BLTP2 is recruited to recycling macropinosomes undergoing fusion with the PM. (A and B) COS-7 cells showing macropinosomes (labeled by internalized fluorescent anti–MHC-I antibodies) that undergo a dramatic morphological change and acquire BLTP2^EGFP as they fuse and collapse with the PM. In B, a newly formed macropinosome at first loses PI(4,5)P₂, but then reacquires PI(4,5)P₂ as it fuses with the PM. (C) Schematic drawing of macropinosome recycling. A nascent macropinosome first loses PI(4,5)P₂ after fission from the PM, then it acquires the identity of early endosomes (PI3P, APPL, and Rab5) or regains PI(4,5)P₂ as it fuses back to the PM. (D) Stimulation of macropinocytosis by expression of HRas G12V in COS-7 cells. BLTP2^Halo is recruited to a newly formed macropinosome that loses PI(4,5)P₂ but does not acquire APPL2 and regains PI(4,5)P₂ signaling during its recycling back to the PM. Scale bar, 2 μm. (E) Schematic drawing depicting the assay to monitor macropinosome fusion with the PM. PM dye is added after the formation of macropinosomes. Preformed macropinosomes will not be labeled by the dye until they fuse with the PM. (F) A BLTP2-positive macropinosome gains access to the PM dye, showing that it fuses with the PM. Macropinocytosis was stimulated by expressing HRas G12V. Scale bar, 2 μm. (G) Acute recruitment of BLTP2^Halo to a macropinosome in the process of fusing with the PM in HeLaM cells. The macropinosome is also positive for mCherry-Rab10. Scale bar, 1 μm. (H) Schematic drawing of the recruitment of BLTP2 to a recycling macropinosome undergoing fusion with the PM. Scale bar, 1 μm.