Nucleation and polymerization properties of Vps60 on membrane. (A) Confocal images of SLBs incubated with indicated concentration of Vps60 (scale bar: 10 μm). (B and C) Quantification of Vps60 puncta per area at different Vps60 concentrations from the experiment described in A. ROI ≥ 50. (C) Time-lapse experiments of SLBs (gray) incubated with Vps60 (red, upper panel) or Snf7 (green, lower panel). Scale bar: 10 μm. (D) Confocal images of SLBs incubated with Vps60 (black) and Snf7 (green) were indicated (scale bar: 10 μm). (E) Quantification of experiments described in D (n = 3, ROI ≥ 6). (F) Confocal images of SLBs (blue) incubated with Snf7 (red) and Vps60 (green; scale bar: 2 μm; n = 3). (G) Plot of fluorescence profile of a representative membrane section from experiments described in F. (H) Confocal images of time-lapse experiment of addition of Vps60 (red) to SLBs (blue) preincubated with Snf7 (green), Vps2, Vps24, Did2, and Ist1 (n = 3). Scale bar: 10 μm. (I) Plot of fluorescence profile of an exemplary membrane section (t = 20 min) from experiments described in H.