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
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