Simultaneous monitoring of PI3P and PI(3,5)P ₂ during recruitment of the PIKfyve ^CC fragment to Rab5 endosomes. (A) Confocal images of HEK293A cells expressing mCherry-FYVE^EEA1, NES^(2x)-mNG^HO-(2x)DdSNXA^PX, iRFP-FKBP-PIKfyve^CC, and a Rab5-targeted FRB fragment (iRFP-FRB-Rab5). Rapamycin addition (100 nM) causes the appearance of the PI(3,5)P₂ reporter and the simultaneous disappearance of the PI3P sensor in the Rab5 compartment. (B) Quantification of the changes in time-lapse images for the cell shown in A. Similar results were recorded in four separate dishes in separate experiments (scale bars = 10 µm).