Griscelli syndrome: a model system to study vesicular trafficking

Griscelli syndrome (GS) is a rare autosomal recessive disorder caused by mutations in either the myosin VA (GS1), RAB27A (GS2) or melanophilin (GS3) genes. The three GS subtypes are commonly characterized by pigment dilution of the skin and hair, due to defects involving melanosome transport in melanocytes. Here, we review how detailed studies concerning GS have contributed to a better understanding of the molecular mechanisms involved in vesicle transport and membrane trafficking processes. Additionally, we demonstrate that the identification and biological analysis of novel disease‐causing mutations highlighted the functional importance of the RAB27A‐MLPH‐MYO5A tripartite complex in intracellular melanosome transport. As the small GTPase Rab27a is able to interact with multiple effectors, including Slp2‐a and Myrip, we report on their presumed role in melanosome transport. Furthermore, we summarize data suggesting that RAB27B and RAB27A are functionally redundant and hereby provide further insight into the pathogenesis of GS2. Finally, we discuss how the gathered knowledge about the RAB27A‐MLPH‐MYO5A tripartite complex can be translated into a possible therapeutic application to reduce (hyper)pigmentation of the skin.     

Characterization of the molecular defects in Rab27a,caused by RAB27A missense mutations found in patients with Griscelli syndrome

Rab27a plays a pivotal role in the transport of melanosomes to dendrite tips of melanocytes and mutations in RAB27A, which impair melanosome transport cause the pigmentary dilution and the immune deficiency found in several patients with Griscelli syndrome (GS). Interestingly, three GS patients present single homozygous missense mutations in RAB27A, leading to W73G, L130P, and A152P transitions that affect highly conserved residues among Rab proteins. However, the functional consequences of these mutations have not been studied. In the present report, we evaluated the effect of overexpression of these mutants on melanosome, melanophilin, and myosin-Va localization in B16 melanoma cells. Then we studied several key parameters for Rab27a function, including GTP binding and interaction with melanophilin/myosin-Va complex, which links melanosomes to the actin network. Our results showed that Rab27a-L130P cannot bind GTP, does not interact with melanophilin, and consequently cannot allow melanosome transport on the actin filaments. Interestingly, Rab27a-W73G binds GTP but does not interact with melanophilin. Thus, Rab27a-W73G cannot support the actin-dependent melanosome transport. Finally, Rab27a-A152P binds both GTP and melanophilin. However, Rab27a-A152P does not allow melanosome transport and acts as a dominant negative mutant, because its overexpression, in B16 melanoma cells, mimics a GS phenotype. Hence, the interaction of Rab27a with melanophilin/myosin-Va is not sufficient to ensure a correct melanosome transport. Our results pointed to an unexpected complexity of Rab27a function and open the way to the search for new Rab27a effectors or regulators that control the transport of Rab27a-dependent vesicles.     

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.     

Functional characterization of two RAB27A missense mutations found in Griscelli syndrome type 2

Human Griscelli syndrome type 2 (GS-2) is characterized by partial albinism and a severe immunologic disorder as a result of RAB27A mutations. In melanocytes, Rab27A forms a tripartite complex with a specific effector Slac2-a/melanophilin and myosin Va, and the complex regulates melanosome transport. Here, we report a novel homozygous missense mutation of Rab27A, i.e. K22R, in a Persian GS-2 patient and the results of analysis of the impact of the K22R mutation and the previously reported I44T mutation on protein function. Both mutations completely abolish Slac2-a/melanophilin binding activity but they affect the biochemical properties of Rab27A differently. The Rab27A(K22R) mutant lacks the GTP binding ability and exhibits cytosolic localization in melanocytes. By contrast, neither intrinsic GTPase activity nor melanosomal localization of Rab27A is affected by the I44T mutation, but the Rab27A(I44T) mutant is unable to recruit Slac2-a/melanophilin. Interestingly, the two mutations differently affect binding to other Rab27A effectors, Slp2-a, Slp4-a/granuphilin-a, and Munc13-4. The Rab27A(K22R) mutant normally binds Munc13-4, but not Slp2-a or Slp4-a, whereas the Rab27A(I44T) mutant shows reduced binding activity to Slp2-a and Munc13-4 but normally binds Slp4-a.     

