Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – C ) The C-terminus of HscA is exposed on the surface of phagosomes. ( A ) Immunostaining of phagosomes containing conidia of hscA-myc or hscA L -myc strains isolated from A549 cells. Scale bars, 2 μm. ( B ) Relative signal intensities of the respective emission fluorescence along the lines drawn across the phagosomes shown in ( A ). ( C ) Quantification of exposure distance from a representative experiment. Data represent the mean ± SD; n = 16 individual phagosomes were analyzed. ( D and E ) Biotinylation of host cell proteins by HscA-miniTurboID (HscA-mT). ( D ) A549 cells incubated with conidia of strains hscA-mT or mT - hscA were stained with streptavidin and an antibody against RAB7. ( E ) Phagosomes with positive streptavidin (Strep + ) signal were quantified. The number of independent experiments is indicated at the bottom of the bars. ( F and G ) GAL3 is recruited to damaged phagosomes containing A. fumigatus conidia in A549 cells after 8 hours of infection. ( F ) A549 cells incubated with A. fumigatus conidia were immunostained with indicated antibodies. Scale bars, 5 μm. ( G ) Quantification of GAL3 + phagosomes containing conidia in A549 cells. ( H and I ) Phagosomal SYTOX is released to the nucleus upon damage of the phagosome membrane. ( H ) Representative A549 cells whose lysosomes were loaded with SYTOX-Green, were incubated with A. fumigatus for 16 h. CellMask and CFW were used to stain the cell membrane and A. fumigatus cell wall, respectively. ( I ) Cells with SYTOX signal in the nuclei were quantified. ( J – L ) HscA-dependent phagosomal damage in hMDMs. ( J ) hMDMs incubated with A. fumigatus conidia were immunostained with indicated antibodies. Scale bars, 5 μm. DIC, differential interference contrast. ( K ) GAL3 + phagosomes and ( L ) RAB7 + phagosomes in hMDMs were quantified. Statistics: Error bars represent the mean ± SD. For C and E, p -values were calculated using unpaired two-tailed t test. For G, I, K, and L, p -values are calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. Gray dots represent the calculated values from individual microscopic images (G, n = 42–47; I, n = 25–28; K and L, n = 46–54), and colored dots represent the summarized result of individual experiments ( n = 3).

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Immunostaining, Isolation, Fluorescence, Incubation, Staining, Infection, Membrane, Two Tailed Test

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – D ) Recruitment of TSG101 and CHMP3 to phagosomes in hMDMs. ( A ) Detection of TSG101 and GAL3, or ( B ) detection of TSG101 and CHMP3 on phagosomes containing A. fumigatus WT conidia in hMDMs. Regions indicated by white or yellow dashed-line frames are enlarged on the right or bottom, respectively. Channel intensity plots show the fluorescence signal across the yellow lines. ( C and D ) Phagosomes positive for ( C ) TSG101 and ( D ) CHMP3 were quantified. ( E – H ) Recruitment of ESCRT components to phagosomes in A549 cells. (E) Immunostaining of A549 cells incubated with A. fumigatus WT conidia, highlighting the indicated ESCRT markers. Yellow arrows mark phagosomes positive for both tested markers. DIC, differential interference contrast. ( F – H) Phagosomes positive for ( F ) CHMP3, ( G ) TSG101, and ( H ) ALG2 were quantified. A549 cells or p11-KO cells were incubated with conidia of WT or Δ hscA strains for 4 hours. Intracellular Ca 2+ was subsequently chelated by adding 25 μM BAPTA-AM to the medium, followed by an additional 4 hours of incubation at 37°C. (I) Chelation of Ca 2+ reduces the recruitment of p11 to phagosomes. ( J – L ) Recruitment of ANXA2 and ANXA1 to phagosomes. (J) A549 cells were incubated with A. fumigatus WT conidia and immunostained with antibodies against p11, ANXA2, and ANXA1. Yellow arrows indicate phagosomes positive for both tested markers, while white arrows denote a phagosome positive for ANXA2 but negative for p11. Phagosomes positive for ( K ) ANXA2 and ( L ) ANXA1 were quantified. ( M ) HscA, p11, and Ca 2+ -dependent recruitment of GAL3 to phagosomes. Statistics: Error bars represent the mean ± SD; p -values were determined using unpaired two-tailed t test (C and D) or one-way ANOVA, followed by Tukey’s multiple comparisons test. The number of individual experiments is indicated below each bar.

