A family of Rab27-binding proteins. Melanophilin links Rab27a and myosin Va function in melanosome transport

The Rab27a GTPase regulates diverse processes involving lysosome-related organelles, including melanosome motility in melanocytes, and lytic granule release in cytotoxic T lymphocytes. Toward an understanding of Rab27a function, we searched for proteins that interact with Rab27a(GTP) using the yeast two-hybrid system and identified JFC1/Slp1, a protein of unknown function. JFC1/Slp1 and related proteins, including melanophilin, contain a conserved amino-terminal domain similar to the Rab3a-binding domain of Rabphilin-3. We used several methods to demonstrate that this conserved amino-terminal domain is a Rab27-binding domain. We show that this domain interacts directly, and in a GTP-dependent manner with Rab27a. Furthermore, overexpression of this domain in melanocytes results in perinuclear clustering of melanosomes, suggesting that this region is sufficient for interaction with, and perturbation of function of, Rab27a in a physiological context. Thus, we identified a novel family of Rab27-binding proteins. We also show that melanophilin associates with Rab27a and myosin Va on melanosomes in melanocytes, and present evidence that a domain within the carboxyl-terminal region of melanophilin interacts with the carboxyl-terminal tail of the melanocyte-specific splice isoform of myosin Va. Thus, melanophilin can associate simultaneously with activated Rab27a and myosin Va via distinct regions, and serve as a linker between these proteins.     

The actin-binding domain of Slac2-a/melanophilin is required for melanosome distribution in melanocytes

Melanosomes containing melanin pigments are transported from the cell body of melanocytes to the tips of their dendrites by a combination of microtubule- and actin-dependent machinery. Three proteins, Rab27A, myosin Va, and Slac2-a/melanophilin (a linker protein between Rab27A and myosin Va), are known to be essential for proper actin-based melanosome transport in melanocytes. Although Slac2-a directly interacts with Rab27A and myosin Va via its N-terminal region (amino acids 1 to 146) and the middle region (amino acids 241 to 405), respectively, the functional importance of the putative actin-binding domain of the Slac2-a C terminus (amino acids 401 to 590) in melanosome transport has never been elucidated. In this study we showed that formation of a tripartite protein complex between Rab27A, Slac2-a, and myosin Va alone is insufficient for peripheral distribution of melanosomes in melanocytes and that the C-terminal actin-binding domain of Slac2-a is also required for proper melanosome transport. When a Slac2-a deletion mutant (DeltaABD) or point mutant (KA) that lacks actin-binding ability was expressed in melanocytes, the Slac2-a mutants induced melanosome accumulation in the perinuclear region, possibly by a dominant negative effect, the same as the Rab27A-binding-defective mutant of Slac2-a or the myosin Va-binding-defective mutant. Our findings indicate that Slac2-a organizes actin-based melanosome transport in cooperation with Rab27A, myosin Va, and actin.     

The leaden gene product is required with Rab27a to recruit myosin Va to melanosomes in melanocytes

The function of lysosome-related organelles such as melanosomes in melanocytes, and lytic granules in cytotoxic T lymphocytes is disrupted in Griscelli syndrome and related diseases. Griscelli syndrome results from loss of function mutations in either the RAB27A (type 1 Griscelli syndrome) or MYO5A (type 2 Griscelli syndrome) genes. Melanocytes from Griscelli syndrome patients and respective murine models ashen (Rab27a mutant), dilute (myosin Va mutant), and leaden exhibit perinuclear clustering of melanosomes. Recent work suggests that Rab27a is required to recruit myosin Va to melanosomes, thereby tethering melanosomes to the peripheral actin network and promoting melanosome retention at the tips of melanocytic dendrites. Here, we characterize the function of the leaden gene product. We show that Rab27a, but not myosin Va, can be localized to melanosomes in leaden melanocytes, suggesting that the leaden gene product acts downstream of, or in parallel to, Rab27a in melanocytes to promote recruitment of myosin Va to melanosomes. We also observed reduced levels of myosin Va protein in leaden and ashen melanocytes, suggesting that myosin Va stability is influenced by the leaden and ashen gene products. In leaden cytotoxic T lymphocytes, we observed that lytic granules polarize towards the immunological synapse and kill target cells normally. However, in contrast to melanocytes, we found that neither the leaden gene product (melanophilin) nor myosin Va was detectable in cytotoxic T lymphocytes. These results suggest that Rab27a interacts with different classes of effector proteins in melanocytes and cytotoxic T lymphocytes.     

