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.
Melanocytes of the skin contain melanosomes, which are specialized lysosome-related organelles with the ability to produce and store a light absorbing pigment called melanin that forms a protective barrier against the damaging effects of UV radiation (reviewed in Raposo and Marks, 2007). Inside the melanocytes, mature melanosomes are transported from the cell centre to the cell periphery. This involves a bi-directional ‘long range’ transport taking place along microtubule tracks. The motor proteins that are responsible for concentrating melanosomes to the centre of the cell are dyneins and move the melanosomes toward the minus end of the microtubule tracks (Vancoillie et al., 2000a). Dispersion of the melanosomes toward the plus end of the microtubules is mediated by kinesins, which eventually directs them to the periphery of the cell and into the dendritic tips (Hara et al., 2000; Vancoillie et al., 2000b). Once melanosomes reach the cortical regions of the cell, they undergo ‘short range’ movements along the sublemmal actin-network through the association with a processive motor protein myosin Va (Myo5a), which attaches to melanosomes through interaction with melanophilin (Mlph) and Rab27a (reviewed in Barral and Seabra, 2004; Nascimento et al., 2003). Studies of the protein–protein interactions occurring between the members of the Rab27a-Mlph-Myo5a tripartite complex have provided major insight into vesicle transport and membrane trafficking processes (Fukuda et al., 2002; Olkkonen and Ikonen, 2006; Strom et al., 2002; Westbroek et al., 2003; Wu et al., 2002a). This is in contrast with the following step in melanosome transport: the release of melanosomes from epidermal melanocytes to their neighbouring keratinocytes occurs through a mainly unsolved mechanism, although different hypotheses have been proposed to date (reviewed in Van Den Bossche et al., 2006). In this review, we focus mainly on intramelanocytic melanosome transport.
Several molecular studies have already revealed that mutations within any member of the RAB27A-MLPH-MYO5A tripartite complex cause one of the three different subtypes of a rare autosomal recessive hereditary disease known as Griscelli syndrome (GS) that develops early in life. GS3 is restricted to a hypopigmentation disorder, while the two other subtypes are primarily and additionally characterized by immunological defects and immunodeficiency (GS2) or neurological dysfunctions and impairments (GS1). They all share distinctive traits resulting in pigmentary dilution of the skin and hair causing silvery gray hair, the presence of large clumps of pigment in hair shafts along with an accumulation of melanosomes around the perinuclear area of melanocytes (reviewed in Huizing et al., 2008).
Griccelli syndrome: identification of melanosome transporter genes
In the past, a subset of mouse coat-color mutants (dilute, ashen, and leaden) which synthesize normal levels of melanin but inefficiently transfer melanosomes to neighboring cells has been identified. This results in clumping of melanin granules in the hair shaft (diluting hair pigmentation) and an abnormal distribution of melanosomes in the cell body of melanocytes. All three loci have been cloned and renamed Myo5a, Rab27a, and Mlph, respectively (Matesic et al., 2001; Mercer et al., 1991; Wilson et al., 2000). Because the phenotype of all three mouse mutants is rescued by the semidominant dilute-suppressor (dsu) (Moore et al., 1988; O’sullivan et al., 2004), it is assumed that their corresponding proteins function in the same or overlapping pathways. Therefore, these mutant mice were subjected to intense studies to identify components functioning in the motility, distribution, and transfer of melanosomes to other cells.
MYO5A functions as a dimeric, actin-based molecular motor protein and is divided into three separate domains termed ‘head’, ‘neck,’ and ‘tail’. Firstly, MYO5A is composed of two separate amino terminal (or ‘head’) motor domains, containing ATP-binding sites as well as having the capability to bind to actin fibers. These ATP-binding sites can convert energy released by multiple catalytic cycles of ATP hydrolysis, generating a mechanical movement along the actin filaments. Following these two separate head domains are two identical heavy chains that dimerize into each other. These two α-helical coils, called the ‘neck’ domains, are comprised of six subsequent amino acid (aa) residues termed IQ motifs with the consensus sequence IQXXXRGXXXR. These IQ motifs act as binding sites for light chains (LC), which are either calmodulin or calmodulin-like LCs and have a regulatory function by controlling the ATPase activity of the globular heads (reviewed in Trybus, 2008).
