The ECLIPSE concept. (A) Schematic diagram showing two-hybrid ECLIPSE assay. (B) In response to rapamycin, FKBP was recruited to the endosome where FRB was already located. (C) Quantitative analysis of colocalization between FKBP and FRB on selected endosomes (n = 5). Rapamycin induction was performed at time = 0 min. (D) Schematic diagram of a three-hybrid ECLIPSE assay showing the Cdc42-WASP interaction as a case of constitutive P-P interaction. (E) ECLIPSE shows binding between constitutively active (GTP) Cdc42 and the CRIB domain of WASP, but no binding between the CRIB domain and the Cdc42 dominant-negative (GDP) form. (F) Quantitative analysis of Cdc42h-WASP interactions between WASP and a constitutively active form of Cdc42 or a dominant-negative form of Cdc42. Colocalization was measured for 5 min after induction. RFU were normalized against the fluorescence before induction (n = 8 for GTP-Cdc42; n = 4 for GDP-Cdc42; error bars indicated S.E.).(Scale bars,10 μm). Abbreviations: R, mCherry; IM, ionomycin.