Journal: The FASEB Journal

Article Title: Autophagy coordinates chondrocyte development and early joint formation in zebrafish

doi: 10.1096/fj.202101167R

Figure Lengend Snippet: Generation of atg13 knock out zebrafish line. (A) Schematic showing location of 5 bp deletion in atg13 knockout line and generation of premature STOP codon, with black lines representing exons. Key domains within atg13 highlighted; LIR, Lc3‐interacting region; MIM, Microtubule interacting motif. (B) Immunoblot showing loss of atg13 expression in atg13 homozygous mutant. (C) Lateral widefield images of atg13 zebrafish larvae at 5 dpf. Orange arrowheads indicate phenotypic differences in development between wt and atg13 mutants (from left to right: bent body axis, uninflated swim bladder and shorter body length). Scale bar = 500 µm. (D) Graph showing whole body length of atg13 larvae from 1–5 dpf. Student's t ‐test performed at 5 dpf between wt and atg13 mutant, * p = .0013. (E) Kaplan–Meier graph showing survival of larvae up to 20 dpf

Article Snippet: Following transfer at 100 V, nitrocellulose membranes were blocked in 5% milk‐TBS‐1% Triton (TBS‐T), then incubated with primary antibodies diluted in 5% milk overnight at 4°C (anti‐ATG13 (SAB4200135, 1:100, Sigma); anti‐GAPDH (10494‐1‐AP, 1:2000, Proteintech, IL, USA); anti‐LC3B (ab51520, 1:300, Abcam, Cambridge, MA, USA); anti‐p62/SQSTM1 (5114, 1:300, Cell Signalling Technologies, MA, USA)).

Techniques: Knock-Out, Western Blot, Expressing, Mutagenesis

Journal: The FASEB Journal

Article Title: Autophagy coordinates chondrocyte development and early joint formation in zebrafish

doi: 10.1096/fj.202101167R

Figure Lengend Snippet: atg13 mutants show reduced autophagy flux. (A) Representative single confocal z‐slices of epidermal cells taken from LysoTracker stained Tg(cmv:gfp‐map1lc3;atg13) wt and mutant larvae, at 4 dpf following treatment with DMSO or 100 nM Bafilomycin for 3 h. Scale bars = 10 µm. (B) Quantification of number of lysosomal (red) and GFP‐Lc3 (green) puncta per cell. Two‐way ANOVA performed for each; **** p < .0001. (C) Representative immunoblot of atg13 wt , heterozygous and mutant larvae at 5 dpf following treatment with DMSO or 100 nM Bafilomycin for 3 h. LE, long exposure; SE, short exposure. Molecular weight markers indicated on right hand side of immunoblots

Article Snippet: Following transfer at 100 V, nitrocellulose membranes were blocked in 5% milk‐TBS‐1% Triton (TBS‐T), then incubated with primary antibodies diluted in 5% milk overnight at 4°C (anti‐ATG13 (SAB4200135, 1:100, Sigma); anti‐GAPDH (10494‐1‐AP, 1:2000, Proteintech, IL, USA); anti‐LC3B (ab51520, 1:300, Abcam, Cambridge, MA, USA); anti‐p62/SQSTM1 (5114, 1:300, Cell Signalling Technologies, MA, USA)).

Techniques: Staining, Mutagenesis, Western Blot, Molecular Weight

Journal: The FASEB Journal

Article Title: Autophagy coordinates chondrocyte development and early joint formation in zebrafish

doi: 10.1096/fj.202101167R

Figure Lengend Snippet: atg13 mutation reduces jaw function. (A) Stills from videos of larval jaw movements taken at 5 dpf of wt and atg13 ‐mutant fish. Red and yellow lines indicate where mouth and buccal width measurements taken from, respectively. Quantification of number (B) and displacement (C) of jaw movements at the mouth and buccal joint. n = 5 for each genotype; three widest jaw openings taken per larvae. Student's unpaired t test performed for (C), **** p < .0001, * p = .0129

Article Snippet: Following transfer at 100 V, nitrocellulose membranes were blocked in 5% milk‐TBS‐1% Triton (TBS‐T), then incubated with primary antibodies diluted in 5% milk overnight at 4°C (anti‐ATG13 (SAB4200135, 1:100, Sigma); anti‐GAPDH (10494‐1‐AP, 1:2000, Proteintech, IL, USA); anti‐LC3B (ab51520, 1:300, Abcam, Cambridge, MA, USA); anti‐p62/SQSTM1 (5114, 1:300, Cell Signalling Technologies, MA, USA)).