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Fluorescence, Immunostaining, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – C ) Deletion of fungal HscA or human host p11 gene, or chelation of Ca 2+ , increased phagosome maturation. ( A ) Immunostaining of A549 cells incubated with A. fumigatus WT conidia, highlighting the indicated phagosomal markers: RAB7 and ANXA2 on the top row, and GAL3 and LAMP1 on the bottom row. Yellow arrows label phagosomes positive for both markers, while white arrows mark phagosomes positive for a single marker. Scale bars, 5 μm. ( B and C ) Phagosomes positive for ( B ) RAB7 and ( C ) LAMP1 were quantified. ( D and E ) Activation of TFEB by p11 deletion or Ca 2+ chelation. ( D ) Immunostaining of A549 cells infected with WT conidia. Dashed-line circles indicate the regions of nuclei. Scale bars, 10 μm. ( E ) Cells with TFEB localized in the nuclei were quantified. Statistics: Error bars represent the mean ± SD; p values were determined using one-way ANOVA, followed by Tukey’s multiple comparisons test. The number of individual experiments for figures B and C is indicated at the base of each bar. For E, grey dots represent the calculated values from individual microscopic images ( n = 41–68) and colored dots represent summarized results from individual experiments ( n = 3 for BAPTA-AM-treated cells and n = 4 for untreated cells).

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Immunostaining, Incubation, Marker, Activation Assay, Infection

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: (A) Presence of Y596/Y αD in HscA/Ssb proteins of human pathogenic fungi. The phylogenic tree was constructed using the alignment of HscA orthologs and Hsp70/Ssa proteins. Branches and names of fungal species are color-coded based on the amino acid residue present in the αD domain: green for Y and yellow for F. Branches corresponding to Hsp70/Ssa are highlighted in pink. ( B and C ) C. albicans and C. glabrata cause damages to phagosomes in hMDMs. (B) hMDMs were incubated with yeast cells of C. albicans (top row) or C. glabrata (bottom row) for 2 hours and immunostained with antibody against GAL3. Scale bars, 5 μm. (C) Phagosomes positive for GAL3 were quantified. CgΔssb1 , the mutant strain of C. glabrata lacking SSB1 . ( D and E ) Increased phagosome damage caused by S. cerevisiae expressing Ssb with an L-to-Y mutation. (D) hMDMs were incubated with yeast cells of Ssb Y protein producing S. cerevisiae ( Sc-SSB Y ) or the Ssb L protein producing S. cerevisiae ( Sc-SSB L ) for 2 hours and immunostained with antibodies against GAL3 and RAB7. Yeast cell wall was stained with CFW. Arrows indicate phagosomes positive for both markers. Regions indicated by dashed-line frames are enlarged on the right. Channel intensity plots show the fluorescence signal across the yellow lines. Scale bars, 5 μm. (E) The yeast cells containing phagosomes that are positive for GAL3 were quantified. Sc , S. cerevisiae WT strain; Sc-AD , S. cerevisiae strain transformed with control vector; Sc-AfHscA , S. cerevisiae strain producing A. fumigatus HscA; Sc-SSB F , S. cerevisiae strain producing Ssb F , Sc-AGA2 , S. cerevisiae strain producing Aga2p. Statistics: Error bars represent the mean ± SD of pooled calculated values, indicated by grey dots, from individual microscopic images ( n = 60 for C. albicans and C. glabrata ; n = 59 for CgΔssb1 ; n = 75–81 for strains of S. cerevisiae ). P- values were calculated using an unpaired two-tailed t test ( C ) or one-way ANOVA followed by Tukey’s multiple comparisons test ( E ). Colored dots represent summarized results from individual experiments ( n = 3 for C and n = 4 for E).