Functional analysis of Slac2-c/MyRIP as a linker protein between melanosomes and myosin VIIa

Slac2-c/MyRIP, an in vitro Rab27A- and myosin Va/VIIa-binding protein, has recently been proposed to regulate retinal melanosome transport in retinal pigment epithelium cells by directly linking melanosome-bound Rab27A and myosin VIIa; however, the exact function of Slac2-c in melanosome transport has never been clarified. In this study, we used melanosome transport in skin melanocytes as a model for retinal melanosome transport and analyzed the in vivo function of Slac2-c in melanosome transport by the ectopic expression of Slac2-c, together with myosin VIIa, in Slac2-a-depleted melanocytes. In vitro binding experiments revealed that myosin VIIa had a greater affinity for Slac2-c, compared with the binding affinity of myosin Va, and that the myosin VIIa-binding domain of Slac2-c is different from the previously characterized myosin Va-binding domain that is conserved between Slac2-a/melanophilin and Slac2-c. Consistent with this result, cyan fluorescent protein-tagged Slac2-c expressed in melanocytes was localized on melanosomes via the specific interaction with Rab27A and recruited co-expressed yellow fluorescent protein-tagged myosin VIIa to the melanosomes without interfering with the normal peripheral melanosome distribution, whereas when myosin VIIa alone was expressed in melanocytes, it was not localized on the melanosomes. Moreover, Slac2-c ectopically expressed in melanocytes did not rescue the perinuclear aggregation phenotype induced by the knockdown of endogenous Slac2-a with a specific small interfering RNA, whereas the expression of the Slac2-c x myosin VIIa complex supported the normal melanosome distribution in Slac2-a-depleted melanocytes, indicating that Slac2-c functions as a myosin VIIa receptor rather than a myosin Va receptor in melanosome transport. Based on these findings, we propose that Slac2-c acts as a functional myosin VIIa receptor and that the Rab27A.Slac2-c x myosin VIIa tripartite protein complex regulates the transport of retinal melanosomes in pigment epithelium cells.     

Slac2-a/melanophilin, the missing link between Rab27 and myosin Va: implications of a tripartite protein complex for melanosome transport

Myosin Va is a member of the unconventional class V myosin family, and a mutation in the myosin Va gene causes pigment granule transport defects in human Griscelli syndrome and dilute mice. How myosin Va recognizes its cargo (i.e. melanosomes), however, has remained undetermined over the past decade. In this study, we discovered Slac2-a/melanophilin to be the "missing link" between myosin Va and GTP-Rab27A present in the melanosome. Deletion analysis and site-directed mutagenesis showed that the N-terminal Slp (synaptotagmin-like protein) homology domain of Slac2-a specifically binds Rab27A/B isoforms and that the C-terminal half directly binds the globular tail of myosin Va. The tripartite protein complex (Rab27A.Slac2-a.myosin Va) in melanoma cells was further confirmed by immunoprecipitation. The discovery that myosin Va indirectly recognizes its cargo through Slac2-a, a novel Rab27A/B effector, should shed light on molecular recognition of its specific cargo by class V myosin.     

In vitro reconstitution of a transport complex containing Rab27a, melanophilin and myosin Va

Rab27a and melanophilin (Mlph) are required in vivo to form a melanosome receptor for myosin Va in which Rab27a anchored in the melanosome membrane recruits Mlph, which in turn recruits myosin Va. Here, we show by reconstitution using purified proteins that Rab27a and Mlph are sufficient to form a transport complex with myosin Va in vitro. These results suggest that additional proteins are not required in vivo for assembly of the myosin Va receptor, although other proteins may associate with this tripartite complex to regulate its activity and/or to assist Rab27a in anchoring the complex to the melanosome membrane.     

Slac2-a/melanophilin contains multiple PEST-like sequences that are highly sensitive to proteolysis