Techniques: Mutagenesis

Journal: The FASEB Journal

Article Title: Autophagy coordinates chondrocyte development and early joint formation in zebrafish

doi: 10.1096/fj.202101167R

Figure Lengend Snippet: Loss of atg13 does not affect size of lower jaw in development. (A) Left , representative confocal z ‐stack projections of the lower jaw at 3, 5, and 7 dpf in wt and atg13 ‐mutant larvae, immunostained for collagen Type II (Col2a1). Scale bar = 50 µm. Right , schematic showing where 3 measurements taken within lower jaw of larvae. (B–D) Quantification of three measurements; (B) length of Merkel's cartilage, (C) length of lower jaw, (D) width of Merkel's cartilage. n = 7 for 3 dpf, n = 6 for 5 dpf and n = 6 and 4 at 7 dpf for wt and atg13 mutant, respectively. Student's unpaired t test performed for each age, ns p > .05

Article Snippet: Following transfer at 100 V, nitrocellulose membranes were blocked in 5% milk‐TBS‐1% Triton (TBS‐T), then incubated with primary antibodies diluted in 5% milk overnight at 4°C (anti‐ATG13 (SAB4200135, 1:100, Sigma); anti‐GAPDH (10494‐1‐AP, 1:2000, Proteintech, IL, USA); anti‐LC3B (ab51520, 1:300, Abcam, Cambridge, MA, USA); anti‐p62/SQSTM1 (5114, 1:300, Cell Signalling Technologies, MA, USA)).

Techniques: Mutagenesis

Journal: The FASEB Journal

Article Title: Autophagy coordinates chondrocyte development and early joint formation in zebrafish

doi: 10.1096/fj.202101167R

Figure Lengend Snippet: atg13 ‐mutant fish show decreased number of chondrocytes and reduced proliferation at joint site. (A) Representative confocal slices of lower jaw joint at 3, 5 and 7 dpf in wt and atg13 ‐mutant fish, immunohistochemically labeled for Col2a1. Scale bars = 20 µm. (B) Left , example slice from confocal image showing Col2a1‐positive cells outlined by modular image analysis program run in Fiji. Right , quantification of Col2a1‐positive cell number normalized to cartilage element volume, (C) and volume of Col2a1‐positive cells within cartilage element for wt and atg13 mutants. Each data point = one larvae. Student's unpaired t test performed where *** p = .0004, * p = .0463. (D) Confocal max projections of larval jaw joint in Tg(atg13; Col2a1aBAC:mcherry) wt and atg13 mutants at 6 dpf following 24‐h treatment with EdU Click‐iT, EdU (cyan) and mCh‐Col2a1 (red). Scale bars = 25 and 20 µm for insets. (E) Quantification of number of EdU positive chondrocytes within jaw joint region (determined as region at 5× zoom on 20× objective, when joint in middle of image plane). EdU positive chondrocytes colocalized to Col2a1 staining and counted by going through z ‐stack. Student's unpaired t test performed, ** p = .0032

Article Snippet: Following transfer at 100 V, nitrocellulose membranes were blocked in 5% milk‐TBS‐1% Triton (TBS‐T), then incubated with primary antibodies diluted in 5% milk overnight at 4°C (anti‐ATG13 (SAB4200135, 1:100, Sigma); anti‐GAPDH (10494‐1‐AP, 1:2000, Proteintech, IL, USA); anti‐LC3B (ab51520, 1:300, Abcam, Cambridge, MA, USA); anti‐p62/SQSTM1 (5114, 1:300, Cell Signalling Technologies, MA, USA)).

Techniques: Mutagenesis, Labeling, Staining

Journal: The FASEB Journal

Article Title: Autophagy coordinates chondrocyte development and early joint formation in zebrafish

doi: 10.1096/fj.202101167R

Figure Lengend Snippet: atg13 mutation affects expression and production of key factors in cartilage development. (A) Left , confocal max projections of lower jaw at 5 dpf in wt and atg13 ‐mutant fish, immunostained for Sox9a (grey) and Col2a1 (red). Scale bars = 50 µm. Right , schematic showing regions of interest selected within lower jaw in modular image analysis program (SoxQuant). Colours correspond to graphs below. (B–D) Quantification of Sox9a expression measured as volume of Sox9a within Col2a1 positive cells from confocal z ‐stack. Student's unpaired t test performed where **** p < .0001, *** p = .0007, * p = .0173; n = 12 for both. (E) Confocal max projections of the lower jaw at 7 dpf in Tg(atg13; Col2a1aBAC:mcherry) wt and atg13 ‐mutant larvae, immunostained for collagen Type X (ColX) (cyan) and mCherry (for Col2a1, red). Scale bars = 50 µm

Article Snippet: Following transfer at 100 V, nitrocellulose membranes were blocked in 5% milk‐TBS‐1% Triton (TBS‐T), then incubated with primary antibodies diluted in 5% milk overnight at 4°C (anti‐ATG13 (SAB4200135, 1:100, Sigma); anti‐GAPDH (10494‐1‐AP, 1:2000, Proteintech, IL, USA); anti‐LC3B (ab51520, 1:300, Abcam, Cambridge, MA, USA); anti‐p62/SQSTM1 (5114, 1:300, Cell Signalling Technologies, MA, USA)).

