Fig. 6: Intracellular effects of phosphomimic MYO6S267E.
a Scheme highlighting the design of MYO6+, the plus-end directed mutant, in which the insert-2 (reverse gear), lever-arm extension and IQ motif are replaced with six IQ motifs of MYO5. b Filopodia numbers per cell expressing either GFP-tagged MYO6+WT, MYO6+S267A or MYO6+S267E are shown for 3 experiments. Statistical significance was determined using one-way ANOVA and post-hoc testing. ***P < 0.01. MYO6+WT n = 2786, average 65 filopodia/cell SD ± 26.834, MYO6+S267A n = 1824, average 52 filopodia/cell SD ± 9.3, MYO6+S267E n = 6163, average 121 filopodia/cell SD ± 18.62. c Filopodia length was measured in 30–50 cells per construct in 3 experiments expressing either GFP-tagged MYO6+WT, MYO6+S267A or MYO6+S267E. The lengths of filopodia were not significantly different between cells expressing MYO6+S267A n = 50, average 4.72 ± 1.82 SD, MYO6+S267E n = 55, average 5.5 ± 4.43 SD and MYO6+WT n = 56, average 5.05 ± 3.02 SD. The lack of statistical significance was determined using a two-sided t-test. d GFP and actin localisation in HeLa cells transfected with GFP-MYO6+S267A or GFP-MYO6+S267E. The accumulation of either construct in filopodia tips was observed in several independent experiments. A representative image is shown. Scale bar, 10 μm and 5 μm for enlarged images. e HeLa cells co-expressing mCherry-MYO6+WT (red) and either GFP-MYO6+WT, GFP-MYO6+S267A or GFP-MYO6+S267E (green) shown in high-resolution images of single filopodia. This experiment has been performed at least 3 times, representative images are shown. Schemes highlight the relative distribution of mCherry-MYO6+WT and GFP-MYO6+WT, GFP-MYO6+S267A or GFP-MYO6+S267E. Scale bar 5 μm. f Degree of co-localization between mCherry-MYO6+WT and GFP-tagged MYO6+WT, GFP-MYO6+S267A or GFP-MYO6+S267E was determined in 3 independent experiments from confocal images using Pearson’s correlation coefficient with automatic Costes threshold. Statistical significance was determined using a two-sided t-test ***P < 0.01. mCherry-MYO6+WT and GFP-MYO6+WT n = 152, average 0.6569 SD ± 0.1054, mCherry-MYO6+WT and GFP-MYO6+S267A n = 150, average 0.7077 SD ± 0.0961, mCherry-MYO6+WT and GFP-MYO6+ S267E n = 145, average 0.2098 SD ± 0.1123. Source data are provided as a Source Data file.
