Targeting of Pan1p to the peroxisome leads to actin polymerization by recruiting other NPFs. (A) Localization of Pex3-GFP and Pex-FRB-mCherry at 0 or 30 min after rapamycin addition. White arrowheads indicate examples of co-localization. (B) Fluid-phase endocytosis in cells expressing Pan1-FKBP. Cells were labeled with 200 μM FM4-64 for 15 min on ice. The images were acquired at 5 or 10 min after FM4-64 internalization in the presence of rapamycin. The lower images show the localization of Abp1-mCherry and Pex-FRB-GFP at 30 min after rapamycin addition. Representative fluorescence intensity profiles along a line are indicated in the right panels. (C) The bar graph represents the percentages of FM4-64- or Abp1-mCherry-labeled puncta localizing at Pex-FRB-mCherry-labeled peroxisome. Data show the mean from at least three experiments, with >50 cells counted for each strain per experiment. (D) Cells expressing GFP-tagged NPF (Las17p, Myo3p, or Myo5p), Pex-FRB-mCherry, and Pan1-FKBP were grown to log phase at 25°C, and each image pair was acquired before rapamycin addition. (E) Cells expressing Abp1-GFP, Pex-FRB-mCherry, and FKBP-tagged NPF were grown and imaged, as described in A. (F) The localization of Pex3-FRB-mCherry and Abp1-GFP in cells expressing Las17-FKBP and/or Myo3/5-FKBP in the absence of rapamycin. Quantification of the percentage of Abp1-GFP co-localizing with Pex-FRB-mCherry-labeled peroxisomes is shown in the lower right panels. Data show the mean ± SD from three experiments (n = 50 puncta for each experiment). (G) The localization of Pex-FRB-mCherry and Abp1-GFP in myo3Δ or myo5Δ cells expressing Pan1-FKBP at 30 min after rapamycin addition. Scale bars, 2.5 μm.