Techniques: Mutagenesis, Expressing

Journal: The FASEB Journal

Article Title: Autophagy coordinates chondrocyte development and early joint formation in zebrafish

doi: 10.1096/fj.202101167R

Figure Lengend Snippet: Ultrastructure and organization of chondrocytes affected in atg13 mutants. (A–D) Electron microscopy of ethmoid plate in wt and atg13 ‐mutant fish at 5 dpf following DMSO or (E and F) BafA1 treatment for 3 h. (B and D) Orange arrow heads highlight areas of non‐uniformity and non‐intercalating chondrocytes in atg13 ‐mutant cartilage. (C) Red asterisks show vesicles fusing with outer membrane in wt , not present in atg13 mutant. Blue dotted boxes and inset show differences in ECM organization and density between wt and atg13 mutants. (E and F) BafA1 treatment increases number of vesicles in both wt and atg13 mutants and ablates vesicle‐membrane fusion events. Blue box and inset in (E) shows autophagosome in BafA1 treated wt chondrocyte, not present in atg13 mutants. Scale bars A, B = 5 µm; C–F = 1.5 µm; C’–E’ = 0.5 µm. (G) Number of chondrocytes on periphery of cartilage and not aligning down central line of stack. Calculated as percentage of total cell number along ethmoid plate in one section. N = 38 chondrocytes total from 3 larvae, per condition and genotype. (H) Number of vesicles fusing with outer cell membrane quantified per cell following DMSO or BafA1 treatment. Two‐way ANOVA performed for each; **** p < .0001. (I) Average number of autophagosomal structures per chondrocyte in bafilomycin A1 treated fish, calculated as average of all chondrocytes per individual fish. Student's unpaired t ‐test performed; * p = .0138

Article Snippet: Following transfer at 100 V, nitrocellulose membranes were blocked in 5% milk‐TBS‐1% Triton (TBS‐T), then incubated with primary antibodies diluted in 5% milk overnight at 4°C (anti‐ATG13 (SAB4200135, 1:100, Sigma); anti‐GAPDH (10494‐1‐AP, 1:2000, Proteintech, IL, USA); anti‐LC3B (ab51520, 1:300, Abcam, Cambridge, MA, USA); anti‐p62/SQSTM1 (5114, 1:300, Cell Signalling Technologies, MA, USA)).

Techniques: Electron Microscopy, Mutagenesis

Journal: ACS Nano

Article Title: Exploiting Mass Spectrometry to Unlock the Mechanism of Nanoparticle-Induced Inflammasome Activation

doi: 10.1021/acsnano.3c05600

Figure Lengend Snippet: Pro-inflammatory responses of silica NPs are iPLA 2 -dependent. (a) Bare silica NPs triggered an elevation in cellular LPC content in THP-1 cells, which was prevented by BEL, a selective iPLA 2 -VIA inhibitor. Cells were exposed for 6 h to bare silica NPs (2.5 μg/mL) with or without BEL (5 μM). (b) Silica NP-triggered IL-1β release in nondifferentiated (monocyte-like) THP-1 cells was effectively blocked by BEL. (c) Silica NP-triggered IL-1β release in primary human CD14+ monocytes was also reduced by BEL. (d) Western blot to confirm PLA2G6 silencing in THP-1 cells using specific siRNAs. GAPDH was used as a loading control. (e) Silencing of PLA2G6 significantly reduced IL-1β production as determined by ELISA. (f) Silica NPs triggered inflammasome assembly at the MTOC, as evidenced by the colocalization of ASC and the centrosomal marker, γ-tubulin (GTU). Samples obtained after 6 h of exposure were visualized by confocal microscopy. For additional results on the impact of BEL, refer to Figure S20 . Data shown as mean values ± SD ( n = 3). ** p < 0.01; **** p < 0.0001; ## p < 0.01; #### p < 0.0001.

Article Snippet: The proteins were then transferred to a Hybond Low-Fluorescent 0.2 μm PVDF membrane (Amersham), blocked for 1 h in Odyssey Blocking Buffer (PBS; LI-COR), and stained overnight at 4 °C with primary antibodies against iPLA 2 (Sigma-Aldrich, SAB4200130).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Marker, Confocal Microscopy

Journal: ACS Nano

Article Title: Exploiting Mass Spectrometry to Unlock the Mechanism of Nanoparticle-Induced Inflammasome Activation

doi: 10.1021/acsnano.3c05600

Figure Lengend Snippet: Understanding sterile inflammation. Schematic of the classical two-signal model of inflammasome activation (a) versus the present model (b). The NLRP3 inflammasome consisting of NLRP3, ASC, and pro-caspase-1 serves as a platform for the activation of caspase-1, leading to the proteolysis of pro-IL-1β and the release of IL-1β. Numerous studies have shown that particulate matter (PM) such as alum and crystalline silica (quartz) as well as high aspect ratio (nano)materials (HARNs), e.g., asbestos and carbon nanotubes, are able to trigger the activation of the NLRP3 inflammasome (panel a). This usually occurs through the internalization of the offending agent into phagosomes that eventually fuse with lysosomes (the phago-lysosomal system is depicted here as an empty circle), leading to the generation of reactive oxygen species (ROS) and the release of lysosomal cathepsin B. Macrophages are usually primed with a Toll-like receptor (TLR) agonist such as LPS (lipopolysaccharide). In contrast, we show that uncoated amorphous silica NPs are capable of triggering NLRP3-dependent IL-1β release in the absence of LPS priming (panel b) and we provide evidence that mitochondrial ROS production may contribute to the effects of silica NPs on membrane lipids (mitochondria are depicted here as a small empty circle). We posit that lipid peroxidation, leading to phospholipase (iPLA 2 ) activation and the generation of lysophosphatidylcholine (LPC), provides a cell autonomous “priming” signal.