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Construct, Residue, Incubation, Mutagenesis, Expressing, Staining, Fluorescence, Transformation Assay, Control, Plasmid Preparation, Two Tailed Test

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) Scheme depicting proximity biotinylation of proteins in response to recruitment of LGALS1, LGALS3, and LGALS8 proteins to damaged lysosomes. ( B ) Experimental workflow for galectin proximity biotinylation. APEX2-tagged LGALS1, LGALS3, and LGALS8 expressed in HeLa cells were subjected to proximity biotinylation 60 min post-LLOMe treatment using 10-plex TMT. ( C ) Volcano plot for LLOMe (60 min)-treated cells versus untreated cells (Log 2 FC versus −Log 10 p-value) for APEX-LGALS8-based proximity biotinylation based on the TMT experiment in (B). Specific categories of proteins are indicated by colored circles. ( D ) Volcano plot for LLOMe (60 min)-treated cells versus untreated cells (Log 2 FC versus −Log 10 p-value) for APEX-LGALS3-based proximity biotinylation based on the TMT experiment in B. Specific categories of proteins are indicated by colored circles. ( E ) Volcano plot for LLOMe (60 min)-treated cells versus untreated cells (Log 2 FC versus −Log 10 p-value) for APEX-LGALS1-based proximity biotinylation based on the TMT experiment in B. Specific categories of proteins are indicated by colored circles. ( F ) Log 2 FC for individual proteins localized to the lysosomal compartment found to be enriched in biotinylated proteins from cells expressing the indicated APEX2-galectin protein. Mean and standard deviation are calculated from two untreated and two treated biological replicates. ( G ) GO: process enrichment categories for APEX2-LGALS8. Figure 3—source data 1. Log 2 FCs for lysosomal proteins from APEX2-LGALS1, 3, and 8 for . Figure 3—source data 2. GO enrichments for APEX2-LGALS8 for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Expressing, Standard Deviation

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) Summary of proteins in proximity to galectins and integration with associations found with APEX2-ATG8 (bold) and Lyso-IP (underline). Other functional classes are indicated. ( B ) Localization of LGALS3 with LAMP1 and MAP1LC3B in response to lysosomal damage. Cells were treated with LLOMe for 1 hr and the LLOMe washed out for 4 hr prior to immunofluorescence using the indicated antibodies and imaging by confocal microscopy. Scale bars 10 μm. Zoom-in panels, 10 μm × 10 μm. ( C ) Cells were left untreated (Untreated), treated with LLOMe for 1 hr and either fixed (LLOMe 1 hr) or the LLOMe was washed out for 4 hr prior to fixation (LLOMe 1 hr+ washout 4 hr). Immunofluorescence was done using α-OPTN/α-LAMP1 and imaging by confocal microscopy. Scale bars 10 μm. Zoom-in panels, 10 μm × 10 μm. ( D ) Cells were treated as in (C). Immunofluorescence was done using α-TAX1BP1/α-LAMP1 and imaging by confocal microscopy. Scale bars 10 μm. Zoom-in panels, 10 μm × 10 μm. ( E ) Cells were treated as in (C). Immunofluorescence was done using α-CALCOCO2/α-LAMP1 and imaging by confocal microscopy. Scale bars 10 μm. Zoom-in panels, 10 μm × 10 μm. ( F ) Quantification of OPTN localization at LAMP1 lysosomes using Mander’s overlap coefficient (MOC). 23 (0 hr), 19 (1 hr), and 22 (4 hr washout) cells were analyzed for MOC. ***p < 0.001. + marks the mean and the line marks the median. The plot represents merged data from three biological replicates for each condition. ( G ) Quantification of TAX1BP1 localization at LAMP1 lysosomes using Mander’s overlap coefficient (MOC). 20 (0 hr), 17 (1 hr), and 22 (4 hr washout) cells were analyzed for MOC. ***p < 0.001. + marks the mean and the line marks the median. The plot represents merged data from three biological replicates for each condition. ( H ) Quantification of CALCOCO2 localization at LAMP1 lysosomes using MOC. 18 (0 hr), 20 (1 hr), and 21 (4 hr washout) cells were analyzed for MOC. **p < 0.01 and ***p < 0.001. + marks the mean and the line marks the median. The plot represents merged data from three biological replicates for each condition. Figure 4—source data 1. Mander’s overlap coefficient (MOC) values for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Functional Assay, Immunofluorescence, Imaging, Confocal Microscopy