Essential Role of RAB27A in Determining Constitutive Human Skin Color

Human skin color is predominantly determined by melanin produced in melanosomes within melanocytes and subsequently distributed to keratinocytes. There are many studies that have proposed mechanisms underlying ethnic skin color variations, whereas the processes involved from melanin synthesis in melanocytes to the transfer of melanosomes to keratinocytes are common among humans. Apart from the activities in the melanogenic rate-limiting enzyme, tyrosinase, in melanocytes and the amounts and distribution patterns of melanosomes in keratinocytes, the abilities of the actin-associated factors in charge of melanosome transport within melanocytes also regulate pigmentation. Mutations in genes encoding melanosome transport-related molecules, such as MYO5A, RAB27A and SLAC-2A, have been reported to cause a human pigmentary disease known as Griscelli syndrome, which is associated with diluted skin and hair color. Thus we hypothesized that process might play a role in modulating skin color variations. To address that hypothesis, the correlations of expression of RAB27A and its specific effector, SLAC2-A, to melanogenic ability were evaluated in comparison with tyrosinase, using human melanocytes derived from 19 individuals of varying skin types. Following the finding of the highest correlation in RAB27A expression to the melanogenic ability, darkly-pigmented melanocytes with significantly higher RAB27A expression were found to transfer significantly more melanosomes to keratinocytes than lightly-pigmented melanocytes in co-culture and in human skin substitutes (HSSs) in vivo, resulting in darker skin color in concert with the difference observed in African-descent and Caucasian skins. Additionally, RAB27A knockdown by a lentivirus-derived shRNA in melanocytes concomitantly demonstrated a significantly reduced number of transferred melanosomes to keratinocytes in co-culture and a significantly diminished epidermal melanin content skin color intensity (ΔL* = 4.4) in the HSSs. These data reveal the intrinsically essential role of RAB27A in human ethnic skin color determination and provide new insights for the fundamental understanding of regulatory mechanisms underlying skin pigmentation.     

Rab27a: A key to melanosome transport in human melanocytes

Normal pigmentation depends on the uniform distribution of melanin-containing vesicles, the melanosomes, in the epidermis. Griscelli syndrome (GS) is a rare autosomal recessive disease, characterized by an immune deficiency and a partial albinism that has been ascribed to an abnormal melanosome distribution. GS maps to 15q21 and was first associated with mutations in the myosin-V gene. However, it was demonstrated recently that GS can also be caused by a mutation in the Rab27a gene. These observations prompted us to investigate the role of Rab27a in melanosome transport. Using immunofluorescence and immunoelectron microscopy studies, we show that in normal melanocytes Rab27a colocalizes with melanosomes. In melanocytes isolated from a patient with GS, we show an abnormal melanosome distribution and a lack of Rab27a expression. Finally, reexpression of Rab27a in GS melanocytes restored melanosome transport to dendrite tips, leading to a phenotypic reversion of the diseased cells. These results identify Rab27a as a key component of vesicle transport machinery in melanocytes.     

Identification and characterization of three forms of glutamine synthetase unique to Rhizobia

Glutamine synthetase (GS) activities of Rhizobia were chromatographically resolved into three distinct forms, GSI, GSII, and GSIII on DEAE cellulose, being eluted with 0.3M, 0.5M and 0.8M KCl, respectively. GSIII was the major form inR. leguminosarum andR. phaseoli. InR. meliloti, however, GSI was the major form. The three forms of GS were also distinguished on the basis of (a) rapid heat inactivation of GSII, (b) insensitivity of GSI to inhibitors, (c) marked inhibition of GSII by thymidine, and (d) inability of Zn⁺⁺ to inhibit GSIII. The three forms of GS are also distinct molecular entities and are unique to Rhizobia.     

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.     

Analysis of the linkage of MYRIP and MYO7A to melanosomes by RAB27A in retinal pigment epithelial cells

The apical region of the retinal pigment epithelium (RPE) typically contains melanosomes. Their apical distribution is dependent on RAB27A and the unconventional myosin, MYO7A. Evidence from studies using in vitro binding assays, melanocyte transfection, and immunolocalization have indicated that the exophilin, MYRIP, links RAB27A on melanosomes to MYO7A, analogous to the manner that melanophilin links RAB27A on melanocyte melanosomes to MYO5A. To test the functionality of this hypothesis in RPE cells, we have examined the relationship among MYRIP, RAB27A and MYO7A with studies of RPE cells in primary culture (including live-cell imaging), analyses of mutant mouse retinas, and RPE cell fractionation experiments. Our results indicate that the retinal distribution of MYRIP is limited to the RPE, mainly the apical region. In RPE cells, RAB27A, MYRIP, and MYO7A were all associated with melanosomes, undergoing both slow and rapid movements. Analyses of mutant mice provide genetic evidence that MYRIP is linked to melanosomes via RAB27A, but show that recruitment of MYRIP to apical RPE is independent of melanosomes and RAB27A. RAB27A and MYRIP also associated with motile small vesicles of unknown origin. The present results provide evidence from live RPE cells that the RAB27A-MYRIP-MYO7A complex functions in melanosome motility. They also demonstrate that RAB27A provides an essential link to the melanosome.     