Article Snippet: The proteins were then transferred to a Hybond Low-Fluorescent 0.2 μm PVDF membrane (Amersham), blocked for 1 h in Odyssey Blocking Buffer (PBS; LI-COR), and stained overnight at 4 °C with primary antibodies against iPLA 2 (Sigma-Aldrich, SAB4200130).

Techniques: Sterility, Activation Assay, Membrane

Journal: bioRxiv

Article Title: Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

doi: 10.1101/200428

Figure Lengend Snippet: BAC GFP-Rab5 cells seeded on a gridded dish were labeled with MitoTracker-Red CMSRox and photoirradiated as before. Cells were fixed after 30 min post-laser treatment and immunostained with specific ZFYVE20 and EEA1 ( A ), and APPL1 and APPL2 antibodies ( B ). ( C ) Quantification of colocalization from untreated and laser-treated cells in ( A ) and ( B ), n = 3. ( D ) Quantification of colocalization between Rab5 and EEA1 as shown in ( A ), n = 3. ( E - F ) BAC GFP-Rab5 cells were treated and processed in the same manner as in ( A ) and immunostained with specific Rabex-5 and Alsin antibodies, respectively. ( G ) Quantification of colocalization from untreated and laser-treated cells in ( E ) and ( F ), n = 3. Scale bars, 10 μm.

Article Snippet: The following antibodies were used by Western blot: anti-mouse cytochrome c (Abcam: ab13575), anti-rabbit gamma tubulin (Sigma-Aldrich: T6557), anti-rabbit Alsin (Sigma Aldrich: SAB4200137), anti-mouse EEA1 (BD Biosciences: 610457), and anti-rabbit TOM20 (Santa Cruz Biotechnology: sc-11415).

Techniques: Labeling

Journal: bioRxiv

Article Title: Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

doi: 10.1101/200428

Figure Lengend Snippet: ( A ) Flow chart depicting the different stages and time (in days) from iPSC, to neuroprogenitor cells (NPC), and to generating mature spinal motor neurons (sMN). Figure legends indicate the various small molecules and compounds that are used at different stages. ( B ) WT and Alsin -/- cells were challenged with 100 μM H 2 O 2 for 1 hour. Cells were fixed and immunostained with Rab5 and TOM20 antibodies. Inset images show the representative of the signals from TOM20 and Rab5 under control (left panel) and H 2 O 2 -treated condition (right panel). Scale bars, 10 μm. ( C ) Subcellular fractionation of cytosolic (Cyto) and mitochondrial (Mito) fractions from WT and Alsin -/- iPSC-sMN treated with either PBS (control) or 200 μM H 2 O 2 for 1 hours at 37°C. Protein samples were loaded onto SDS-PAGE and imunoblotted with Rab5, tubulin (cytoplasmic loading control), and TOM20 antibodies by Western blot. ( D ) Cytosolic fractions were prepared as in from WT and Alsin -/- iPSC-sMN challenged with 200 μM H 2 O 2 for 1 hours at 37μC. Densitometric quantification of cytosolic cytochrome c from Western blot probed cytochrome c antibody. Data were collected from three independent experiments. Y-axis corresponds to normalized ratio intensity of experimental to loading control (* p <0.05; ** p <0.005).

Article Snippet: The following antibodies were used by Western blot: anti-mouse cytochrome c (Abcam: ab13575), anti-rabbit gamma tubulin (Sigma-Aldrich: T6557), anti-rabbit Alsin (Sigma Aldrich: SAB4200137), anti-mouse EEA1 (BD Biosciences: 610457), and anti-rabbit TOM20 (Santa Cruz Biotechnology: sc-11415).

Techniques: Fractionation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

doi: 10.1101/200428

Figure Lengend Snippet: In normal condition, mitochondria (Mito, red) are elongated and tubular (top left). Rab5 (green) are localized on early endosomes (EE) to assemble Rab5 machinery for endosomal maturation and membrane trafficking. At steady state, we observed some EE transiently making contacts with mitochondria. During oxidative stress (eg. laser- or chemically-induced) that leads to MOMP, mitochondria undergo a dramatic morphological transformation into rounded and swollen structures (top right). This is accompanied by an increase in Rab5-positive endosomes forming MCS with mitochondria of less than <5 nm. Release of apoptotic signal such as cytochrome c from mitochondria into the cytosol is associated with Rab5 translocation to the OMM, which is involved in blocking cytochrome c release and caspase activation. The recruitment and activation of Rab5 on mitochondria depend on the Rab5 GEF Alsin (blue), resulting in a selective recruitment of Rabenosyn-5 (light green), in order to regulate apoptosis and confer cytoprotection.

Article Snippet: The following antibodies were used by Western blot: anti-mouse cytochrome c (Abcam: ab13575), anti-rabbit gamma tubulin (Sigma-Aldrich: T6557), anti-rabbit Alsin (Sigma Aldrich: SAB4200137), anti-mouse EEA1 (BD Biosciences: 610457), and anti-rabbit TOM20 (Santa Cruz Biotechnology: sc-11415).