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) Scheme depicting measurement of lysophagic flux using Lyso-Keima (Keima-LGALS3). Cells stably expressing Keima-LGALS3 are treated with LLOMe (1 hr), and the Keima-LGALS3 is recruited from the cytosol to damaged lysosomes, representing the initial recruitment step (green dot). After removing LLOMe (washout), damaged lysosomes undergo autophagy-dependent trafficking to a healthy lysosome, leading to a red-shift in Keima fluorescence (red dots) due to the acidic environment of the lysosome. Cells can be analyzed by imaging, flow cytometry or SDS–PAGE for processed Keima. ( B ) Keima-LGALS3 in untreated HeLa cells or in cells that were treated with LLOMe for 1 hr and the LLOMe washed out for 4 or 12 hr and imaged using excitation at 442 or 561 nm. Scale bar 10 μm. Zoom-in panels, 10 μm × 20 μm. ( C ) Keima-LGALS3 HeLa cells were either left untreated or treated for 1 hr followed by washout (12 hr) with or without prior addition of TBK1i or BafA. Cells were imaged using excitation at 442 or 561 nm. A ratio of the 561 nm/442 nm images was taken and puncta were identified from this 561 nm/442 nm image. Scale bar 10 μm. Zoom-in panels, 10 μm × 20 μm. ( D ) Quantification of Keima-positive lysosomes. 69 (untreated), 83 (BafA), and 66 (TBKi) cells were analyzed ****p < 0.0001. + marks the mean and the line is at the median. The plot represents merged data from three biological replicates. ( E ) Triplicate HeLa cells expressing Keima-LGALS3 were either left untreated or treated for 1 hr followed by washout (12 hr) with or without addition of BafA. Cells were then subjected to flow cytometry to measure the 561 nm/488 nm ratio. All values are normalized to the untreated sample. ****p < 0.0001. The plot represents mean and standard deviation from three biological replicates. ( F ) Triplicate HeLa cells expressing Keima-LGALS3 were either left untreated or treated for 1 hr followed by washout (12 hr) with or without prior addition of TBK1i, ULK1i, TAK243, and p97i. Cells were then subjected to flow cytometry to measure the 561 nm/488 nm ratio. All values are normalized to the untreated sample. ****p < 0.0001. The plot represents mean and standard deviation from three biological replicates. ( G ) HeLa cells expressing Keima-LGALS3 were either left untreated or treated for 1 hr followed by washout followed by harvesting at the indicated times. Lysed cells were then subjected to immunoblotting with the indicated antibodies. ( H ) Cells were left untreated (Untreated), treated with LLOMe for 1 hr and either fixed (LLOMe 1 hr) or the LLOMe was washed out for 4 hr prior to fixation (LLOMe 1 hr + washout 4 hr). Immunofluorescence was done using α-pTBK1/α-LAMP1 and imaging by confocal microscopy. Scale bar = 10 μm. Zoom-in panels, 10 μm × 10 μm. Right: quantification of localization using Mander’s overlap coefficient (MOC). 23 (0 hr), 21 (1 hr), and 18 (4 hr washout) cells were analyzed for MOC. ***p < 0.001. + marks the mean and the line is at the median. The plot represents merged data from three biological replicates. ( I ) HeLa cells expressing Keima-LGALS3 were either left untreated or treated with LLOMe for 1 hr and then incubated for four or 12 hr post-washout in the presence or absence of either BafA or TBK1i. Cell lysates were subjected to immunoblotting using the indicated antibodies. ( J ) Triplicate WT, ATG5 −/− , or TBK1 −/− HeLa cells expressing Keima-LGALS3 were either left untreated or treated for 1 hr followed by washout (4 hr) prior to flow cytometry to measure the 561 nm/488 nm ratio. All values are normalized to the untreated sample within each genotype. The plot represents mean and standard deviation from three biological replicates. Figure 5—source data 1. Quantification of Keima-positive lysosomes. Figure 5—source data 2. 561/488 Keima ratios for . Figure 5—source data 3. 561/488 Keima ratios for . Figure 5—source data 4. Uncropped blots for . Figure 5—source data 5. MOC values for . Figure 5—source data 6. 561/488 Keima ratios for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Stable Transfection, Expressing, Fluorescence, Imaging, Flow Cytometry, SDS Page, Standard Deviation, Western Blot, Immunofluorescence, Confocal Microscopy, Incubation

