Leep1 interacts with the Scar complex to regulate leading edge activity. (A) Under cross-linking conditions, PirA and ScrA coimmunoprecipitated specifically with GFP-Leep1. (B) The interaction between GFP-Leep1 and Scar depended on cross-linking. (C) PirA and ScrA coimmunoprecipitated with GFP-Leep1^AA. (D and E) Time-lapse imaging of cells expressing GFP-Leep1 or GFP-Leep1^1-623 from a stable copy integrated into the genome of leep1⁻ via restriction enzyme-mediated integration. Arrows point to macropinocytic cups. (F–H) Flow cytometry analysis of cells incubated with TRITC-dextran for 30 min. Each sample represents ∼50,000 cells. (I) Time-lapse imaging of PirA-GFP during under-agarose chemotaxis. PirA-GFP was expressed from a stable single copy integrated into the genome. (J) Quantification of the average lifetimes of PirA-GFP patches in PirA-GFP^REMI/pirA⁻ (n = 33) and PirA-GFP^REMI/pirA⁻leep1⁻ (n = 30) cells. (K and L) Time-lapse imaging of PirA-GFP^REMI/pirA⁻ cells expressing RFP-Leep1 during under-agarose chemotaxis. Arrows point to the accumulation of fluorescent fusion proteins. Graphs in F–H are from one representative experiment out of three independent experiments. Data in J were obtained from two independent experiments, and n represents the number of cells quantified. The scatter plots show data points with means and SEM. Significance was determined by t test with Welch's correction (**, P < 0.01). Scale bar = 10 µm. IP, immunoprecipitation; A.U., arbitrary units.