Techniques: Transformation Assay, Translocation Assay, Blocking Assay, Activation Assay

Journal: The Journal of Cell Biology

Article Title: Atlastin 2/3 regulate ER targeting of the ULK1 complex to initiate autophagy

doi: 10.1083/jcb.202012091

Figure Lengend Snippet: ATL2/3 contribute to the recruitment of ULK1 to the autophagosome formation sites specified by FIP200/ATG13. (A and B) Compared with control cells (A), puncta formed by endogenous FIP200 (detected by anti-FIP200) show no evident change in ATL2/3 DKO COS-7 cells (B) after 1 h of starvation. Scale bars, 5 µm. (C and D) Compared with control cells (C), puncta labeled by anti-ATG13 antibody are not changed in ATL2/3 DKO COS-7 cells (D) after 1 h of starvation. Scale bars, 5 µm. (E and F) ULK1-GFP forms fewer puncta in ATL2/3 DKO COS-7 cells (F) compared with control cells (E) after 1 h of starvation. Scale bars, 5 µm. (G and H) Compared with control cells (G), fewer puncta are labeled by anti-ATG101 antibody in ATL2/3 DKO COS-7 cells (H) after 1 h of starvation. Scale bars, 5 µm. (I) Quantitative data for A–H are shown as mean ± SEM ( n = 30 cells in each group). *, P < 0.05; ***, P < 0.001. (J–L) The relationship between ATG13 puncta and ULK1-GFP puncta in control (K) and ATL2/3 DKO COS-7 cells (L) after 1 h of starvation. The percentage of ATG13 puncta positive for ULK1-GFP is decreased in ATL2/3 DKO cells (L) compared with control cells (K) after 1 h of starvation. Quantitative data are shown in J as mean ± SEM ( n = 20 cells in each group). ***, P < 0.001. Scale bars, 5 µm; inset scale bars, 0.5 µm. (M) Levels of endogenous ULK1 and ATG101 coprecipitated by GFP-ATG13 are decreased in ATL2/3 DKO COS-7 cells compared with control cells in GFP-Trap assays, while levels of endogenous FIP200 precipitated by GFP-ATG13 are not reduced. Quantifications of ULK1, ATG101, and FIP200 levels (normalized by GFP-ATG13 levels) are also shown. (N) Levels of endogenous FIP200, ULK1, and ATG13 coprecipitated by GFP-ATG101 are dramatically decreased in ATL2/3 DKO COS-7 cells compared with control cells in GFP-Trap assays. Quantifications of FIP200, ULK1, and ATG13 levels (normalized by GFP-ATG101 levels) are also shown. (O–Q) A subset of ULK1-GFP punctate structures accumulate at distinct Myc-ATL2 (O) and Myc-ATL3 (P) puncta in control cells after 1 h of starvation. Myc-ATL2 and Myc-ATL3 puncta are defined as having a fluorescence intensity that is clearly stronger than the surrounding area. Quantitative data are shown in Q as mean ± SEM ( n = 15 cells in each group). Scale bars, 5 µm; inset scale bars, 0.5 µm. (R) In cells treated with wortmannin, levels of endogenous ATL2 and ATL3 precipitated by ULK1-GFP are dramatically decreased compared with untreated cells. Quantification of ATL2 and ATL3 levels (normalized by ULK1-GFP levels) are also shown. (S) Levels of endogenous ATL2 and ATL3 precipitated by ULK1-GFP are increased in VMP1 KO COS-7 cells compared with control cells in GFP-Trap assays. Quantifications of ATL2 and ATL3 levels (normalized by ULK1-GFP levels) are also shown. ctrl, control; Strv, starved.

Article Snippet: The following antibodies were used for immunostaining or immunoblotting assays: mouse anti-LC3 (MBL, M152-3), rabbit anti-LC3 (2775S; Cell Signaling Technology), rabbit anti-p62 (MBL, PM045), rabbit anti-FIP200 (SAB4200135; Sigma-Aldrich), rabbit anti-FIP200 (17250-1-AP; Proteintech), rabbit anti-ULK1 (8054; Cell Signaling Technology), rabbit anti-Atg13 (13468S; Cell Signaling Technology), rabbit anti-ATL2 (16688-1-AP; Proteintech), rabbit anti-ATL3 (ab117819; Abcam), mouse anti-WIPI2 (ab105459; Abcam), mouse anti-VAPB (66191-1-IG; Proteintech), rabbit anti-VAPA (15275-1-AP; Proteintech), mouse anti-LAMP1 (553792; BD Biosciences), rabbit anti-PTPIP51 (20641-1-AP; Proteintech), rabbit anti-ATG101 (13492; Cell Signaling Technology), rabbit anti-Myc (2278S; Cell Signaling Technology), mouse anti-Myc (M5546; Sigma-Aldrich), mouse anti-GFP (11814460001; Roche), mouse anti-actin (60008-1-IG; Proteintech), and rabbit anti-HA (H6908; Sigma-Aldrich).

