Figure 7.

The Arf1-PI4KIIIβ positive vesicle localized protein SEC14L2 contributes to the fission of lysosomal tubules. (A) Representative image of a HeLa cell expressing SEC14L2-mCherry, GFP-PI4KIIIβ, and Arf1-SNAP. Excess cytosolic signal was removed by permeabilization with digitonin before fixation. Red circles show SEC14L2 colocalization with Arf1-PI4KIIIβ positive vesicles. Scale bar: 10 and 1 µm (inset). (B) Normalized SEC14L2 mRNA levels in cells treated with the indicated siRNAs. (C and D) Representative images of Lamp1 positive organelles morphology in HeLa cells expressing Lamp1-mCherry and treated with the indicated siRNAs (C) and quantification of the number of Lamp1 tubules in these cells (D). Scale bar: 10 and 1 µm (inset). BFA treatment (10 µg/ml for 1 h) was used as a positive control. One-way ANOVA with Dunnett’s multiple comparison test. (E and F) Representative images of lysosomes (Cresyl violet positive Lamp1 organelles) in HeLa cells treated with the indicated siRNAs (E) and quantification of the number of lysosomal tubules (F). Scale bar: 10 and 1 µm (inset). Two-sided unpaired t test. (G) Quantification of the number of Lamp1 positive tubules in HeLa cells expressing cytosolic mCherry or the zebrafish SEC14L2 homologue zSec14l3-mCherry after treatment with the indicated siRNAs. Two-way ANOVA with Tukey’s multiple comparisons test. ns: P value = 0.8998. (H) Representative images of HeLa cells treated with the indicated siRNAs and incubated with 30 mM sucrose for 24 h to promote the formation of endolysosomes (sucrosomes) before and after 2 h Invertase (0.5 mg/ml) as indicated. Scale bars: 10 and 1 µm (inset). (I) Quantification of the number of Lamp1 positive tubules in these cells in H. Two-way ANOVA with Tukey’s multiple comparisons test. (J and K) Normalized rate of Lamp1 tubule fission in cells treated with the indicated siRNAs after prolonged starvation (HBSS 8 h; J) or 1 h inhibition of PIKfyve (YM201636) and washout of the drug (K). Two-sided unpaired t test. (L) Quantification of the number of Lamp1 tubule fission events marked by an Arf1 positive vesicle in cells treated with indicated siRNAs and starved (HBSS) for 8 h. (M) Representative images of cells expressing CFP-LC3 and Lamp1-mCherry treated with the indicated siRNAs and incubated in HBSS for the indicated time. Scale bars: 10 and 1 µm. (N) Quantification of the number of enlarged autolysosomes (>1 µm²) of cells in M. Two-way ANOVA with Tukey’s multiple comparison tests. All graphs (B, D, F, G, I–K, and N) show the mean ± SEM, cells from three independent experiments.