The synaptotagmin-like protein homologue lacking C2 domains-a (Slac2-a)/melanophilin was recently identified as the "missing link" between the small GTPase Rab27A and the actin-based motor protein myosin Va. Although formation of a tripartite protein complex by three molecules had been shown to be required for proper melanosome distribution in melanocytes (Kuroda, T. S., Ariga, H., and Fukuda, M. (2003) Mol. Cell. Biol. 23, 5245-5255), the regulatory mechanisms of the complex (i.e. assembly and disassembly of the complex) had never been elucidated. In this study, we discovered that Slac2-a and a closely related isoform, Slac2-c/MyRIP, contain multiple PEST-like sequences (potential signals for rapid protein degradation) in the myosin Va- and actin-binding domains at the C terminus. We found that the C-terminal domain of Slac2-a is highly sensitive to low concentrations of proteases, such as trypsin and calpain, in vitro, whereas the N-terminal Rab27A-binding domain is highly resistant to these proteases. We further found that endogenous calpains selectively cleave Slac2-a, but not Rab27A or myosin Va, in melanocytes. A mutant Slac2-a lacking one of the PEST-like sequences located at the interface between the myosin Va- and actin-binding domains (DeltaPEST; amino acids 399-405) is more stable than the wild-type protein, both in vitro and in melanocytes. Expression of the mutant Slac2-a-DeltaPEST with an N-terminal green fluorescence protein tag often induced perinuclear aggregation of melanosomes ( approximately 40% of the transfected cells) compared with the wild-type Slac2-a. Our findings suggest that protein degradation of Slac2-a is an essential process for proper melanosome distribution in melanocytes.     

Identification of a minimal myosin Va binding site within an intrinsically unstructured domain of melanophilin

Myosin V is a molecular motor that transports a variety of cellular cargo, including organelles, vesicles, and messenger RNA. The proper peripheral distribution of melanosomes, a dense pigment-containing organelle, is dependent on actin and the activity of myosin Va. The recruitment of myosin Va to the melanosome and proper transport of the melanosome requires melanophilin, which directly binds to myosin Va and is tethered to the melanosome membrane via Rab27a. Here we use highly purified proteins to demonstrate that the globular tail domain of myosin Va binds directly to an intrinsically unstructured domain of melanophilin. The myosin Va binding domain of melanophilin lacks stable secondary structure, and (1)H NMR measurements indicate that the protein is unfolded. This domain is extremely sensitive to mild proteolysis and has a hydrodynamic radius that is consistent with a random coil-like polypeptide. We show that myosin Va binding does not induce the global folding of melanophilin. Truncations of melanophilin were utilized to define a short peptide sequence (26 residues) within melanophilin that is critical for myosin Va binding. We demonstrate that a peptide corresponding to these residues binds directly to the globular tail domain with the same affinity as melanophilin. We discuss the possible implications of protein intrinsic disorder in recruitment and maintenance of myosin Va on melanosome membranes.     

The role of Rab27a in the regulation of melanosome distribution within retinal pigment epithelial cells

Melanosomes within the retinal pigment epithelium (RPE) of mammals have long been thought to exhibit no movement in response to light, unlike fish and amphibian RPE. Here we show that the distribution of melanosomes within the mouse RPE undergoes modest but significant changes with the light cycle. Two hours after light onset, there is a threefold increase in the number of melanosomes in the apical processes that surround adjacent photoreceptors. In skin melanocytes, melanosomes are motile and evenly distributed throughout the cell periphery. This distribution is due to the interaction with the cortical actin cytoskeleton mediated by a tripartite complex of Rab27a, melanophilin, and myosin Va. In ashen (Rab27a null) mice RPE, melanosomes are unable to move beyond the adherens junction axis and do not enter apical processes, suggesting that Rab27a regulates melanosome distribution in the RPE. Unlike skin melanocytes, the effects of Rab27a are mediated through myosin VIIa in the RPE, as evidenced by the similar melanosome distribution phenotype observed in shaker-1 mice, defective in myosin VIIa. Rab27a and myosin VIIa are likely to be required for association with and movement through the apical actin cytoskeleton, which is a prerequisite for entry into the apical processes.     

A coiled-coil domain of melanophilin is essential for Myosin Va recruitment and melanosome transport in melanocytes

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F-binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.     

