) is shown. (d) Anti-myc immunoprecipitates (IP) of lysates from 293T cells transfected with combinations of myc-tagged PGAM5 and PINK1-V5 with or without CCCP treatment and immunoblotting with anti-PINK1 or anti-myc. (e) Four constructs expressing either: (1) no PGAM5 (empty vector) or (2) amino acids 1–98, (3) 1–110 and (4) 98–289 truncated versions of PGAM5 (left panel). Immunoblot of HEK293T cells expressing PINK1-GFP and myc-tagged PGAM5 truncation mutants as indicated with anti-PINK1 or anti-myc for the input proteins as well as the anti-GFP IP (right panel). (f) Co-transfection of GFP, WT PGAM5, 2RA mutant (R98A and R104A) or the 2HA mutant (H99A and H105A) with a PINK1 expression plasmid into HEK293T cells and the input cell lysates as well as the anti-myc IP were immunoblotted with antibodies against PINK1, PGAM5 or actin. WT PINK1 was expressed in HEK293T cells with or without PGAM5 or a GFP control plasmid after (g) DMSO or (h) CCCP treatment. Cells were permeabilized with digitonin or Triton X-100 separately, followed by treatment with the indicated amounts of trypsin on ice for 30 min. Cell lysates were immunoblotted for PINK1, PGAM5, Tomm20 or HSP60 as indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are shown.
Genetic deficiency of the mitochondrial protein PGAM5 causes a Parkinson’s-like movement disorder (2014)
f4

(a) Body weight was measured in 5-month-old and 12-month-old female Pgam5 WT and KO mice. Behavioral measurements on 12-month-old female WT and KO mice (WT n=10, KO n=8) were as follows: (b) Open-field tests including time spent mobile, time spent immobile, whole area distance and centre area distance; (c) Gait length, gait frequency in steps/ss and step angle; (d) rearing test, (e) akinesia test and (f) ladder climb test and (g) vertical pole test. T-turn indicates the time mice spent to turn their bodies 180 degrees from the head up start position before descending the pole. T-down indicates the time mice spent to descend to the bottom of pole. Graphs show the mean and s.d. values and were analysed by the student’s t-test, *P0.05, **P0.01, ***P0.001, NS, not significant.
Genetic deficiency of the mitochondrial protein PGAM5 causes a Parkinson’s-like movement disorder (2014)
f5

High-performance liquid chromatography-electrochemical detection quantitation of (a) striatal DA and its metabolites (b) dihydroxyphenylacetic acid (DOPAC) (c) Homovanillic acid (HVA) and (d) 3MT levels at three different time points (1, 2 and 12-month-old) in Pgam5 WT or KO mice. In 1, 2 and 12 mo cohorts, TH-positive neurons were quantified in the SNpc region of WT and KO mice by unbiased stereological counting. (e) Representative images of TH-immunohistochemistry from WT and KO mice. The VTA region is indicated. Stereological quantitation of (f) TH and (g) Nissl-positive neurons in the SNpc (n=4 mice per group). Error bars represent the mean±s.e.m., n=4 mice per group. (two-way analysis of variance, ***P0.01, *P0.05, NS, not significant). mo, month old.
Genetic deficiency of the mitochondrial protein PGAM5 causes a Parkinson’s-like movement disorder (2014)
f6

The 18-month-old Pgam5 WT and KO mice received either water as control (n=4), or 5 mg kg−1 L-DOPA and 12.5 mg kg−1 Benserazide (n=5) by intraperitoneal injection. Twenty minutes later, mice were subjected to the open-field test and the centre area time in seconds (s) as well as the centre area distance in centimeters (cm) were measured as described in the Materials and Methods. Graphs show the mean and s.d. values and were analysed by the student t-test, NS, no significance; *P0.05; **P0.01.
Disruption of neuronal autophagy by infected microglia results in neurodegeneration. (2008)
Figure 4

Western blot analysis of autophagy related proteins.