Techniques: Labeling, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Atlastin 2/3 regulate ER targeting of the ULK1 complex to initiate autophagy

doi: 10.1083/jcb.202012091

Figure Lengend Snippet: ATL2/3 contribute to the recruitment of ULK1 by ATG13. (A–D) The percentage of FIP200 puncta positive for ATG13 or the percentage of ATG13 puncta positive for FIP200 is not changed in ATL2/3 DKO COS-7 cells (B) compared with control cells (A) after 1 h of starvation. Quantitative data are shown in C and D as mean ± SEM ( n = 17 cells for A, n = 19 cells for B). Scale bars, 5 µm; inset scale bars, 0.5 µm. (E) Levels of endogenous FIP200 and ATG13 coprecipitated by ULK1-GFP are decreased in ATL2/3 DKO COS-7 cells compared with control cells in GFP-Trap assays. Control or ATL2/3 DKO COS-7 cells were transfected with ULK1-GFP. Cell lysates were immunoprecipitated using GFP-Trap and analyzed by immunoblotting with anti-FIP200 or anti-ATG13 antibody. Quantifications of ATG13 and FIP200 levels (normalized by ULK1-GFP levels) are also shown. (F) Levels of ULK1 protein are decreased in ATL2/3 DKO cells compared with control cells. Quantification of ULK1 levels (normalized by actin levels) is also shown. (G) In ER fractions, the levels of endogenous ULK1 and ATG101 proteins are not evidently changed in ATL2/3 DKO cells. Compared with control cells, the interactions of GFP-ATG13 with ULK1 and ATG101 are decreased in ATL2/3 DKO cells. Quantification of endogenous ULK1 and ATG101 are normalized by protein disulfide isomerase (PDI) levels. Quantifications of ULK1 and ATG101 trapped by GFP-ATG13 are first normalized by GFP-ATG13 protein levels and further normalized by their respective input samples. (H) In soluble fractions, the endogenous ULK1 protein level is decreased in ATL2/3 DKO cells, while the ATG101 protein level is only slightly reduced. Compared with control cells, the interactions of GFP-ATG13 with ULK1 and ATG101 are not changed obviously in ATL2/3 DKO cells. Quantifications of endogenous ULK1 and ATG101 are normalized by actin levels. Quantifications of ULK1 and ATG101 trapped by GFP-ATG13 are first normalized by GFP-ATG13 protein levels and further normalized by their respective input samples. (I) Levels of endogenous ATG13 precipitated by ULK1-GFP are not decreased in cells expressing Myc-RTN4a compared with control cells in GFP-Trap assays. Quantification of ATG13 (normalized by ULK1-GFP levels) is also shown. (J) Levels of ATL2 and ATL3 precipitated by GFP-FIP200 are increased in VMP1 KO COS-7 cells compared with control cells in GFP-Trap assays. Control or VMP1 KO cells were transfected with GFP-FIP200. Lysates were immunoprecipitated using GFP-Trap and analyzed by immunoblotting with anti-ATL2 or anti-ATL3 antibody. Quantifications of ATL2 and ATL3 levels (normalized by GFP-FIP200 levels) are also shown. (K) Endogenous ATL2 and ATL3 are precipitated by GFP-ATG13 in GFP-Trap assays. (L) GFP-ATG101 fails to precipitate endogenous ATL2 and ATL3 in GFP-Trap assays. ctrl, control; Strv, starved.

Article Snippet: The following antibodies were used for immunostaining or immunoblotting assays: mouse anti-LC3 (MBL, M152-3), rabbit anti-LC3 (2775S; Cell Signaling Technology), rabbit anti-p62 (MBL, PM045), rabbit anti-FIP200 (SAB4200135; Sigma-Aldrich), rabbit anti-FIP200 (17250-1-AP; Proteintech), rabbit anti-ULK1 (8054; Cell Signaling Technology), rabbit anti-Atg13 (13468S; Cell Signaling Technology), rabbit anti-ATL2 (16688-1-AP; Proteintech), rabbit anti-ATL3 (ab117819; Abcam), mouse anti-WIPI2 (ab105459; Abcam), mouse anti-VAPB (66191-1-IG; Proteintech), rabbit anti-VAPA (15275-1-AP; Proteintech), mouse anti-LAMP1 (553792; BD Biosciences), rabbit anti-PTPIP51 (20641-1-AP; Proteintech), rabbit anti-ATG101 (13492; Cell Signaling Technology), rabbit anti-Myc (2278S; Cell Signaling Technology), mouse anti-Myc (M5546; Sigma-Aldrich), mouse anti-GFP (11814460001; Roche), mouse anti-actin (60008-1-IG; Proteintech), and rabbit anti-HA (H6908; Sigma-Aldrich).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing

