Regardless of the mechanism, PIKfyve seems to have an impact on the number of acidified organelles in the cell and therefore on the cellular capacity to down-regulate certain biomolecules in lysosomes . It will be important to analyse the impact of PIKfyve inhibition on the endosomal system in greater detail in future work if we are to establish which aspects of endo/lysosomal dysfunction lead to the profound neurodegeneration observed in patients with PIKfyve pathway deficiency. Acknowledgments We thank Dr Xu for kindly providing the GFP-ML1Nx2 probe. restricted to the rim of vacuoles. Additionally, Ho et al.  found that endo/lysosomal pH appeared to be unaffected by PIKfyve inhibition using ratiometric pH detection with FITC dextran. As the question whether PIKfyve controls endo/lysosomal acidification is important, we attempted to clarify whether this depends on PIKfyve. We utilized lysotracker DND-99 to analyse acidification of organelles and performed PIKfyve inhibition using Apilimod at 25?nM and 250?nM for 4?h. From the lysotracker staining it was evident that small, acidic vesicles remained in the cytoplasm of PIKfyve inhibited cells, consistent with the analysis of Ho et al. . However, there was a marked reduction in their number. Using image segmentation, we automatically detected and quantified the average number of lysotracker positive vesicles in cells and found them to be reduced by approximately 50% with both Apilimod concentrations (Figures 4A and ?and4B).4B). We also noted a slight decrease in the average vesicle intensity using 25?nM Apilimod and a somewhat more pronounced reduction using 250?nM Apilimod (Figure 4C). It should be noted that under both conditions there is abundant vacuole formation apparent under the light microscope, particularly using the higher Apilimod concentration (results not shown). However, we did not detect significant lysotracker staining in these vacuoles. This seems to be an important difference between the RAW macrophage line and HeLa cells utilized in the present study. Open in a separate window Figure 4 Inhibition of PIKfyve using Apilimod affects the number of acidified organelles and LampI positive late endosomes and lysosomes(A) Inhibition of PIKfyve using two different concentrations of Apilimod (25?nM and 250?nM) led to a strong decrease in the number of acidified organelles labelled with lysotracker Red DND-99. (B) Quantification of the average cellular number of lysotracker positive vesicles measured using Squassh analysis in the MOSAIC suite in ImageJ. Both 25?nM and 250?nM Apilimod significantly reduced the number of lysotracker positive vesicles. (C) Quantification of average vesicular lysotracker intensity showed a small but significant reduction in vesicular lysotracker staining with 25?nM Apilimod and a stronger reduction with 250?nM. (ACC) Data were pooled from three independent experiments, yielding a total of 309 cells per condition. Error bars are S.E.M. (D) Analysis of the LampI positive compartment upon inhibition of the PIKfyve complex using 25?nM and 250?nM Apilimod. Both concentrations of Apilimod reduced the number of LampI positive vesicles. (E) Quantification of the average number of H3F3A LampI positive vesicles per cell and its dependence on PIKfyve showed that its inhibition reduced the number of LampI positive late endosomes and lysosomes. (F) Analysis of average size of LampI positive late endosomes/lysosomes showed that although the number of LampI vesicles per cell Ligustilide was reduced, their size increased, suggesting swelling or aggregation of Ligustilide the compartment. Data presented in (E) and (F) were pooled from three independent experiments with 240 cells analysed per condition. Error bars are S.E.M. Statistical analysis in (B), (C), (E) and (F) was performed using one-way ANOVA with Tukey’s post-hoc test,  showed very clear mCherryCLampI localization to the membrane of these. Currently, it is unclear whether the difference stems from the different cell models used (HeLa compared with COS1) or whether from endogenous compared with overexpressed LampI. To test whether the observed effect on acidification as analysed by lysotracker staining is indeed PIKfyve specific, we also utilized YM201636 at 100? nM and 1?M for 4?h. YM201636 also reduced the number of lysotracker positive vesicles. The effect was less pronounced than with Apilimod (Figures 5A and ?and5B),5B), entirely consistent with our experience with the two PIKfyve inhibitors in which Apilimod consistently leads to a stronger phenotype than YM201636. We also used ammonia, a weak base that Ligustilide is trapped in acidic compartments and thereby reduces the concentration of free protons, increasing the organellar pH . As expected the addition of 10?mM ammonium sulfate for 4?h strongly decreased the number of acidified vesicles in cells (Figures 5A and ?and5B),5B), confirming that.
Regardless of the mechanism, PIKfyve seems to have an impact on the number of acidified organelles in the cell and therefore on the cellular capacity to down-regulate certain biomolecules in lysosomes