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) Raw flow cytometry data. HeLa cells expressing Keima-LGALS3 were either left untreated (red), or treated for 1 hr followed by washout (12 hr) with (orange) or without (blue) addition of BafA. Cells were then subjected to flow cytometry to measure the 561 nm/488 nm ratio. ( B ) HeLa cells expressing Keima-LGALS3 were treated with LLOMe (1 hr) prior to washout for 4 or 8 hr. In one set of samples, the E1 inhibitor TAK243 at 2 μM was added prior to damage. Cell extracts at the indicated time were subjected to immunoblotting with the indicated antibodies. Figure 5—figure supplement 1—source data 1. Uncropped blots.

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Flow Cytometry, Expressing, Western Blot

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) Triplicate WT; ATG5 −/− ; or OPTN −/− ; TAX1BP1 −/− ; CALCOCO2 −/− (TKO) HeLa cells expressing Keima-LGALS3 were either left untreated or treated for 1 hr followed by washout (12 hr) prior to flow cytometry to measure the 561 nm/488 nm ratio. All values are normalized to the untreated sample within each genotype. The plot represents mean and standard deviation from three biological replicates. ( B ) Triplicate WT or TKO HeLa cells expressing Keima-LGALS3 were reconstituted with lentivirally expressed EGFP-FLAG-HA, EGFP-CALCOCO2, EGFP-OPTN, or EGFP-TAX1BP1. Cells were either left untreated or treated for 1 hr followed by washout (12 hr) prior to flow cytometry to measure the 561 nm/488 nm ratio. As a control for lysophagic flux, some samples were also treated with BafA during the washout. All values are normalized to the untreated sample within each genotype. ****p < 0.0001. The plot represents mean and standard deviation from three biological replicates. ( C ) Cells from panel B were lysed and subjected to immunoblotting with the indicated antibodies. ( D ) HeLa cells expressing Keima-LGALS3 (with or without deletion of ATG7, TAX1BP1, OPTN, CALCOCO2, or SQSTM1) were either left untreated or treated for 1 hr followed by washout (12 hr) prior to flow cytometry to measure the 561 nm/488 nm ratio. All values are normalized to the untreated sample within each genotype. ****p < 0.0001. The plot represents mean and standard deviation from three biological replicates. ( E ) HeLa cells (with or without deletion of TAX1BP1, OPTN, CALCOCO2, or SQSTM1) were either left untreated or treated for 1 hr followed by washout (10 hr) prior to immunostaining with α-LAMP1 (green) and α-LGALS3 (magenta). The number of LGALS3 puncta per cell present after washout is plotted (right top panel). The block to lysophagic flux was rescued by expression of EGFP-TAX1BP1 but not EGFP (lower right panel). 41 (WT), 21 (TAX1BP1), 25 (OPTN), 21 (CALCOCO2), and 27 (SQSTM1) cells were analyzed in the upper graph. 29 (WT), 28 (EGFP), and 32 (EGFP-TAX1BP1) cells were analyzed in the bottom graph. ****p < 0.0001. Scale bar 10 μm. Zoom-in panels, 10 μm × 10 μm. + marks the mean and the line is at the median. The plot represents merged data from three biological replicates. Figure 6—source data 1. 561/488 Keima ratios for . Figure 6—source data 2. 561/488 Keima ratios for . Figure 6—source data 3. 561/488 Keima ratios for . Figure 6—source data 4. The number of galectin puncta per cell post-washout for . Figure 6—source data 5. Uncropped blots for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Expressing, Flow Cytometry, Standard Deviation, Control, Western Blot, Immunostaining, Blocking Assay