Journal: The Journal of Cell Biology

Article Title: Atlastin 2/3 regulate ER targeting of the ULK1 complex to initiate autophagy

doi: 10.1083/jcb.202012091

Figure Lengend Snippet: ATL2/3 interact with ULK1 and ATG13. (A) Schematic illustration of the domains in ATL2 and ATL3. ATL2/3 contain an N-terminal GTPase domain, a 3HB, two membrane-embedded regions (TMs), and a C-terminal tail (CT). (B and C) In GFP-Trap assays, the N terminus of Myc-ATL2(1–373) is precipitated by ULK1-GFP (B) and GFP-ATG13 (C). HEK293T cells were transfected with ULK1-GFP (B) or GFP-ATG13 (C) together with Myc-tagged full-length or fragments of ATL2. Cell lysates were immunoprecipitated using GFP-Trap and analyzed by immunoblotting with anti-Myc antibody. (D and E) Schematic illustration of the domains in ULK1 (D) and ATG13 (E). ULK1 contains an N-terminal kinase domain, a long IDR, and a C-terminal EAT domain. ATG13 contains a HORMA domain in the N-terminal region, which dimerizes with the HORMA domain of ATG101, and a long C-terminal IDR. The IDR of ATG13 interacts with FIP200 and ULK1. The dotted line (348–373) indicates the FIP200-binding motif, and the last 3 aa in the C terminus constitute the ULK1-binding motif. (F and G) Endogenous ATL2/3 are precipitated by the kinase domain of ULK1 and the 1–346 fragment of ATG13. HEK293T cells were transfected with full-length or fragments of ULK1-GFP or GFP-ATG13. Cell lysates were immunoprecipitated using GFP-Trap and analyzed by immunoblotting with anti-ATL2 or anti-ATL3 antibody.

Article Snippet: The following antibodies were used for immunostaining or immunoblotting assays: mouse anti-LC3 (MBL, M152-3), rabbit anti-LC3 (2775S; Cell Signaling Technology), rabbit anti-p62 (MBL, PM045), rabbit anti-FIP200 (SAB4200135; Sigma-Aldrich), rabbit anti-FIP200 (17250-1-AP; Proteintech), rabbit anti-ULK1 (8054; Cell Signaling Technology), rabbit anti-Atg13 (13468S; Cell Signaling Technology), rabbit anti-ATL2 (16688-1-AP; Proteintech), rabbit anti-ATL3 (ab117819; Abcam), mouse anti-WIPI2 (ab105459; Abcam), mouse anti-VAPB (66191-1-IG; Proteintech), rabbit anti-VAPA (15275-1-AP; Proteintech), mouse anti-LAMP1 (553792; BD Biosciences), rabbit anti-PTPIP51 (20641-1-AP; Proteintech), rabbit anti-ATG101 (13492; Cell Signaling Technology), rabbit anti-Myc (2278S; Cell Signaling Technology), mouse anti-Myc (M5546; Sigma-Aldrich), mouse anti-GFP (11814460001; Roche), mouse anti-actin (60008-1-IG; Proteintech), and rabbit anti-HA (H6908; Sigma-Aldrich).

Techniques: Transfection, Immunoprecipitation, Western Blot, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Atlastin 2/3 regulate ER targeting of the ULK1 complex to initiate autophagy

doi: 10.1083/jcb.202012091

Figure Lengend Snippet: ATLs and VAPs act partially redundantly in mediating the formation of FIP200/ULK1 puncta. (A and B) Compared with ATL2/3 DKO COS-7 cells (A), the number of LC3 puncta is further decreased by simultaneous KD of VAPA/B (B). Scale bars, 5 µm. (C) Immunoblotting assays showing LC3 levels in control and ATL2/3 DKO COS-7 cells with or without KD of VAPA/B . Quantifications of LC3-I and LC3-II/LC3-I ratios are also shown (normalized by actin level). The level of LC3-I and the ratio of LC3-II/LC3-I in control cells is set to 1.00. (D and E) Compared with ATL2/3 DKO COS-7 cells (D), the number of ULK1-GFP puncta is much lower after simultaneous KD of VAPA/B (E). Scale bars, 5 µm. (F–K) Compared with ATL2/3 DKO COS-7 cells (F, H, and J), the numbers of ATG101 puncta, FIP200 puncta, and ATG13 puncta are dramatically decreased by simultaneous KD of VAPA/B (G, I, and K). Scale bars, 5 µm. (L) Quantitative data for A and B and D–K are shown in L as mean ± SEM ( n = 32 cells in A and B, n = 26 cells in D, n = 21 cells in E, n = 20 cells in F–K). ***, P < 0.001. (M–Q) EM micrographs showing autophagic structures in ATL2/3 DKO COS-7 cells (M and O) and ATL2/3 DKO cells with simultaneous depletion of VAPA/B (N and P) after 1 h of starvation (M and N) or after Bfa.A1 treatment (O and P). Arrowheads indicate autophagosomes. Quantifications of the numbers of autophagosomes are shown in Q as mean ± SEM ( n = 50 sections in each group, one section for each cell). *, P < 0.05; **, P < 0.01. Scale bars, 0.5 µm. ctrl, control; NC, negative control; Strv, starved.