The ‘tail’ (proximal/medial) domain commences at the site where both α-helical coils dimerize to form a homodimer made up of a series of coiled-coil segments interrupted by small flexible globular regions (∼500aa). The length and composition of the medial tail region vary depending on alternative splicing. In humans, six different isoforms have been identified that are expressed in certain cell types, whereby each isoform plays a unique role in specifying which cargo is bound and transported. Transcripts containing the exon F were found to be the isoforms intervening in melanosome transport (Lambert et al., 1998a).
Finally, the last ∼ 400aa distinguish the distal/globular tail region (globular tail domain: GTD), also known as the cargo-binding site, where the interaction with different cargos is mediated by organelle-specific receptors.
Recent studies have revealed that this GTD is involved in correct motor functioning of Myo5a taking on a folded compact conformation resembling a triangular structure, when inactive. The region where the two α-helical coils of the neck domain connect is flexible enough to allow the two head domains to fold over along this jointed region and make contact with the GTD residing on each side of the tail domain. The two neck regions resemble two sides of the triangle with the flexible joint region resembling one point, while the GTD resembles the last remaining side. It has also been shown that the GTD simultaneously binds to the motor domain and the C-terminal residues of the first coiled-coil segment of the tail. The end of this first coiled-coil region is critical in stabilizing the folded triangular structure of Myo5a and prevents the conformational changes of the motor domain during the ATP turnover cycle, consequently inhibiting the actin-activated ATPase activity of the motor domain (Li et al., 2006, 2008; Sato et al., 2007). Very recently, Li et al. (2008) identified a conserved acidic residue (D136) in the motor domain and two conserved basic residues (K1706 and K1779) in the GTD as critical residues involved in this inhibition of Myo5a.
In an activated state, myosin V takes on an elongated conformation and disrupts its inhibiting triangular conformation. Cargo binding and high Ca2+ concentrations reduce the interaction between the GTD and head domain, thereby disabling the inhibiting triangular conformation and stimulating the extended conformation of MYO5A. This transition is accompanied by an increase in actin-activated ATPase activity and initiates the motor activity of myosin V (reviewed in Taylor, 2007).
Griscelli disease caused by mutations in MYO5A is termed GS1 (OMIM #214450). In general, these patients show hypomelanosis with a primary neurologic deficit and without immunologic impairment or manifestations of hemophagocytic syndrome (HS). MYO5A mutations were identified in only two GS patients (Pastural et al., 1997, 2000). The Arg-Cys substitution at codon 1246, described by Pastural and co-workers in 1997, appeared to be a polymorphism that also occurred in healthy individuals (Lambert et al., 2000). Intriguingly, in 2003 a form of GS1 was found in which the phenotype was restricted to hypopigmentation (Ménasché et al., 2003a) and associated with a homozygous 2439 bp genomic deletion spanning exon F of the MYO5A gene as well as part of the flanking 5′ and 3′ intronic sequences. Neurologic impairment was not diagnosed in this form of GS1. The other described cases did have hypomelanosis associated with severe neurologic impairment (Pastural et al., 1997, 2000; Sanal et al., 2002). It is known that Myo5a aids in the actin-based transport of smooth endoplasmic reticulum (sER) in the dendritic spines of neurons (Langford, 1999; Takagishi et al., 1996). The absence of sER from the Purkinje cell spines of Myo5a null mutant rats (dilute-opisthotonus) causes impairment of inositol 1,4,5-triphosphate-mediated Ca2+ release, resulting in a defect of long-term synaptic depression underlying cerebellar motor learning (Takagishi and Murata, 2006). Moreover, not much is known about how MYO5A functions in a molecular complex in the brain.