Alternative Splicing in Class V Myosins Determines Association with Rab10

Rab proteins influence vesicle trafficking pathways through the assembly of regulatory protein complexes. Previous investigations have documented that Rab11a and Rab8a can interact with the tail region of myosin Vb and regulate distinct trafficking pathways. We have now determined that a related Rab protein, Rab10, can interact with myosin Va, myosin Vb, and myosin Vc. Rab10 localized to a system of tubules and vesicles that have partially overlapping localization with Rab8a. Both Rab8a and Rab10 were mislocalized by the expression of dominant-negative myosin V tails. Interaction with Rab10 was dependent on the presence of the alternatively spliced exon D in myosin Va and myosin Vb and the homologous region in myosin Vc. Yeast two-hybrid assays and fluorescence resonance energy transfer studies confirmed that Rab10 binding to myosin V tails in vivo required the alternatively spliced exon D. In contrast to our previous work, we found that Rab11a can interact with both myosin Va and myosin Vb tails independent of their splice isoform. These results indicate that Rab GTPases regulate diverse endocytic trafficking pathways through recruitment of multiple myosin V isoforms.Eukaryotic cells are comprised of networks of highly organized membranous structures that require the efficient and timely movement of diverse intracellular proteins for proper function. Molecular motors provide the physical force needed to move these materials along microtubules and actin microfilaments. Unconventional myosin motors, such as those belonging to classes V, VI, and VII, have roles in the trafficking and recycling of membrane-bound structures in eukaryotic cells (1) and are recruited to discrete vesicle populations. Myosin VI is involved in clathrin-mediated endocytosis (2), whereas myosin VIIa participates in the proper development of stereocilia of inner ear hair cells and the transport of pigment granules in retinal pigmented epithelial cells (3, 4). Similarly, the three members of vertebrate class V myosins, myosin Va, myosin Vb, and myosin Vc, are required for the proper transport of a wide array of membrane cargoes, such as the melanosomes of pigment cells, synaptic vesicles in neurons, apical recycling endosomes in polarized epithelial cells, and bulk recycling vesicles in non-polarized cells (5).Members of the Rab family of small GTPases regulate many cellular systems, including membrane trafficking (6, 7). Certain Rab proteins associate with and regulate the function of class V myosins. Rab27a, in a complex with the adaptor protein melanophilin/Slac2-a, is required to localize myosin Va to the surface of melanin-filled pigment granules in vertebrates (8-10), whereas Rab27a and Slac2-c/MyRIP associate with both Myosin Va and myosin VIIa (3, 11). Rab11a, in a complex with its adaptor protein Rab11-FIP2, associates with myosin Vb on recycling endosomes (12-14) where the tripartite complex regulates the recycling of a variety of cargoes (15-19). In addition, Rab8a associates with both myosin Vb (20) and myosin Vc (21) as part of the non-clathrin-mediated tubular recycling system (20). Recently, Rab11a has also been shown to associate with myosin Va in the transport of AMPA receptors in dendritic spines (22), contributing to the model of myosin V regulation by multiple Rab proteins.Previous investigations have documented alternative splicing of myosin Va in a tissue-specific manner (23-28). Alternate splicing occurs in a region lying between the coiled-coil region of the neck of the motor and the globular tail region. Three exons in particular are subject to alternative splicing: exons B, D, and F (23-25). Exon F is critical for association with melanophilin/Slac2 and Rab27a (8, 9, 29, 30). Additionally, exon B is required for the interaction of myosin Va with dynein light chain 2 (DLC2) (27, 28). Currently no function for the alternatively spliced exon D has been reported. Similar to myosin Va, myosin Vb contains exons A, B, C, D, and E, whereas no exon F has yet been identified in myosin Vb (Fig. 1A). In addition, exon B in myosin Vb does not resemble the dynein light chain 2 (DLC2) binding region in myosin Va (27, 28), and therefore, it likely does not interact with DLC2. On the other hand, exon D is highly conserved among Myosin Va, myosin Vb, and myosin Vc, suggesting a common function in these molecular motors.Open in a separate windowFIGURE 1.Tissue distribution of human myosin Va and myosin Vb splice isoforms. A, schematic of the alternative exon organization in the tails of myosin Va and myosin Vb. It is known that exons B, D, and F are subject to alternative splicing in myosin Va, whereas there is only evidence that exon D is alternatively spliced in myosin Vb, which does not contain exon F. B, alignment of exon D sequences from mouse and human myosin V''s. myosin Va and myosin Vb both contain exon D (amino acids 1320-1346 of myosin Va and 1315-1340 of myosin Vb), whereas myosin Vc contains an exon D-like region (amino acids 1124-1147 of human myosin Vc) that is not known to be alternatively spliced. Alignment of the exon D regions from all three motors reveals a high degree of homology, especially in the center of the exon. Asterisks indicate amino acid identities. C, PCR-based analysis of human tissue panels reveals the alternative splicing pattern of exon D in myosin Va and myosin Vb. Primers flanking the region encoding exon D for both motors were used to amplify cDNA from human MTC™ panels (Clontech). cDNA amplified from HeLa cell RNA as well as myosin Va and myosin Vb tail constructs were used as positive controls. Variants expressing exon D (upper bands) and lacking exon D (lower bands) were visible. Per., peripheral; Pos., positive.Here we report that Rab10, a protein related to Rab8a and thought to have similar function (31-35), localizes to a system of tubules and vesicles overlapping in distribution with Rab8a in HeLa cells. Utilizing dominant-negative myosin V tail constructs, we show that Rab8a and Rab10 can interact with Myosin Va, myosin Vb, and myosin Vc in vivo. In addition, we have determined that the alternatively spliced exon D in both myosin Va and myosin Vb is required for interaction with Rab10. In contrast to our previous findings, we demonstrate that Rab11a is able to interact with both myosin Va and myosin Vb tails in an exon independent-manner. These results reveal that multiple Rab proteins potentially regulate all three class V myosin motors.     