The occurrence of eukaryotic type III glutamine synthetase in the marine diatom Chaetoceros compressum

Glutamine synthetase (GS) has been described as one of the oldest functioning genes and thus a good molecular clock protein. GS is diverged into three distinct forms, type I (GSI), type II (GSII) and type III (GSIII), the last type of which is a member of the most recently discovered family among GSs and thus has been reported from a limited number of prokaryotes. In the present study, we determined the full-length sequence of GSIII from the marine diatom Chaetoceros compressum. The 3′ untranslated region of the diatom GSIII gene was composed of a polyadenylation signal followed by a poly (A)⁺ tail, clearly demonstrating that its mRNA is transcribed from the eukaryotic genome. We also screened available genome databases and identified full-length GSIII sequences from 5 eukaryotic species. These eukaryotic GSIIIs specifically contained regions A–D and a long additional sequence flanking region V toward the C-terminal site, both being specific to GSIII. Phylogenic analysis revealed that eukaryotic GSIIIs are not within a monophyletic relationship with the possible occurrence of lateral gene transfer in GSIII during evolution.     

The ternary Rab27a-Myrip-Myosin VIIa complex regulates melanosome motility in the retinal pigment epithelium

The retinal pigment epithelium (RPE) contains melanosomes similar to those found in the skin melanocytes, which undergo dramatic light-dependent movements in fish and amphibians. In mammals, those movements are more subtle and appear to be regulated by the Rab27a GTPase and the unconventional myosin, Myosin VIIa (MyoVIIa). Here we address the hypothesis that a recently identified Rab27a- and MyoVIIa-interacting protein, Myrip, promotes the formation of a functional tripartite complex. In heterologous cultured cells, all three proteins co-immunoprecipitated following overexpression. Rab27a and Myrip localize to the peripheral membrane of RPE melanosomes as observed by immunofluorescence and immunoelectron microscopy. Melanosome dynamics were studied using live-cell imaging of mouse RPE primary cultures. Wild-type RPE melanosomes exhibited either stationary or slow movement interrupted by bursts of fast movement, with a peripheral directionality trend. Nocodazole treatment led to melanosome paralysis, suggesting that movement requires microtubule motors. Significant and similar alterations in melanosome dynamics were observed when any one of the three components of the complex was missing, as studied in ashen- (Rab27a defective) and shaker-1 (MyoVIIa mutant)-derived RPE cells, and in wild-type RPE cells transduced with adenovirus carrying specific sequences to knockdown Myrip expression. We observed a significant increase in the number of motile melanosomes, exhibiting more frequent and prolonged bursts of fast movement, and inversion of directionality. Similar alterations were observed upon cytochalasin D treatment, suggesting that the Rab27a-Myrip-MyoVIIa complex regulates tethering of melanosomes onto actin filaments, a process that ensures melanosome movement towards the cell periphery.     

The lavender plumage colour in Japanese quail is associated with a complex mutation in the region of MLPH that is related to differences in growth, feed consumption and body temperature

ABSTRACT: BACKGROUND: The lavender phenotype in quail is a dilution of both eumelanin and phaeomelanin in feathers that produces a blue-grey colour on a wild-type feather pattern background. It has been previously demonstrated by intergeneric hybridization that the lavender mutation in quail is homologous to the same phenotype in chicken, which is caused by a single base-pair change in exon 1 of MLPH. RESULTS: In this study, we have shown that a mutation of MLPH is also associated with feather colour dilution in quail, but that the mutational event is extremely different. In this species, the lavender phenotype is associated with a non-lethal complex mutation involving three consecutive overlapping chromosomal changes (two inversions and one deletion) that have consequences on the genomic organization of four genes (MLPH and the neighbouring PRLH, RAB17 and LRRFIP1). The deletion of PRLH has no effect on the level of circulating prolactin. Lavender birds have lighter body weight, lower body temperature and increased feed consumption and residual feed intake than wild-type plumage quail, indicating that this complex mutation is affecting the metabolism and the regulation of homeothermy. CONCLUSIONS: An extensive overlapping chromosome rearrangement was associated with a non-pathological Mendelian trait and minor, non deleterious effects in the lavender Japanese quail which is a natural knockout for PRLH.     

