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. Author manuscript; available in PMC: 2011 Sep 10.
Published in final edited form as: Science. 2010 Sep 10;329(5997):1341–1345. doi: 10.1126/science.1191710

Figure 2. Membrane requirements for FLS formation.

Figure 2

(A) Time course of FLS appearance shows negatively-charged lipids are essential for FLS formation and there is specificity for PI(4,5)P2 and PS. Cyan: 60% PC/30% PS/10% PI(4,5)P2, purple: 60% PC/30% PI/10% PI(4,5)P2, orange:40% PC/60% PS, red: 55% PC/45% PS, green: 70% PC/30% PS. (B) Time course of FLS appearance. All PIPs can nucleate FLSs but there is preference for PI(4,5)P2. Compositions: 60% PC/30% PS/10% PIP, where lime:PI(4,5)P2, gray:PI(3,4)P2 magenta:PI(3,5)P2 navy:PI(3,4,5)P3, brown:PI. Data are the mean of 3 timecourses normalized to the average number of structures from the 3 experiments from 5 or more pictures over each supported bilayer. (C) Rate of actin addition (using the pulse-chase approach) for the different PIPs shows PI(4,5)P2 specificity, error bars are s.d., n = 18, 19, 16, 20. (4-way ANOVA, p<0.001). (D) Confocal microscopy of supported bilayer surface. GFP-PH domain (green) addition to supported bilayers including rhodamine-PE (red) shows membrane domains. Bar: 2 µm. The lipid composition was 45% PC, 45% PI, 10% PI(4,5)P2, similar data was obtained with 60% PC, 30% PS, 10% PI(4,5)P2. (E) FLSs grow preferentially from rhodamine-PE depleted (PH domain binding) regions. Alexa-647 actin is in green, rhodamine-PE in red. Bar: 2 µm (F) Contour plot of the number of FLSs per field of view at steady state (20 min) from fluid membranes in response to PI(4,5)P2 and extract concentrations. The lipid composition was 45% PC, 45% PI, 10% PI(4,5)P2. For comparison, overlaid single points show the number of FLSs formed from the gel phase. Data is plotted logarithmically. Example pictures and control data are in Fig. S4.