Effect of PIKfyve ^CC expression on subcellular PI3P levels. (A and B) Confocal images of HEK293A cells transfected with the PI3P sensor, EGFP-(2x)FYVE^Hrs (A) or EGFP-FYVE^EEA1 (B), together with the recruitable mRFP-FKBP-PIKfyve^CC fragment. Note that cells expressing the PIKfyve^CC fragment show no PI3P-positive structures (red arrows), while those with no detectable enzyme (green arrows and outlined with dotted yellow lines) show the characteristic subcellular PI3P signal. Apilimod treatment (1 µM, 10 min) causes the reappearance of the PI3P-positive structures, suggesting that the disappearance of PI3P is due to the presence of the catalytically active PIKfyve^CC fragment. Enlarged images of cell-1 and cell-2 before and after apilimod are also shown on the right. (B) HEK293A cells were transfected with the PI3P sensor, EGFP-FYVE^EEA1, together with the recruitable mRFP-FKBP-PIKfyve^CC enzyme and a Rab5-targeted FRB fragment (iRFP-FRB-Rab5). Cells that express all three constructs and still show PI3P signal associated with their endosomes were subjected to rapamycin treatment (100 nM, 5 min), which results in rapid loss of the membrane-bound PI3P signal from the endosomes as the FKBP-PIKfyve^CC fragment is recruited to the Rab5 compartment. Apilimod treatment (1 µM, 5 min) reverses the effect of the recruited enzyme. (C) Quantification of these changes from five individual cells is shown from one representative recording (mean ± SEM). Note that PI3P (green) rapidly disappears even when the amount of recruited enzyme (red) is barely detectable (scale bars = 10 µm).