Effect of PIKfyve ^CC expression on cellular PI(3,5)P ₂ levels as monitored by the PX domain of D. discoideum (Dd) SNXA. (A) Confocal images of HEK293A cells transfected with the PI(3,5)P₂ sensor, mNG^HO-DdSNXA^PX, together with the recruitable mRFP-FKBP-PIKfyve^CC enzyme and Rab5-targeted FRB fragment (iRFP-FRB-Rab5). Expression of the mNG^HO-DdSNXA^PX shows enrichment associated with the nucleus and nucleolus, as well as in the centrosomal area, but no endosomal localization. The addition of rapamycin (100 nM, 5 min) causes rapid association of the enzyme with the Rab5-positive endosomes and a corresponding increase in the local mNG^HO-DdSNXA^PX signal. Endosomal association of the mNG^HO-DdSNXA^PX signal is reversed by treatment with apilimod (1 µM, within 10 min), but the nuclear- and centrosome-associated signals remain. The left panels show higher magnification images of the boxed inset. (B) Experiment performed as in A but using the NES^(2x)-mNG^HO-(2x)SNXA^PX construct as the PI(3,5)P₂ reporter. (C) Quantification of the changes from six cells obtained in one representative time-lapse recording (mean ± SEM). Note that PI(3,5)P₂ (green) already appears even when the amount of recruited enzyme (red) is barely detectable (scale bars = 10 µm).