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) RFP-EGFP-LGALS3 is trafficked to lysosomes in iNeurons. ES cells expressing RFP-EGFP-LGALS3 via a PiggyBac vector were converted to iNeurons using inducible NGN2 (see Materials and methods) and imaged for EGFP, RFP, and LAMP1 using α-LAMP1 antibodies. While EGFP signal was diffusely localized in the soma, RFP-positive puncta colocalized with lysosomes based on colocalization with LAMP1 staining, indicating that a subset of the RFP-EGFP-LGALS3 protein is trafficked to the lysosome under basal conditions. Scale bar = 20 μm. iN soma zoom-in panels, 30 μm × 40 μm. ( B ) iNeurons expressing RFP-EGFP-LGALS3 were either left untreated, treated with LLOMe for 1 hr, or treated with LLOMe for 1 hr followed by a 12 hr washout. Cells were imaged for EGFP and RFP and the number of EGFP puncta per cell quantified. Loss of EGFP puncta during the washout period is indicative of lysophagic flux. Scale bar = 20 μm. ( C ) Quantification of EGFP puncta per cell after washout from experiments in panel B. The average EGFP puncta per cell was 0.289 at 0 hr (45 cells), 6.46 at 1 hr LLOMe (55 cells) and 0.652 at 12 hr washout after LLOMe (66 cells). ****p < 0.0001. + marks the mean and the line is at the median. The plot represents merged data from three biological replicates. ( D ) iNeurons were subjected to LLOMe treatment and washout as in panel B but treated with or without TBK1i, VPS34i, or BafA during the washout period. Cells were imaged for EGFP and RFP. Scale bar = 20 μm. iN soma zoom-in panels, 30 μm × 40 μm. ( E ) Quantification of EGFP puncta per cell from the experiment in panel D. The average EGFP puncta per cell at 12 hr washout was 0.65 with no inhibitor (66 cells), 5.14 with BafA (49 cells), 11.95 with VPS34i (44 cells), and 5.84 with TBKi (63 cells). ****p < 0.0001, ***p < 0.001. + marks the mean and the line is at the median. The plot represents merged data from three biological replicates. ( F ) WT, TAX1BP1 −/− , or TAX1BP1 −/− ; OPTN −/− iNeurons were subjected to LLOMe treatment and washout as in panel B. Cells were imaged for EGFP and RFP. Scale bar = 10 μm. iN soma zoom-in panels, 20 μm × 20 μm. ( G ) Quantification of EGFP puncta per cell from the experiment in panel F. The average EGFP puncta per cell at 12hr washout after LLOMe for wild-type cells was 0.474 (38 cells), for TAX1BP1 −/− cells was 4.36 (62 cells) and for TAX1BP1 −/− ; OPTN −/− cells was 4.03 (39 cells). ****p < 0.0001, **p< 0.01. + marks the mean and the line is at the median. The plot represents merged data from three biological replicates. Figure 7—source data 1. The number of GFP puncta per cell for . Figure 7—source data 2. The number of GFP puncta per cell for . Figure 7—source data 3. The number of GFP puncta per cell for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Expressing, Plasmid Preparation, Staining

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) iNeurons stably expressing RFP-EGFP-LGALS3 R186S were either left untreated, treated with LLOMe for 1 hr, or treated with LLOMe for 1 hr followed by a 12hr washout. Cells were imaged for EGFP and RFP. Scale bar = 10 μm. Zoom-in panels, 10 μm × 10 μm. ( B ) Quantification of GFP puncta per cell after washout from experiments in panel A demonstrates the absence of GFP-positive puncta in response to lysosomal damage. + marks the mean and the line is at the median. The plot represents data from one replicate. ( C ) RFP-positive puncta in cells expressing RFP-EGFP-LGALS3 WT or the R186S mutant demonstrates comparable number of puncta. + marks the mean and the line is at the median. The plot represents data from one replicate. ( D ) TAX1BP1 lesion read by Illumina Miseq analysis. ( E ) Extracts from WT or TAX1BP1 −/− ES cells were immunoblotted with the indicated antibodies to demonstrate deletion of TAX1BP1. ( F ) OPTN lesion read by Illumina Miseq analysis. ( G ) Extracts from WT, TAX1BP1 −/− , or TAX1BP1 −/− ; OPTN −/− ES cells were immunoblotted with the indicated antibodies to demonstrate deletion of OPTN and TAX1BP1 . Figure 7—figure supplement 1—source data 1. The number of EGFP puncta per cell for . Figure 7—figure supplement 1—source data 2. Uncropped blots for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Stable Transfection, Expressing, Mutagenesis

