SLO pores trigger rapid endocytosis in the presence of Ca²⁺. (A–C) Live confocal imaging showing rapid endocytosis in HeLa cells expressing YFP-GPI (green) on the plasma membrane. (A) After exposure to 250 ng/ml SLO and PI (red), the two cells analyzed initiated endocytosis 5–15 s after pore formation (indicated by PI influx). The graph shows quantification of PI fluorescence and newly formed YFP-GPI intracellular spots. See Video 4. (B) Kymographs of YFP-GPI fluorescence along a line drawn across the periphery of cells imaged for 3 min. The red dashed line indicates when 100 μg/ml SLO was locally added. Endosomes (dark pixels) were first detected 5–10 s after SLO addition. See Video 5. (C) HeLa cells expressing YFP-GPI (green) and exposed to Cherry-SLO (red) locally with or without Ca²⁺. Individual spots containing both YFP-GPI and Cherry-SLO seen moving into the cell are indicated (the arrowheads, short arrows, and long arrows point to each of three examples of these spots). See Videos 6 and 7 . Bars: (A and B) 10 μm; (C) 5 μm.