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Fig. 6. Deletion of the myosin-Va-GT-binding site of Slac2-a creates a dilute-like phenotype in wild-type melanocytes. Melan-a cells were transfected with a vector encoding GFP-tagged wild-type Slac2-a (AE) or mutant GFP-Slac2-a-GT (F-J). After the cells were fixed and permeabilized, they were stained with anti-Rab27A (green) and anti-myosin-Va (red) antibodies followed by Alexa Fluor secondary antibody conjugates as described previously (Kuroda et al., 2002a; Kuroda et al., 2003). The cells were examined for fluorescence of GFP-Slac2-a proteins (A and F), Rab27A (B and G) and myosin-Va (C and H) by confocal microscopy. Bright-field images (E and J) show the melanosome distribution in the cells, and the mutant GFPSlac2-a-GT-expressing cell is outlined in yellow (J). D and I are merged images of Rab27A and myosin-Va. Note that expression of GFP-Slac2-a-GT induced melanosome aggregation in the perinuclear region (J), segregation of myosin-Va from Rab27A (I) and attenuation of myosin-Va-immunoreactivity (H), whereas the wild-type Slac2-a-expressing cell exhibited peripheral melanosome distribution (E), with both Rab27A and myosin-Va being colocalized on melanosomes (D). Bars, 10 µm.

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