The adaptor protein melanophilin regulates dynamic myosin-Va:cargo interaction and dendrite development in melanocytes

Regulation of organelle transport by the cytoskeleton is fundamental for eukaryotic survival. Cytoskeleton motors are typically modular proteins with conserved motor and diverse cargo binding domains. Motor:cargo interactions are often indirect and mediated by adaptor proteins e.g. Rab GTPases. Rab27a, via effector melanophilin (Mlph), recruits myosin-Va to melanosomes and thereby disperses them into melanocytes dendrites. To better understand how adaptors regulate motor:cargo interaction we used single melanosome fluorescence recovery after photo-bleaching (smFRAP) to characterise the association kinetics between myosin-Va, its adaptors and melanosomes. We found that myosin-Va and Mlph rapidly recovered after photo-bleaching, while Rab27a did not, indicating that myosin-Va and Mlph dynamically associate with melanosomes and Rab27a does not. This suggests that dynamic Rab27a:effector interaction rather than Rab27a melanosome:cytosol cycling regulates myosin-Va:melanosome association. Accordingly a Mlph-Rab27a fusion protein reduced myosin-Va smFRAP, indicating that it stabilised melanosomal myosin-Va. Finally, we tested the functional importance of dynamic myosin-Va:melanosome interaction. We found that while a myosin-Va-Rab27a fusion protein dispersed melanosomes in myosin-Va deficient cells, dendrites were significantly less elongated than in wild-type cells. Given that dendrites are the prime sites of melanosome transfer from melanocytes to keratinocytes we suggest that dynamic myosin-Va:melanosome interaction is important for pigmentation in vivo. Movie S1 Movie S1 MVa-tail expressed in wild-type (melan-a) cells (supports Figure 1). Movie S2 Movie S2 MVa-tail expressed in myosin-Va -/- (melan-d) cells (supports Figure S1A). Movie S3 Movie S3 MVa-FL expressed in myosin-Va -/- (melan-d) cells (supports Figure S1C). Movie S4 Movie S4 Rab27a expressed in wild-type (melan-a) cells (supports Figure 2A). Movie S5 Movie S5 Rab27a expressed in Rab27a -/- (melan-ash) cells (supports Figure 2B). Movie S6 Movie S6 Rab27a expressed in Mlph -/- (melan-ln) cells (supports Figure 2C). Movie S7 Movie S7 Rab27aSF1F4 expressed in wild-type (melan-a) cells (supports Figure 2D). Movie S8 Movie S8 Mlph expressed in wild-type (melan-a) cells (supports Figure 3A). Movie S9 Movie S9 Mlph expressed in Mlph -/- (melan-ln) cells (supports Figure 3B). Movie S10 Movie S10 Mlph expressed in myosin-Va -/- (melan-d) cells (supports Figure 3C). Movie S11 Movie S11 MVa-tail co-expressed with mCherry-Mlph expressed in Mlph -/- (melan-ln) cells (supports Figure 4B). Movie S12 Movie S12 MVa-tail co-expressed with mCherry-Mlph-Rab27aSF1F4 expressed in Mlph -/- (melan-ln) cells (supports Figure 4C). Movie S13 Movie S13 GFP-Mlph- Rab27aSF1F4 expressed in Mlph -/- (melan-ln) cells (supports Figure S4). Movie S14 Movie S14 Mlph R27BD expressed in wild-type (melan-a) cells (supports Figure S5). Movie S15 Movie S15 Myo-Rab expressed in myosin-Va -/- (melan-d) cells (supports Figures 5 and S7). Movie S16 Movie S16 Sytl2 (R27BD) expressed in wild-type (melan-a) cells (supports Figures S8).