The Ras super family of small GTPases monomeric G proteins (20–25 kDa) is essential in regulating a wide variety of cell processes including vesicular transport pathways, cellular differentiation, and motility (reviewed in Corbeel and Freson, 2008; Seabra et al., 2002). RAB27A is a member of this family and has the ability to act as a molecular switch by cycling between two conformations through the binding of guanosine triphosphate (GTP), rendering it in an ‘active’ state or hydrolyzing GTP to guanosine diphosphate (GDP), translating it to its ‘inactive’ form. Two regions have been shown to change conformation upon GDP or GTP binding and have been termed switch I and switch II regions (Pereira-Leal and Seabra, 2000). A guanine nucleotide exchange factor (GEF), called Rab3GEF, is responsible for the activation of Rab27a in melanocytes (Figueiredo et al., 2008).
Rab27a is additionally a substrate for prenylation by an enzyme called Rab geranylgeranyl transferase. These prenyl groups are anchored to mature melanosomes and bind to two conserved cysteine residues located at the C-terminal end, also referred to as geranylgeranylation motifs of Rab27a (Anant et al., 1998; Larijani et al., 2003). This interaction can be considered similar to the labeling or tagging of membranes of vesicles, thus defining the identity and future routing of these vesicles. This implies that Rab27a is involved in targeting, docking, and fusion of transport vesicles with their appropriate acceptor membranes, causing it to be essential in membrane trafficking in different mammalian cell types (reviewed in Fukuda, 2005).
Table 1. Summary of reported mutations in Griscelli patients
p0 = no protein detected, fs = frameshift, X = stop codon.
aNomenclature of mutations is performed according to the ‘Human Genome Variation Society’ guidelines (Den Dunnen and Antonarakis, 2000) and based on GenBank Accession Numbers NM_000259 (MYO5A), NM_004580 (RAB27A), and NM_024101 (MLPH), where the ‘A’ from the start codon ATG is nucleotide number 1.
Reexpression of RAB27A in GS melanocytes (or ashen melanocytes) restored the proper distribution of melanosomes to the dendritic tips, once again stressing its importance in melanosome trafficking (Bahadoran et al., 2001; Wu et al., 2001). In parallel, coimmunoprecipitation studies with antibodies against Rab27a proved an association, although indirectly, between Rab27a and Myo5a (Hume et al., 2001). Although Rab27a was postulated to serve as a Myo5a receptor, these latter data strongly imply the existence of a linker protein connecting both Rab27a and Myo5a.
As mentioned earlier, the second type of Griscelli syndrome (GS2, OMIM #607624) is caused by mutations in RAB27A, which is also located on chromosome 15q21. In addition to its role in melanocytes, the RAB27A protein also functions in granule release within cytotoxic T lymphocytes (Bizario et al., 2004). Next to pigmentary dilution of the skin and silvery gray hair (Figure 1A) with clumps of pigment in hair shafts (Figure 1Bversus C (normal hair)), GS2 patients also develop immunodeficiency (due to impaired lytic granule exocytosis) leading to episodes of a life-threatening uncontrolled T lymphocyte and macrophage activation syndrome also known as HS or hemophagocytic lymphohistiocytosis (HLH). During HS, activated T cells and macrophages infiltrate various organs. Leukocyte infiltration in the brain probably leads to secondary neurologic impairment with convulsions and/or cerebellar manifestations. Primary central nervous system defects are not observed in patients diagnosed with GS2. This could be explained by the fact that RAB27A is scarcely expressed in the brain (Chen et al., 1997; Ramalho et al., 2001). There is one report describing a GS2 patient not displaying HS (Aksu et al., 2003).
Very recently, Pachlopnik Schmid et al. (2008) described a murine model of HLH in human GS. By infecting Rab27a−/− (ashen) mice with the lymphocytic choriomeningitis virus (LCMV strain WE), the authors were able to demonstrate that these infections triggered HLH. Of interest was that the clinical and laboratory features developed in the mice corresponded to the diagnostic criteria of human HLH. The LCMV-infected Rab27a−/− mice also showed a substantially better survival rate as compared with perforin-deficient mice (Jordan et al., 2004) and therefore represent a useful model to study the pathophysiology of HLH in more detail. Novel insights into the pathogenesis of HLH could eventually contribute to the development of new treatments for GS2 patients.
