(A) Principle of the reversible chemical dimerizer system (rCDS). Translocation of the Enzyme 5Ptase to the PM by the reversible chemical dimerizer rCD1 leads to breakdown of PI(4,5)P2 to PI(4)P. Addition of the competing ligand FK506 displaces the enzyme from the PM, which restores PI(4,5)P2 levels rapidly. See also Figure 1—figure supplement 1 and Video 1, which show reversible PI(4,5)P2 depletion by the rCDS in living cells. (B–E) Representative SDC fluorescence images of the ventral PM of HeLa Kyoto cells transfected with the Anchor (LCK-ECFP-SNAP), Enzyme (mRFP-FKBP-5Ptase) and the indicated HIV-1 derived constructs. Cells were treated with 1% DMSO (left panels) or 1 µM rCD1 (right panels) at 4 hpt. Gag was detected at 22 hpt at the ventral membranes of cells via EGFP (in living cells, pCHIV derived constructs in (B) and (C)) or by indirect immunolabeling (in fixed cells, NL4-3 derived constructs in (D) and (E)). Arrows indicate Gag clusters. Scale bar represents 20 µm. (F) Number of Gag clusters detected at the ventral membrane per 1000 µm² membrane area. Error bars represent the standard error of the mean (statistical significance was assessed with the two-tailed unpaired Student‘s t-test; ***p≤0.001) of n = 27/26; 15/18; 35/33; 29/22 cells (from n = 3; 2; 2; 2 independent experiments), respectively.