A Highlights from MBoC Selection: Identification and characterization of multiple novel Rab–myosin Va interactions

Myosin Va is a widely expressed actin-based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To our knowledge, myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end, we report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A′, 6B, 11B, 14, 25, 39B), which include interactions with three new Rab subfamilies (Rab6, Rab14, Rab39B). Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. We demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively spliced exon, and the globular tail). Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.     

Myrip uses distinct domains in the cellular activation of myosin VA and myosin VIIA in melanosome transport

Myrip is a Rab27a and MyosinVIIa (MyoVIIa) linking protein that may regulate melanosome transport in the retinal pigment epithelium (RPE). Myrip also binds MyosinVa (MyoVa) in vitro however it is unclear whether this interaction is of sufficient affinity to be physiologically relevant. Here, we addressed the questions of whether Myrip interacts with MyoVa in cells and the molecular basis of cellular activation of MyoVa and MyoVIIa by Myrip. To answer these questions we used melanosome transport in skin melanocytes and RPE cells as read‐outs of MyoVa and MyoVIIa activity. We found that Myrip recruits and activates MyoVa on skin melanosomes with similar efficiency to the established MyoVa activator Melanophilin (Mlph). Mutagenesis showed that a Myrip–Mlph conserved amphipathic helix (MMAH) is essential for MyoVa interaction while other Myrip regions, including the MyoVa exon F binding domain equivalent, play non‐essential roles in this interaction. This suggests that, in contrast to Mlph, Myrip interacts with MyoVa lacking melanocyte‐specific exon F. Parallel studies of RPE melanosome transport reveal that Myrip‐specific inserts, but not the MMAH, are essential for MyoVIIa activation. We conclude that Myrip is a versatile Rab27a‐associated myosin‐activating protein that mediates cellular activation of MyoVa and MyoVIIa via non‐overlapping domains.     

Rab7 and Rab27a control two motor protein activities involved in melanosomal transport

Melanosomes are lysosome-related organelles that synthesize, store and transport melanin. In epidermal melanocytes, melanosomes mature and are transferred to surrounding keratinocytes, which is essential for skin and coat colour. Mouse coat colour mutants reveal a critical role for the small GTPase Rab27a, which recruits myosin Va through its effector protein melanophilin/Slac2a. Here we have studied how two different Rab GTPases control two motor proteins during subsequent phases in transport of melanosomes. We show that the small GTPase Rab7 mainly associates with early and intermediate stage melanosomes and Rab27a to intermediate and mature melanosomes. Rab27a is found in an active state on mature melanosomes in the tips of the dendrites. The Rab7-Rab7-interacting lysosomal protein-dynein pathway only controls early and intermediate stage melanosomes because the mature melanosomes lack Rab7 and associate with the actin network through Rab27a recruited MyoVa. Thus two Rab proteins regulate two different motor proteins, thereby controlling complementary phases in melanosome biogenesis: Rab7 controls microtubule-mediated transport of early and Rab27a the subsequent actin-dependent transport of mature melanosomes.     

Rab27A-binding protein Slp2-a is required for peripheral melanosome distribution and elongated cell shape in melanocytes

The synaptotagmin-like protein (Slp) family is implicated in regulating Rab27A-mediated membrane transport, but how it might do this is unknown. Here we report that Slp2-a, a previously uncharacterized Rab27A-binding protein in melanocytes, controls melanosome distribution in the cell periphery and regulates the morphology of melanocytes. Slp2-a is the most abundantly expressed of the Slp- and Slac2-family proteins in melanocytes and colocalizes with Rab27A on melanosomes. Knockdown of endogenous Slp2-a protein by small-interfering RNAs (siRNAs) markedly reduced the number of melanosomes in the cell periphery of mouse melanocytes ('peripheral dilution'). Expression of siRNA-resistant Slp2-a (Slp2-a(SR)) rescued the peripheral dilution of melanosomes induced by Slp2-a siRNAs, but Slp2-a(SR) mutants, which failed to interact with either phospholipids or Rab27A, did not. Loss of Slp2-a protein also induced a change in melanocyte morphology, from their normal elongated shape to a more rounded shape, which depended on the phospholipid-binding activity of Slp2-a, but not on its Rab27A-binding activity. By contrast, knockdown of Slac2-a (also called melanophilin), another Rab27A-binding protein in melanocytes, caused perinuclear aggregation of melanosomes alone without altering cell shape. These results reveal the differential and sequential roles of Rab27A-binding proteins in melanosome transport in melanocytes.     