Several years ago, Matesic et al. (2001) identified melanophilin (Mlph) as the mutated gene in leaden (ln) mice. As the phenotype of the leaden mouse is similar to the ashen and dilute mouse, Mlph was considered as a potential new candidate gene that was mutated in GS. Interestingly, the only defect observed in leaden melanocytes was perinuclear clustering of melanosomes. This suggested that Mlph might function as part of a transport complex with Myo5a and Rab27a (Matesic et al., 2001).
The newly identified Mlph or the synaptotagmin-like protein (Slp) lacking C2 domains-a (Slac2-a) represented a novel class of the Slp family. All members of the Slp family contain an N-terminal Slp homology domain (SHD) consisting of two conserved potential α-helical regions (SHD1 and SHD2) often separated by two zinc finger (ZnF) motifs. In contrast to other Slp proteins, the Slac2 family (Slac2-a/Mlph, Slac2-b, and Slac2-c/MyRIP) lacks C-terminal tandem C2 domains (termed C2A and C2B). Instead, MLPH contains two unique coiled-coil domains (Coil 1 and Coil 2) at its C-terminal end (Nagashima et al., 2002). Biochemical and cell biological analyses have now revealed that MLPH is the specific linker protein between MYO5A and RAB27A (for more details, see below).
In 2003, the sole mutation in the human MLPH gene (located on 2q37), giving rise to GS type 3 (GS3, OMIM #609227), was documented (Ménasché et al., 2003a). A homozygous C103T transition was identified in exon 1 of the patient’s DNA, resulting in a putative R35W substitution. Interestingly, phenotypic expression of GS3 is restricted to the characteristic hypopigmentation of this syndrome. GS3-associated hypomelanosis is indistinguishable from that of GS1 and GS2. Loss of function of MLPH does not cause neurologic or immunologic defects. The same pigment dilution phenotype caused by MLPH mutations can also occur as a result of MYO5A exon F deletion, as described previously in GS1.
Intramelanocytic melanosome transport
The RAB27A-MLPH-MYO5A tripartite protein complex
In the past, we have reported that human MYO5A undergoes tissue-specific alternative splicing located at its medial tail region, leading to an alternate usage of three exons designated as B, D, and F (Lambert et al., 1998a). Of the six known human isoforms (ABCDEF, ABCEF, ACDEF, ABCDE, ABCE, and ACE), three contain exon F. These exon F-containing isoforms are most abundantly expressed in melanocytes and were shown to colocalize with melanosomes (Lambert et al., 1998a; Westbroek et al., 2003; Wu et al., 2002b). Detailed molecular studies (use of dominant negative constructs, rescue experiments, yeast two-hybrid screenings) revealed that both the C-terminal globular tail domain and the exon F sequence are essentially required for Myo5a to colocalize with and influence the position of melanosomes in melanocytes (Westbroek et al., 2003; Wu et al., 2002b). In addition, it has become clear that Rab27a and Myo5a exon F-containing isoforms function as a receptor complex involving a specific linker protein. Yeast two-hybrid screenings containing exon F and globular tail constructs have revealed specific interactions with a Rab-effector protein, termed melanophilin (Westbroek et al., 2003; Wu et al., 2002a). Other groups have also confirmed these observations by performing colocalization studies in leaden or ashen melanocytes (Hume et al., 2002; Provance et al., 2002) or in vitro binding assays (Fukuda et al., 2002). Further proof that Mlph functions as a linker between Rab27a and Myo5a was given by the fact that the region of Mlph responsible for binding of both Rab27a and Myo5a was mapped to its amino- and carboxy-termini, respectively (Fukuda et al., 2002; Nagashima et al., 2002; Wu et al., 2002a). Based on the results summoned above, the recruitment of Myo5a to melanosomes occurs as follows: activated Rab27a first binds to the surface of the melanosomes and then recruits Mlph, which subsequently recruits the Myo5a exon F-containing isoforms (Figure 2A). In general, it has recently been accepted that the Rab27a-Mlph-Myo5a tripartite complex is essential for the transportation of melanosomes from the microtubles to the actin filaments followed by their capturing within the distal, actin-rich regions of the dendrites (Fukuda et al., 2002; Goud, 2002; Strom et al., 2002; Westbroek et al., 2003; Wu et al., 2002a). Moreover, in vitro reconstitution of the Myo5a receptor complex by use of dominant active GFP-tagged Rab27a and Mlph demonstrated that they were not only required, but also sufficient, to form a transport complex that moves processively on actin (Wu et al., 2006). Consequently, loss of one of the components of the tripartite complex results in GS, biologically characterized by a perinuclear accumulation of melanosomes in the melanocytes (Figure 2B).
