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. 2023 Jun 8;222(9):e202205128. doi: 10.1083/jcb.202205128

Figure 6.

Figure 6.

Arf1 positive vesicle-mediated Lamp1 tubule fission events are associated with a PI(3)P increase and depletion of lysosomal PI(3)P impairs tubule fission. (A–C) (A) Normalized fluorescence intensity of PX (PI(3)P biosensor) at Lamp1 positive organelles during tubule fission events. MEFs expressing Lamp1-mCherry treated with HBSS, 8 h. (B) Events monitored in A were classified according to the presence or absence of an Arf1 positive vesicle at the fission site. Two-way ANOVA with Tukey’s multiple comparison test. (C) Representative time-lapse image of a Lamp1 fission event marked by an Arf1 positive vesicle analyzed in MEFs. Red arrows indicate fission. Scale bar: 1 µm. (D) Cartoon illustration of the GAI-GID1 dimerization system used to acutely recruit the PI(3)P phosphate MTM1 to lysosomes. (E) Representative Airyscan images of MEFs expressing LysoYFP-GID1, PX-SNAP, and CFP-GAI or CFP-GAI-MTM1 and treated with GA3-AM (10 µM for 1 h). Scale bar: 10 and 2 µm (insets). (F) Quantification of the mean fluorescence intensity of PX at LysoYFP-GID1 positive organelles normalized to cytosolic levels. Two-sided unpaired t test. (G) Representative Airyscan images of MEFs expressing LysoYFP-GID1 and CFP-GAI or CFP-GAI-MTM1 before and after treatment with GA3-AM (10 µM for 1 h) and stained with the acidic marker Cresyl violet. Scale bars: 10 and 2 µm (inset). Yellow arrows indicate tubules. (H) Quantification of the number of lysosomal tubules in cells in G. Two-way ANOVA with Tukey’s multiple comparison tests. (I) Normalized rate of tubule fission of LysoYFP-GID1 positive organelles after recruitment of indicated construct by GA3-AM treatment in starved MEFs (HBSS). Two-sided unpaired t test. (J) Representative Airyscan images of phagolysosomes from RAW 264.7 cells phagocyting SRBCs expressing iRFP-GID1-Rab7 (not imaged), Lamp1-mCherry, and CFP-GAI or CFP-GAI-MTM1 after treatment with GA3-AM (10 µM for 1 h; yellow arrows show tubules. Scale bar: 2 µm) and quantification of the number of tubules per phagolysosome. n = number of phagolysosomes. Two-sided unpaired t test. (K–M) Representative images of HeLa cells expressing Lamp1-mCherry and PX-GFP and treated with the VPS34 inhibitor VPS34-IN1 (1 µM for 1 h) or DMSO as vehicle control. Scale bar: 10 and 1 µm (inset). (L) Quantification of the levels of PX at Lamp1 positive organelles normalized to the cytosolic level of the probe. Two-sided unpaired t test. (M) Quantification of the number of Lamp1 positive tubules in these cells. BFA treatment (1 h) was used as a positive control. One-way ANOVA with Dunnett’s multiple comparison test. All graphs (A, B, F, and H–M) show the mean ± SEM, cells from three independent experiments.