Investigating the role of proteins in melanosome transport

The specialized epidermal cells produce melanin within the organelles referred to as melanosomes. These melanosomes are believed to be transported along the microtubules from the perinuclear region to the actin and then the tri-partile complex of myosin Va, Melanophilin and Rab27a move the melanosomes along the actin filaments to the plasma membrane. Any defectiveness or disruption of these proteins leads to several disorders such as Hermansky-Pudlak Syndrome (HPS), Chediak-Higashi Syndrome and Griscelli Syndrome which are caused due to defects in genes that control organelle biogenesis, organelle motility or cargo transport within cells. The key aim of this project is to study the involvement of the proteins myosin Va, Rab27a and its effector proteins melanophilin and slp2-a. The experiment fluorescence Recovery after photobleaching(FRAP) was performed in different mutant cell lines such as melan-leaden (lacks melanophilin), melan-d (lacks myosin Va) and melan-ash (lacks Rab27a). We then transfected members of the tri-partile complex into these cell cells to observe their effect on melanosome transport. A mutant Rab27a which highly reduces the binding of its effectors slp2 and melanophilin was also used to investigate the binding dynamics of these effector proteins with Rab27a. Finally, these findings led to the idea of constructing a chimeric molecule where the RBD region of melanophilin been replaced with the RBD region of Slp2 and was expected to have a stable interaction with Rab27a. From these experiments, it was identified that melanophilin has a dynamic interaction with Rab27a and Slp2 has a stable interaction. It was understood that the Rab binding region(RBD) of slp2 and melanophilin perform a major part in controlling the nature of movement with Rab27a

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).publishersversionpublishe

Investigating the role of proteins in melanosome transport

The specialized epidermal cells produce melanin within the organelles referred to as melanosomes. These melanosomes are believed to be transported along the microtubules from the perinuclear region to the actin and then the tri-partile complex of myosin Va, Melanophilin and Rab27a move the melanosomes along the actin filaments to the plasma membrane. Any defectiveness or disruption of these proteins leads to several disorders such as Hermansky-Pudlak Syndrome (HPS), Chediak-Higashi Syndrome and Griscelli Syndrome which are caused due to defects in genes that control organelle biogenesis, organelle motility or cargo transport within cells. The key aim of this project is to study the involvement of the proteins myosin Va, Rab27a and its effector proteins melanophilin and slp2-a. The experiment fluorescence Recovery after photobleaching(FRAP) was performed in different mutant cell lines such as melan-leaden (lacks melanophilin), melan-d (lacks myosin Va) and melan-ash (lacks Rab27a). We then transfected members of the tri-partile complex into these cell cells to observe their effect on melanosome transport. A mutant Rab27a which highly reduces the binding of its effectors slp2 and melanophilin was also used to investigate the binding dynamics of these effector proteins with Rab27a. Finally, these findings led to the idea of constructing a chimeric molecule where the RBD region of melanophilin been replaced with the RBD region of Slp2 and was expected to have a stable interaction with Rab27a. From these experiments, it was identified that melanophilin has a dynamic interaction with Rab27a and Slp2 has a stable interaction. It was understood that the Rab binding region(RBD) of slp2 and melanophilin perform a major part in controlling the nature of movement with Rab27a