During the last years, several binding domains (and critical residues) of Mlph have been identified as being essential for the correct binding of Rab27a and proper recruitment of Myo5a. The amino-terminal part of Mlph, containing SHD1, SHD2, and an intervening ZnF domain, is responsible for interaction with Rab27a and is termed R27BD (RAB27A-binding domain). Within this region, SHD1 binds directly with the switch II region of the GTP-bound active form of Rab27a present on melanosomes (Fukuda, 2002; Strom et al., 2002). The globular tail and exon F sequence of Myo5a bind two distinct domains of Mlph; Myo5a-GT binds to a newly characterized region adjacent to SHD2 (mouse aa 147–240, termed GTBD). In comparison, Myo5a-exon F has been proven to bind to the middle region of Mlph (mouse aa 320–406, termed EFBD) (see also Figure 2A). The interaction between Mlph and Myo5a-GT seems to be less stable and much weaker compared with the interaction between Mlph and Myo5a-exon F. Interestingly, studies with dilute missense mutations in Myo5a-GT impaired only the former interaction and not the latter, indicating that the Mlph-Myo5a-GT is physiologically relevant. As a consequence, both domains are essential and probably function in a synergistic manner during melanosome transport in melanocytes (Fukuda and Kuroda, 2004). Hume et al. (2006) extended the results shown above by demonstrating that melanosomal Rab27a-GTP recruits Mlph to the melanosomes via interaction with both SHD1 and SHD2. In addition, the authors identified a coiled-coil structure in the C-terminal end (mouse aa 440–483), termed the actin-binding domain (ABD) of Mlph, which is essential for the recruitment of Myo5a to the melanosomes by increasing the interaction of the Myo5a-binding domain (MBD) of Mlph with Myo5a.
It is noteworthy to mention that the binding site of Myo5a-GTBD has been recently narrowed down to a 26 amino acid region of Mlph (mouse aa 176–201) (Geething and Spudich, 2007). In the same study, it was demonstrated that this short peptide sequence corresponded to an intrinsically unstructured domain of Mlph. The reasons of using such an unfolded binding region have not been resolved yet, but it may offer some functional advantages. One possibility is that it provides a site for rapid degradation following cargo delivery, in addition to the PEST site present in Mlph (Fukuda and Itoh, 2004).
In addition, the functional involvement of the C-terminal ABD of Mlph was further studied in detail by Kuroda and Fukuda and colleagues. Strong exogenous expression of different Mlph-truncated proteins in intact cells revealed that the ABD interacts with actin, suggesting that the ABD of Mlph may be required for melanosome transport from microtubules to actin filaments in addition to the formation of the tripartite complex (Fukuda and Kuroda, 2002; Kuroda et al., 2003). However, the latter hypothesis is not further supported by other studies as colocalization of Mlph with actin, for example, is only evident at high levels of exogenous Mlph expression (Fukuda and Itoh, 2004; Hume et al., 2006). Further research will be necessary to determine if endogenous Mlph is able to interact with actin.
