Vps60 recruits ESCRT-III subunits to membrane. (A and B) Quantification of Vps2 fluorescence (A, n ≥ 3, ROI ≥ 23) and confocal images (B) of Vps60-covered SLBs incubated with Alexa488-Vps2 and the indicated protein mixture. (C and D) Quantification of Vps24 fluorescence (C, n = 3, ROI ≥ 24) and confocal images (D) of Vps60-covered SLBs incubated with Alexa488-Vps24 and the indicated protein mixture. (E) Quantification of Did2 fluorescence of Vps60-covered SLBs incubated with Alexa488-Vps24 and the indicated protein mixture (n = 3, ROI ≥ 24). (F) Quantification of Ist1 fluorescence of Vps60-covered GUVs incubated with Alexa488-Vps24 and the indicated protein mixture (n = 3, ROI ≥ 24). (G) Kymographs and quantification of time-lapse experiments of Vps60-covered SLBs incubated with Alexa488-Vps24 (purple), Alexa488-Vps2 (red), Alexa488-Did2 (yellow), and Alexa488-Ist1 (cyan; n ≥ 3, ROI ≥ 24). (H) Schematic representation of the model for sequential binding of ESCRT-III subunits to Vps60-polymers (n ≥ 3, ROI ≥ 29). Scale bar: 10 μm.