(A) The LC3-II level is reduced after 3 or 24 hr exposure to SIV-infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin (2 µM). The protein level of elongation complex Atg12-Atg5 is also reduced, and again the effect is blocked in the presence of rapamycin. The protein level of p62 is increased for similar conditions with SIV-infected microglia supernatant, and the increase is blocked in the presence of rapamycin. GAPDH protein was used in these experiments as the loading control. One representative experiment of n = 4 is shown. (B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.
Disruption of neuronal autophagy by infected microglia results in neurodegeneration. (2008)
Figure 5

AV counting and distribution for neurons exposed to different treatments.

(A) Total AV counting results show that a significant drop occurs in neurons exposed to TNF-α or NMDA for 3 or 24 hr. *** P<0.001, ** P<0.01 for n = 6 experiments. All values are mean±SEM. (B and C) Distribution of AV number in soma and neurites respectively. (B) The number of AV in neurites decreased significantly in neurons exposed to both TNF-α or NMDA for 3 or 24 hr. *** P<0.001, for n = 6 experiments. (C) The AV number in soma is increased in neurons after exposure to TNF-α for 3 or 24 hr, however it remained unaffected in neurons after exposure to NMDA. ** P<0.01 for n = 6 experiments. (D) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The four panels display a typical sample image of a neuron under 3 hr and 24 hr exposure to TNF-α or NMDA at 25 ng/ml and 35 µM respectively. Scale bar, 20 µm.
Disruption of neuronal autophagy by infected microglia results in neurodegeneration. (2008)
Figure 7

Transcriptional regulation of p62 in SIVE monkey brains, and its presence in human and monkey neurons associated with ubiquitin.

(A) Relative values (RV) of p62 mRNA level in frontal lobe of SIVE versus SIV and uninfected (UN) monkeys, n = 7 for SIVE, n = 12 for SIV and n = 9 for UN, P<0.05. (B) RV of p62 mRNA level in frontal cortex of HAD versus non neurological diseased subjects (NNDS), n = 8 for HAD and n = 10 for NNDS, P<0.0001. (C) p62 and ubiquitin immunoreactivity on serial sections in SIVE monkeys, as well as those with HAD in brain sections from hippocampus. p62 has a diffuse immunoreactivity in UN monkeys and in human NNDS and a dot profile structures in SIVE and HAD brain tissues. Higher magnification views are shown in green insets. Black arrows indicate p62- or ubiquitin-immunoreactive structures within the neuronal cell body or proximal processes, and red arrowheads indicate a dot-like immunoreactivity in the neuropil. Scale bar, 20 µm.
Distinct retrograde microtubule motor sets drive early and late endosome transport (2020)
Figure 3

Figure 3. STIM1 regulates early endosome retrograde transport.

(A) Confocal microscopy images of ECs silenced with a control siRNA (siCTL) or one targeting STIM1 (siSTIM1) and stained for endogenous Rab5 (in green) and EEA-1 (in red) to visualize EEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.

(B) Average size, normalized on cell size, of EEA-1+ endosomes as in a. Results are the average ± SEM of three independent experiments for a total 4483 early endosomes in 18 siCTL cells (299 ± 33 endosomes per cell) and 2454 early endosomes in 13 siSTIM1 cells (189 ± 17 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.

(C) Average number, normalized on cell size, of EEA-1+ endosomes as in a. Results are the average ± SEM of three independent experiments for a total 4483 early endosomes in 18 siCTL cells (299 ± 33 endosomes per cell) and 2454 early endosomes in 13 siSTIM1 cells (189 ± 17 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,01 **.

(D) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV1 and EV2) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 60 seconds. Results are from three independent experiments for a total of 903 early endosomes in 18 siCTL cells (50 ± 5 endosomes per cell) and 935 early endosomes in 20 siSTIM1 cells (47 ± 9 endosomes per cell). Data are analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,01 **.