In another in vitro study, Passeron et al. (2004) demonstrated that physiological melanocyte-differentiating agents such as α-melanocyte stimulating hormone, by means of activating the cAMP pathway, rapidly induce an increase in the interaction of Mlph with actin, resulting in a fast accumulation of melanosomes around the actin-rich region of the dendrite extremities. Additionally, cAMP stimulates the expression of Rab27a that could facilitate the interaction of melanosomes with cortical actin. In melanocytes, the effects of cAMP on melanin synthesis are mediated by microphtalmia-associated transcription factor (MITF) that plays an essential role in survival, migration, proliferation, and differentiation of melanocytes during development (reviewed in Hou and Pavan, 2008). MITF controls the expression of genes essential for melanin synthesis (such as TYR, TYRP1, and DCT) and for the maturation of melanosomes (MART1, PMEL17, and GPR143) (Gaggioli et al., 2003; review: Levy et al., 2006). Based on the knowledge described above, Ballotti and colleagues decided to examine the possible role of MITF in melanosome transport. Silencing of MITF in human and mouse melanoma cells (treated with forskolin to stimulate cAMP) induced perinuclear aggregation of melanosomes, including a relocalization of RAB27A, MLPH, and MYO5A to the cell body caused by a dramatic decrease in RAB27A expression. Functional analysis of the RAB27A promoter revealed that MITF binds directly to two E-boxes in the proximal region of the RAB27A promoter, thereby stimulating the expression of RAB27A and facilitating its interaction with MLPH (Chiaverini et al., 2008). In this manner, loss of MITF inhibits the expression of RAB27A (and blocks the effect of cAMP on RAB27A), which consequently results in the impairment of the RAB27A-MLPH-MYO5A tripartite complex, subsequently explaining the abnormal melanosome distribution in MITF-deficient melanoma cells. The identification of RAB27A as a new MITF target gene links MITF to actin-dependent melanosome transport, an important step in melanocyte differentiation and skin pigmentation.
Considering that RAB27A is also involved in numerous biological processes other than skin pigmentation, the data above illustrate how newly obtained information, based on a melanosome transport model, could inspire others to investigate the transcriptional regulation of RAB27A in cell types such as cytotoxic T-lymphocytes or pancreas β-cells.
Role of EB1 in (intracellular) melanosome transport?
The microtubule (MT) end-binding EB1 protein is mainly located at the growing plus ends of MTs where it stimulates MT growth and interacts with other proteins, allowing them to be transported to the cell periphery (Vaughan, 2005). Wu et al. (2005) demonstrated that the last 100 residues at the C-terminus ABD of Mlph (mouse aa 490–590) can interact with EB1. A physical interaction between EB1 and Mlph seems to play a role in the transport of melanosomes to the peripheral dendrites and the retention thereof. More specifically, an EB1-Mlph-Myo5a complex is formed at the growing plus end of MTs that is targeted to the periphery by MT polymerization. In the meantime, melanosomes carrying Rab27a are transported along MT tracks toward the periphery of the dendritic tips by the action of processive MT motors. This conceives an image showing melanosomes reaching the plus end of their MT tracks followed by a Rab27a interaction with MT-associated Mlph-Myo5a, allowing melanosomes to be released from MTs, succeeded by an immediate capture into the surrounding cortical actin cytoskeleton (Wu et al., 2005).
Recently, Hume et al. (2007) studied in depth the functional relationship between Mlph and EB1 by measuring MT-plus-end tracking function of EB1 in the presence or absence of Mlph in mouse melanocytes. The results of their yeast two-hybrid and biochemical binding assays suggested that Mlph and EB1 indeed interact with each other in vitro (confirming the observations of Wu et al., 2005). However, colocalization studies and several functional tests of the interaction of Mlph with EB1 did not show a strong effect of the loss of either protein on the function of the other. The authors were able to illustrate that siRNA knockdown of EB1 does not affect melanosome distribution in wild-type cells or Mlph-dependent rescue of melan-ln defects. In addition, overexpression of dominant negative truncated EB1 molecules comprising the Mlph-binding site does not affect melanosome distribution in the dendrites of wild-type melanocytes. From these and other performed experiments, Hume et al. (2007) concluded that there is no physical or functional evidence that the Mlph-EB1 interaction is important for initial targeting of Mlph or its function in melanosome transport to peripheral dendrites. Parallel studies to investigate the role of other Mlph-interacting proteins pointed out that Rab27a is the primary Mlph targeting and stabilization factor, whereas Myo5a also stabilizes Mlph and allows peripheral retention of melanosomes via direct interaction with actin. This means that the model of sequential recruitment of Mlph and Myo5a to the melanosome membrane by Rab27a, as proposed previously by others (Fukuda et al., 2002; Hume et al., 2002; Nagashima et al., 2002; Provance et al., 2002; Strom et al., 2002; Westbroek et al., 2003; Wu et al., 2002a), is supported. As EB1 does not appear to be involved in melanosome transport, one can only speculate on the role of Mlph-EB1 interaction in Mlph function. According to Hume et al. (2007), the Mlph-EB1 interaction could, for example, be involved in other specialized aspects of melanosome transport, such as the intercellular transfer of pigment from melanocytes to keratinocytes in skin and hair. Further studies of the Mlph-EB1 interaction in co-culture systems of melanocytes and keratinocytes are warranted to support this hypothesis.
