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

Christopher L Robinson, Richard D Evans, Kajana Sivarasa, Jose S Ramalho, Deborah A Briggs, Alistair N Hume

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Abstract

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).

Original languageEnglish
Pages (from-to)742-752
Number of pages11
JournalMolecular Biology of the Cell
Volume30
Issue number6
Early online date30 Jan 2019
DOIs
Publication statusPublished - 15 Mar 2019

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