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.     

GLUTAMINE SYNTHETASE IN MARINE ALGAE: NEW SURPRISES FROM AN OLD ENZYME

Glutamine synthetase (GS), which catalyzes the formation of glutamine from ammonium and glutamate in the presence of ATP, is encoded by three distinct gene families: GSI, GSII, and GSIII. Genes encoding GSI are found in the Bacteria and Archaea, whereas GSII genes are found in eukaryotes and a few species of Bacteria. Members of the third family, GSIII, have been described from a limited number of bacteria; however, recent biochemical and molecular data suggest that this type of enzyme is broadly distributed among the algae. Peptide fragments obtained from GS purified from the marine diatom Skeletonema costatum (Greville) Cleve are 77% identical to a partial sequence of GSIII from Chaetoceros compressum Lauder, which permits the unambiguous assignment of the biochemically characterized enzyme to the GSIII gene family. The N-terminal sequence was 43% identical to the GSIII-like enzyme purified from the haptophyte Emiliania huxleyi (Lohm.) Hay et Miller and several residues were conserved among bacterial and eukaryotic GSIII enzymes. The presence of genes encoding GSIII in diatoms and haptophytes indicates that this enzyme family is more broadly distributed in eukaryotes than previously suspected.     

MOLECULAR EVOLUTION OF GLUTAMINE SYNTHETASE II AND III IN THE CHROMALVEOLATES1

Glutamine synthetase (GS) is encoded by three distinct gene families (GSI, GSII, and GSIII) that are broadly distributed among the three domains of life. Previous studies established that GSII and GSIII isoenzymes were expressed in diatoms; however, less is known about the distribution and evolution of the gene families in other chromalveolate lineages. Thus, GSII cDNA sequences were isolated from three cryptophytes (Guillardia theta D. R. A. Hill et Wetherbee, Cryptomonas phaseolus Skuja, and Pyrenomonas helgolandii Santore), and GSIII was sequenced from G. theta. Red algal GSII sequences were obtained from Bangia atropurpurea (Mertens ex Roth) C. Agardh; Compsopogon caeruleus (Balbis ex C. Agardh) Mont.; Flintiella sanguinaria F. D. Ott and Porphyridium aerugineum Geitler; Rhodella violacea (Kornmann) Wehrmeyer and Dixoniella grisea (Geitler) J. L. Scott, S. T. Broadwater, B. D. Saunders, J. P. Thomas et P. W. Gabrielson; and Stylonema alsidii (Zanardini) K. M. Drew. In Bayesian inference and maximum‐likelihood (ML) phylogenetic analyses, chromalveolate GSII sequences formed a weakly supported clade that nested among sequences from glaucophytes, red algae, green algae, and plants. Red algal GSII sequences formed two distinct clades. The largest clade contained representatives from the Cyanidiophytina and Rhodophytina and grouped with plants and green algae. The smaller clade (C. caeruleus, Porphyra yezoensis, and S. alsidii) nested within the chromalveolates, although its placement was unresolved. Chromalveolate GSIII sequences formed a well‐supported clade in Bayesian and ML phylogenies, and mitochondrial transit peptides were identified in many of the sequences. There was strong support for a stramenopile‐haptophyte‐cryptophyte GSIII clade in which the cryptophyte sequence diverged from the deepest node. Overall, the evolutionary history of the GS gene families within the algae is complex with evidence for the presence of orthologous and paralogous sequences, ancient and recent gene duplications, gene losses and replacements, and the potential for both endosymbiotic and lateral gene transfers.     

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.     

Myosin-V,a versatile motor for short-range vesicle transport

Myosin-V is a versatile motor involved in short-range transport of vesicles in the actin-rich cortex of the cell. It binds to several different kinds of vesicles, and the mechanism by which it interacts with the vesicle surface is being unraveled, primarily in melanocytes. Members of the Rab family of G-proteins are required for the recruitment of myosin-V to vesicles. Rab27a and its rabphilin-like effector protein, Melanophilin, recruit myosin-Va to melanosomes and appear to serve as the membrane receptor. Myosin-V is also involved in fast axonal/dendritic transport and, interestingly, it forms a complex with kinesin, a microtubule-based motor. This kinesin/myosin-V heteromotor complex allows long-range movement of vesicles within axons and dendrites on microtubules and short-range movement in the dendritic spines and axon terminals on actin filaments. The direct interaction of motors from both filament systems may represent the mechanism by which the transition of vesicles from microtubules to actin filaments is regulated .     

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.