Manassantin B inhibits melanosome transport in melanocytes by disrupting the melanophilin–myosin Va interaction

Human skin hyperpigmentation disorders occur when the synthesis and/or distribution of melanin increases. The distribution of melanin in the skin is achieved by melanosome transport and transfer. The transport of melanosomes, the organelles where melanin is made, in a melanocyte precedes the transfer of the melanosomes to a keratinocyte. Therefore, hyperpigmentation can be regulated by decreasing melanosome transport. In this study, we found that an extract of Saururus chinensis Baill (ESCB) and one of its components, manassantin B, inhibited melanosome transport in Melan‐a melanocytes and normal human melanocytes (NHMs). Manassantin B disturbed melanosome transport by disrupting the interaction between melanophilin and myosin Va. Manassantin B is neither a direct nor an indirect inhibitor of tyrosinase. The total melanin content was not reduced when melanosome transport was inhibited in a Melan‐a melanocyte monoculture by manassantin B. Manassantin B decreased melanin content only when Melan‐a melanocytes were co‐cultured with SP‐1 keratinocytes or stimulated by α‐MSH. Therefore, we propose that specific inhibitors of melanosome transport, such as manassantin B, are potential candidate or lead compounds for the development of agents to treat undesirable hyperpigmentation of the skin.     

Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes

Rab GTPases regulate discrete steps in vesicular transport pathways. Rabs require activation by specific guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP. Rab27a controls motility and regulated exocytosis of secretory granules and related organelles. In melanocytes, Rab27a regulates peripheral transport of mature melanosomes by recruiting melanophilin and myosin Va. Here, we studied the activation of Rab27a in melanocytes. We identify Rab3GEP, previously isolated as a GEF for Rab3a, as the non-redundant Rab27a GEF. Similar to Rab27a-deficient ashen melanocytes, Rab3GEP-depleted cells show both clustering of melanosomes in the perinuclear area and loss of the Rab27a effector Mlph. Consistent with a role as an activator, levels of Rab27a-GTP are decreased in cells lacking Rab3GEP. Recombinant Rab3GEP exhibits guanine nucleotide exchange activity against Rab27a and Rab27b in vitro, in addition to its previously documented activity against Rab3. Our results indicate promiscuity in Rab GEF action and suggest that members of related but functionally distinct Rab subfamilies can be controlled by common activators.     

Expression of constructs of the neuronal isoform of myosin-Va interferes with the distribution of melanosomes and other vesicles in melanoma cells

Myosin-Va has been implicated in melanosome translocation, but the exact molecular mechanisms underlying this function are not known. In the dilute, S91 melanoma cells, melanosomes move to the cell periphery but do not accumulate in the tips of dendrites as occurs in wild-type B16 melanocytes; rather, they return and accumulate primarily at the pericentrosomal region in a microtubule-dependent manner. Expression of the full-length neuronal isoform of myosin-Va in S91 cells causes melanosomes to disperse, occupying a cellular area approximately twice that observed in non-transfected cells, suggesting a partial rescue of the dilute phenotype. Overexpression of the full tail domain in S91 cells is not sufficient to induce melanosome dispersion, rather it causes melanosomal clumping. Overexpression of the head and head-neck domains of myosin-Va in B16 cells does not alter the melanosome distribution. However, overexpression of the full tail domain in these cells induces melanosome aggregation and the appearance of tail-associated, aggregated particles or vesicular structures that exhibit variable degrees of staining for melanosomal and Golgi beta-COP markers, as well as colocalization with the endogenous myosin-Va. Altogether, the present data suggest that myosin-Va plays a role in regulating the direction of microtubule-dependent melanosome translocation, in addition to promoting the capture of melanosomes at the cell periphery as suggested by previous studies. These studies also reinforce the notion that myosin-V has a broader function in melanocytes by acting on vesicular targeting or intracellular protein trafficking.