Lifeact sequence design.
(A) Frequency of amino acids in the top 100 designs produced by Rosetta (left panel) and predicted structure of the E16R mutant (right panel). The WT structure is included for comparison. (B) Cosedimentation of F-actin and 3- μM Lifeact–mCherry proteins detected by SDS-PAGE. The upper band corresponds to Lifeact–mCherry, and the lower band corresponds to actin. Representative stain-free gels are shown. The uncropped gels can be found in S4 Fig. (C) The fractions of Lifeact–mCherry that cosedimented with F-actin were quantified by densitometry and plotted versus actin concentrations. The error bars in panel C correspond to standard deviations of 3 independent experiments. (D) Representative images of cells expressing the indicated Lifeact variants are shown on the left panel. The total fluorescence signal of Lifeact–mCherry from the 1- μm2 square region consisting of an F-actin-rich patch was divided by the total fluorescence signal of phalloidin in the same region and plotted on the right panel. (E) The total fluorescence signal of Lifeact–mCherry from the 0.25- μm2 square region (as shown on the left panel) consisting of an F-actin-rich patch was divided by the total fluorescence signal of Lifeact–mCherry in the F-actin-lacking region (“background”) and plotted on the graph on the right panel. A total of 15 patches from 2 independent experiments were used to prepare right panels on (D) and (E). For statistical analysis, the unpaired t test was used. ****p < 0.0001; ns, not significant. Scale bars, 1 μm. Data points that were used to create graphs are reported in S2 Table. F-actin, filamentous actin; pel, pellet; sup, supernatant; WT, wild-type.