Biological relevance of mutations in the RAB27A-MLPH-MYO5A tripartite complex
Several mutations and deletions have been pinpointed in each component of the RAB27A-MLPH-MYO5A tripartite complex. A summary of all known GS mutations reported in the literature is listed in Table 1. Molecular and functional analyses of these mutations are a helpful tool to understand the underlying mechanisms by which each mutation can cause a defect in melanosome transport. Some of these mutations in GS patients have been studied in great depth. The biological relevance of these mutations is briefly explained next. The few MYO5A mutations detected in GS patients occur mostly as either a ‘R779X’ nonsense mutation or ‘a 47 bp insertion’. The nonsense mutation R779X is located directly in front of the calmodulin/light chain-binding domain which contains the ‘IQ’ motifs, conceiving a truncated protein lacking the calmodulin domain, the neck, and the tail region (Pastural et al., 1997). Therefore, this specific mutation ultimately leads to a complete loss of function of MYO5A. On the other hand, the 47 bp insertion that is located at the beginning of the carboxy-end of MYO5A predicts a truncated protein lacking most of the globular domain (Pastural et al., 2000). The absence of the globular tail domain of MYO5A explains the GS phenotype in melanocytes as this domain is important for the proper assembly and function of the RAB27A-MLPH-MYO5A tripartite complex. In one certain GS patient, a deletion of MYO5A-exon F has been identified (Ménasché et al., 2003a). Due to the loss of exon F, which is absolutely required for the correct capturing of the melanosomes in the subcortical actin-network, intracellular melanosome transport was inhibited as the formation of a functional RAB27A-MLPH-MYO5A-exon F tripartite complex was impaired. The phenotype of this particular patient was restricted to hypopigmentation, comparable with GS type 3, and could likely be contributed to the fact that MYO5A-exon F isoforms are abundantly expressed in melanocytes but not in the brain.
Most RAB27A mutations are homozygous nonsense or frameshift mutations leading to a premature stop codon predicting a truncation of the C-terminal geranylgeranylation motif, which is required for correct vesicular targeting. Therefore, the resulting RAB27A proteins are not functional. Alternatively, some of these mutations altered mRNA or protein stability and resulted in the absence of a RAB27A protein (Ménasché et al., 2000). It is obvious that a nonfunctional RAB27A or a complete loss of this protein leads to a disruption of the RAB27A-MLPH-MYO5A protein complex, finally resulting in perinuclear aggregation of melanosomes in the melanocytes of GS patients.
