Fig. 4.

Nutrients control lysosomal positioning by modulating pH_i and lysosomal levels of KIF2 and ARL8B. (a) Starvation increases pH_i in HeLa cells allowed to starve using three different protocols (see Methods) with or without subsequent recovery. (b-d) Changing pH_i from 7.1 to 7.7 is sufficient to affect localisation of lysosomes and mTORC1 activity. pH_i was titrated in full tissue culture medium containing nigericin, which allows one to force changes in pH_i by altering pH in the medium, followed by immunostaining (b, c) or western blotting (d) using antibodies as shown. (e) Changes in lysosomal localisation have no effect on pH_i. ARL8B and KIF2 were overexpressed or knocked down in HeLa cells followed by pH_i measurement. (f, g) Nutrients and pH_i affect levels of ARL8 and KIF2 in lysosomal fractions. Protein levels and their quantification in total cellular lysates or in isolated lysosomal fractions from HeLa cells subjected to 1 h nutrient deprivation/recovery (f) or to 1 h changes of pH_i in full tissue culture medium containing nigericin (g) are shown. (h, i) Effect of nutrients and pH_i on binding of ARL8 and KIF2 to polymerised microtubules. HeLa cells treated as in panels (f) and (g), followed by isolation of polymerised microtubule fraction and western blotting. Asterisk indicates a nonspecific band.

For all panels values are means ± s.e.m. of three independent experiments performed in triplicate. All comparisons are with the control within each treatment condition, * p < 0.05, ** p < 0.01, *** p < 0.005 t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. 7.