Fig. 8: Expression of choanoflagellate (Monosiga brevicollis) Mb-SPIRE protein rescues melanosome dispersion in mouse melanocytes depleted of endogenous SPIRE1/2.

Melan-a cells (wild-type melanocytes) were depleted of endogenous SPIRE1/2 by siRNA transfection and 72 h later infected with adenoviruses expressing the indicated proteins. Cells were fixed after 24 h and imaged using bright-field and fluorescence optics to assess localisation on melanosomes (see experimental procedures). a Bright-field microscopic images of wild-type (untransfected) melanocytes (left panel) and of those depleted of endogenous SPIRE1/2 (SPIRE1/2 knockdown, right panel). Images in the lower panel show higher magnification. The scale bar in the upper panel represents 50 μm and the scale bar in the lower panel represents 10 μm. b Representative images show distribution of melanosomes (phase-contrast microscopy) and eGFP (GFP) (fluorescence microscopy) in fields of cells expressing respective protein. Scale bar represents 10 μm. Yellow and green dotted lines in the images showing eGFP-expressing cells indicate cell edges. c Scatter plot showing the percentage of melanocytes in each population in which melanosome distribution is classified as dispersed and hyper-dispersed. Each data point indicates the percentage of cells in each class in each of 4 independent experiments and represents the average percentage of cells with the indicated phenotype. One hundred cells were scored for each expressed protein in each experiment. ****, ** and ns indicate significant differences p = < 0.0001, <0.01 and >0.05 compared with eGFP-expressing cells as determined by one-way ANOVA. Coloured asterisks indicate the class of data tested. No significant differences were seen between other datasets and GFP or Hs-SPIRE1. Bars indicate the mean and 25th and 75th percentile of data. d Cross-species interactions of Monosiga brevicollis and mammalian SPIRE and FMN proteins were analysed by GST-pulldown experiments. The SPIRE KIND domains were fused to GST and purified from bacteria. The FMN-FH2-FSI proteins were transiently expressed as AcGFP1 (GFP) fusion proteins in HEK 293 cells. Western blots (WB) of the loaded and pulled proteins and a Ponceau S stain of the GST-KIND proteins are shown.