Article Snippet: The following antibodies were used for immunostaining or immunoblotting assays: mouse anti-LC3 (MBL, M152-3), rabbit anti-LC3 (2775S; Cell Signaling Technology), rabbit anti-p62 (MBL, PM045), rabbit anti-FIP200 (SAB4200135; Sigma-Aldrich), rabbit anti-FIP200 (17250-1-AP; Proteintech), rabbit anti-ULK1 (8054; Cell Signaling Technology), rabbit anti-Atg13 (13468S; Cell Signaling Technology), rabbit anti-ATL2 (16688-1-AP; Proteintech), rabbit anti-ATL3 (ab117819; Abcam), mouse anti-WIPI2 (ab105459; Abcam), mouse anti-VAPB (66191-1-IG; Proteintech), rabbit anti-VAPA (15275-1-AP; Proteintech), mouse anti-LAMP1 (553792; BD Biosciences), rabbit anti-PTPIP51 (20641-1-AP; Proteintech), rabbit anti-ATG101 (13492; Cell Signaling Technology), rabbit anti-Myc (2278S; Cell Signaling Technology), mouse anti-Myc (M5546; Sigma-Aldrich), mouse anti-GFP (11814460001; Roche), mouse anti-actin (60008-1-IG; Proteintech), and rabbit anti-HA (H6908; Sigma-Aldrich).

Techniques: Western Blot, Negative Control

Journal: The Journal of Cell Biology

Article Title: Atlastin 2/3 regulate ER targeting of the ULK1 complex to initiate autophagy

doi: 10.1083/jcb.202012091

Figure Lengend Snippet: Depletion of ATL2/3 impairs the formation of the ER–IM contact. (A and B) Compared with control cells, levels of WIPI2 precipitated by GFP-FIP200 (A) and ULK1-GFP (B) are dramatically decreased in ATL2/3 DKO COS-7 cells in GFP-Trap assays. Quantification of WIPI2 levels (normalized by GFP-FIP200 levels and ULK1-GFP levels, respectively) is also shown. (C and D) Levels of endogenous ULK1 and WIPI2 precipitated by GFP-VAPA (C) and GFP-VAPB (D) are dramatically decreased in ATL2/3 DKO COS-7 cells compared with control cells in GFP-Trap assays. Quantifications of ULK1 and WIPI2 levels (normalized by GFP-VAPA levels and GFP-VAPB levels, respectively) are also shown. (E–G) EM micrographs showing the contacts between the ER and autophagosomes in control (E) and ATL2/3 DKO (F) COS-7 cells with Baf.A1 treatment. The arrowheads indicate the ER. For individual autophagosomes, the percentage of the perimeter in contact with the ER was determined. Data are shown in G as mean ± SEM ( n = 50 autophagosomes in each group). Scale bars, 200 nm. (H) Compared with VMP1 KD cells, levels of WIPI2 precipitated by ULK1-GFP are dramatically decreased in VMP1 KD cells with simultaneous depletion of ATL2/3 in GFP-Trap assays. Quantification of WIPI2 levels (normalized by ULK1-GFP levels) is also shown. (I and J) Model for the role of ATL2/3 in targeting and stabilizing the ULK1 complex on the ER. In early steps of autophagy, FIP200 and ATG13 are recruited to the autophagosome formation site. The C-shaped NTD of FIP200 is shown here. ULK1 is recruited to the FIP200 puncta with the aid of ATL2/3 and VAPA/B. VAPA/B contribute to stabilizing FIP200-ULK1, while ATL2/3 contribute to stabilizing ATG13-ULK1. ATL2/3 and VAPA/B also contribute to the establishment and/or stabilization of the ER–IM contacts (I). In ATL2/3 DKO cells, formation of FIP200 and ATG13 puncta is unaffected, while targeting of ULK1 and ATG101 is severely impaired. The ER–IM contacts are also impaired (J). ctrl, control.

Article Snippet: The following antibodies were used for immunostaining or immunoblotting assays: mouse anti-LC3 (MBL, M152-3), rabbit anti-LC3 (2775S; Cell Signaling Technology), rabbit anti-p62 (MBL, PM045), rabbit anti-FIP200 (SAB4200135; Sigma-Aldrich), rabbit anti-FIP200 (17250-1-AP; Proteintech), rabbit anti-ULK1 (8054; Cell Signaling Technology), rabbit anti-Atg13 (13468S; Cell Signaling Technology), rabbit anti-ATL2 (16688-1-AP; Proteintech), rabbit anti-ATL3 (ab117819; Abcam), mouse anti-WIPI2 (ab105459; Abcam), mouse anti-VAPB (66191-1-IG; Proteintech), rabbit anti-VAPA (15275-1-AP; Proteintech), mouse anti-LAMP1 (553792; BD Biosciences), rabbit anti-PTPIP51 (20641-1-AP; Proteintech), rabbit anti-ATG101 (13492; Cell Signaling Technology), rabbit anti-Myc (2278S; Cell Signaling Technology), mouse anti-Myc (M5546; Sigma-Aldrich), mouse anti-GFP (11814460001; Roche), mouse anti-actin (60008-1-IG; Proteintech), and rabbit anti-HA (H6908; Sigma-Aldrich).

Techniques:

Journal: Experimental eye research

Article Title: Retinal gliosis and phenotypic diversity of intermediate filament induction and remodeling upon acoustic blast overpressure (ABO) exposure to the rat eye

doi: 10.1016/j.exer.2023.109585

Figure Lengend Snippet: Antibodies, sources, hosts/clonality, and working dilutions of stocks.

Article Snippet: Synemin , SAB4200138 , Millipore , Sigma Rabbit polyclonal , 1:200 IHC 1:1000 WB.

Techniques: