BRET-based measurements of PI3P and PI(3,5)P ₂ changes within Rab5-positive endosomes at the cell population scale. (A) Cartoon showing the design of the BRET construct used for monitoring PI3P levels in the Rab5-positive endosomes (also see Baba et al. [2019]; Kim et al. [2022]; Pemberton et al. [2020]). (B) HEK293A cells were transfected with the high-affinity BRET reporter monitoring PI3P in the Rab5-positive compartments (sLuc-(2x)FYVE^Hrs-tPT2A-mVenus-Rab5) together with the iRFP-FRB-Rab5 and either iRFP-FKBP-PIKfyve^CC enzyme or a catalytically inactive (K1877A) variant (see Materials and methods for further details and the cartoon presented on the right). BRET values from rapamycin-treated cells were normalized to the identical transfection regime in cells treated only with DMSO. Times of treatment with rapamycin (100 nM) or apilimod (1 µM) are indicated on each trace. Note that the active enzyme (green traces) already decreases PI3P levels compared with cells expressing the inactive version (blue traces), but their PI3P level returns to the same baseline level after apilimod treatment. Recruitment of the FKBP-PIKfyve^CC enzyme to Rab5-positive compartments quickly decreases the residual PI3P levels (red traces), while apilimod treatment rapidly reverses these effects. Grand averages (± SEM) are presented from three experiments, with distinct treatments performed in triplicate within each replicate. (C) Comparison of the single and tandem DdSNXA^PX BRET-based biosensors (see cartoon on the right). BRET measurements were performed as described for B but replacing the (2x)FYVE^Hrs domain with either the NES^(2x)-equipped single (sLuc-DdSNXA^PX-tPT2A-mVenus-Rab5) or tandem (sLuc-(2x)DdSNXA^PX-tPT2A-mVenus-Rab5) versions of the DdSNXA^PX domain in the Rab5-targeted BRET construct. Note the larger total signal obtained with the tandem version of the reporter. Grand averages (± SEM) are presented from three experiments, with distinct treatments performed in triplicate within each replicate. (D) BRET-based measurements of PI(3,5)P₂ generation within Rab5-positive compartments in response to the FKBP-PIKfyve^CC recruitment using the NES^(2x)-(2x)DdSNXA^PX reporter. Note the rapid reversal of the PI(3,5)P₂ increase after the administration of apilimod and the lack of effects associated with recruitment of the catalytically inactive enzyme. (E) Similar experiment showing that pretreatment of cells with apilimod (1 µM, 30 min) completely prevents the ability of the FKBP-PIKfyve^CC recruitment to increase the levels of the PI(3,5)P₂ reporter. Grand averages (± SEM) are presented from three experiments, with distinct treatment performed in triplicate within each replicate.