PI4P regulates BLTP2-dependent contacts of the ER with tubular endosomes. (A) PM-connected tubular endosomes in HeLaM cells are positive for PI4P (labeled by iRFP-P4M) and PI(4,5)P₂ (labeled by GFP-PH_PLCδ1) markers. (B) Design of the rapamycin-dependent dimerization assay to recruit the 4-phosphatase domain of Sac1 (target PI4P) or the 5-phosphatase domain of INPP5E (target PI(4,5)P₂) to PM-connected tubular endosomes. (C and D) BLTP2^Halo disassociates from tubular endosomes after PI4P depletion on their membranes following the recruitment of RFP-FKBP-Sac1. In contrast, BLTP2^Halo shows no clear change after PI(4,5)P₂ depletion following the recruitment of RFP-FKBP-INPP5E. In the quantification graphs shown at the bottom of the two panels, pairs of dots connected by a dashed line represent the total tubular BLTP2 fluorescence in each cell before and after the addition of rapamycin. Paired t test. Sac1: n = 31 cells. INPP5E: n = 37 cells. Data are from two independent experiments. (E) The loss of BLTP2 tubular fluorescence after phosphatase recruitment was quantified by dividing the total tubular fluorescence intensity in each cell after the addition of rapamycin by the value obtained before rapamycin. Points below the dashed line at y = 1 indicate a decrease of the intensity after phosphatase recruitment. Unpaired t test. n = 31 cells for Sac1 and n = 37 cells for INPP5E. Both are from two independent experiments. Box and whiskers indicate min to max. (F) Endogenous BLTP2 also undergoes disassociation from tubular endosomes in response to treatment with A1. (G) Cells in Fare quantified for the presence of BLTP2 tubules. Two-tailed t test. Mean ± SEM. n = 3 independent experiments. Non-treated (NC): 85 cells; A1 treated: 118 cells. (H) BLTP2^Halo gradually disassociates from tubular endosomes after PI4KIIIα inhibition in response to addition of the A1 compound. (I) mCh-Rab10 tubular endosomes persist after BLTP2^Halo disassociation from them in response to A1 treatment.