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet: ( A ) Domain structure of TAX1BP1 showing the location of mutations examined in this study. ( B ) Domain structure of OPTN showing the location of mutations examined in this study. ( C ) HeLa TKO cells expressing Keima-LGALS3 were infected with lentiviruses expressing GFP-tagged WT or mutant TAX1BP1 proteins to obtain stable expression. Cells in biological triplicate were either left untreated or treated for 1 hr followed by washout (12 hr) prior to flow cytometry to measure the 561 nm/488 nm ratio. All values are normalized to the untreated sample within each genotype. The plot represents mean and standard deviation from three biological replicates. ****p < 0.0001 ( D ) Immunoblot of cell extracts from panel C probed with α-TAX1BP1 or α-actin as a loading control. Note that some mutants are highly stabilized, as reported previously . The EGFP-TAX1BP1 CC2Δ mutant is not detected by western blot due to the loss of the epitope-binding site of the antibody, nevertheless is detected by FACS . ( E ) HeLa TKO cells expressing Keima-LGALS3 were infected with lentiviruses expressing GFP-tagged WT or mutant OPTN proteins to obtain stable expression. Cells in biological triplicate were either left untreated or treated for 1 hr followed by washout (12 hr) prior to flow cytometry to measure the 561 nm/488 nm ratio. The plot represents mean and standard deviation from three biological replicates. ****p < 0.0001. ( F ) Immunoblot of cell extracts from panel E probed with α-GFP or α-actin as a loading control. ( G ) HeLa TKO cells were infected with lentiviruses expressing EGFP-tagged WT or mutant TAX1BP1 proteins to obtain stable expression. Cells were either left untreated or treated for 1 hr with LLOMe followed by washout. Cells were harvested at the indicated times and subjected to immunoblotting with the indicated antibodies. ( H ) HeLa TKO cells were infected with lentiviruses expressing EGFP-tagged WT or mutant OPTN proteins to obtain stable expression. Cells were either left untreated or treated for 1 hr with LLOMe followed by washout (12 hr). Cells were harvested at the indicated times and subjected to immunoblotting with the indicated antibodies. ( I ) Model figure. Lysosomal rupture leads to the parallel recruitment of galectins and unleashes a wave of ubiquitination on the lysosome (Steps 1a and b). In step 2, ubiquitination promotes the recruitment of both OPTN-TBK1 and TAX1BP1-TBK1-RB1CC1 complexes to the damage lysosome, thereby promoting de novo phagophore formation and local TBK1 activation to drive efficient lysophagy (Steps 3–5). Figure 8—source data 1. 561/488 Keima ratios for . Figure 8—source data 2. 561/488 Keima ratios for . Figure 8—source data 3. Uncropped blots for .

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Expressing, Infection, Mutagenesis, Flow Cytometry, Standard Deviation, Western Blot, Control, Binding Assay, Ubiquitin Proteomics, Activation Assay

Journal: eLife

Article Title: Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

doi: 10.7554/eLife.72328

Figure Lengend Snippet:

Article Snippet: Antibody , Galectin-3/ LGALS3 (Rabbit Antibody, polyclonal) , Proteintech , 60207–1-I; RRID: AB_10951109 , IF (1:300), WB (1:1000).

Techniques: Immunofluorescence, Magnetic Beads, Peptide Fractionation, Protease Inhibitor, Recombinant, Staining, Plasmid Preparation, Software