Fig. 5: The AF assembly (FH1-FH2) and SPIRE interaction (FSI) domains of FMN1 are essential for melanosome dispersion.

a A schematic representation of the domain structure of murine FMN1 and the composition of truncation mutants, and chimeric proteins used in functional studies (b, c). White asterisks indicates the site of point mutations. Numbers indicate amino acid boundaries. b melan-f cells were plated on glass coverslips and infected with adenoviruses expressing the indicated proteins. Cells were fixed 24 h later, processed for immunofluorescence and the intracellular distribution of expressed protein and melanosomes (bright-field) was observed using a fluorescence microscope (see Experimental procedures). Scale bars = 20 μm and 2 μm for magnified region. Dashed boxes in FH1-FH2-Rab27a images indicate the region of the image shown in high magnification below. The merged image show melanosomes and FH1-FH2-Rab27a coloured magenta and green. c A bee swarm plot showing the extent of pigment dispersion in cells expressing the indicated proteins. n = 30 (GFP), 59 (FMN1), 18 (N-term), 70 (FH1-FH2-FSI), 31 (ΔFSI), 20 (FH2-FSI), 37 (I1074A), 35 (K1229D), 28 (K1418E) and 51 (FH1-FH2-Rab27a). ****, **, * and n.s. indicate significant differences of p = <0.0001, 0.01, 0.05 and no significant difference as determined by one-way ANOVA. Significance indicators above and below each dataset indicate differences between that dataset and the positive (FMN1 wild type) and negative (GFP alone) controls. Results shown are representative of three independent experiments. Bars indicate the mean and 25th and 75th percentile of data. FH formin homology, FSI formin-SPIRE interaction sequence. Source data for c are provided in the Supplementary Source data file.