So far, only a few missense mutations of RAB27A have been identified. Functional characterization of the W73G mutant revealed that although this mutant was able to bind to GTP or GDP, it was incapable of hydrolyzing GTP. Consequently, this resulted in abrogation of its interaction with the SHD domain of MLPH. These results indicated that a conservation of tryptophan at position 73 is essential for the intrinsic GTP hydrolysis activity of the RAB27A protein, independent of the nucleotide binding (Bahadoran et al., 2003; Ménasché et al., 2003a). The other two mutants, L130P and A152P, seemed to act as dominant negative mutants because they are able to impair the function of RAB27A and block melanosome transport to dendrite extremities when overexpressed in B16 melanoma cells (Bahadoran et al., 2003). Interestingly, the mutations L130P and A152P affect highly conserved residues among Rab proteins. Ménasché et al. (2003b) additionally reported that insertion of a proline residue in either the α4 (A152P) or the ß5 (L130P) loop of these mutants dramatically impairs both GTP and GDP nucleotide-binding activity of RAB27A, probably by disrupting protein folding. Such conformational changes of the RAB27A protein apparently have an effect on its association with MLPH or other effectors. For example, the L130P did not have the ability to interact with MLPH. On the other hand, the A152P mutation was not able to be targeted to melanosomes but did interact with MLPH. The latter interaction was, however, not sufficient to ensure correct melanosome transport pointing out the complexity of the RAB27A function (Bahadoran et al., 2003).
The RAB27A (G43S) homozygous missense mutation was identified in an Afghani GS2 patient and further analyzed in detail by Westbroek et al. (2008). Expression of this mutant RAB27A-GFP in normal melanocytes induced a perinuclear aggregation of melanosomes comparable with that of WT RAB27A-GFP (presence of melanosomes in the cell periphery and dendrite tips). In addition, transfection of RAB27A (G43S) did not restore the normal distribution of melanosomes in GS2 melanocytes. Co-immunoprecipitation studies revealed that RAB27A (G43S) was unable to interact with MLPH. As this mutation was located in the switch I region of RAB27A, it was hypothesized that this amino acid residue, and probably other conserved amino acids, are also involved in the recruitment of MLPH alongside the already known involvement of the switch II region (Fukuda, 2002). This illustrates even more the complexity of RAB27A functioning and stresses the importance of a functional mutation analysis, so as to obtain a better insight into the mechanisms of RAB27A-dependent melanosome transport.
To date only one MLPH mutation has been identified in a GS3 patient. A homozygous C103T transition (located in exon 1 of MLPH) was identified at the genomic level. On protein level this causes the substitution of arginine with tryptophan at position 35. The R35W mutation is located in the SHD domain of MLPH and is highly conserved among the members of the Slp family. Through functional assays Ménasché et al. (2003a) were able to show that the R35W mutation completely inhibits the interaction of MLPH with RAB27A. As the arginine residue (R35) is situated at the interface of the MLPH-RAB27A interaction, the authors have hypothesized that the replacement of a positively charged side chain residue (R) by one with a bulky aliphatic side chain (W) may affect the stereochemical constraint of the interaction. These results point at the necessity of the conserved R residue at position 35 for RAB27A-MLPH binding.
Role and hierarchy of Rab27a effectors in melanosome transport
Several putative Rab27a effectors existing in human and mice have been classified into three distinct groups (Slp family (Slp1-5 and rabphilin), Slac2 family (Slac2 a-c and Noc2), and Munc13-4) based on their structure (reviewed in Fukuda, 2005). With regard to melanocyte cells, SLAC2-A (MLPH) is the best characterized RAB27A effector present on melanosomes and as a member of the RAB27A-MLPH-MYO5A tripartite complex, it is involved in the capturing of melanosomes in the actin-rich dendritic tips. Several years ago, Kuroda et al. (2002) demonstrated that Slp2-a, another Rab27a-effector, colocalized to melanosomes and as a consequence associates with Rab27a. These results assumed that Rab27a was likely to have at least one additional role in melanosome transport. Additional immunofluorescence studies of mouse melan-a cells confirmed the presence of both Slp2-a and Slac2-a on well-matured melanosomes (Kuroda and Fukuda, 2004). Interestingly, reduction of endogenous Slp2-a protein by RNAi caused a rounded cell shape and a pronounced decrease in melanosomes in the cell periphery (i.e., peripheral dilution of melanosomes), in contrast to Slac2-a deficient cells where an elongated cell shape and a perinuclear melanosome accumulation was observed. These observations demonstrated that Slp2-a controls cell shape and is additionally involved in the regulation of the peripheral distribution of melanocytes. In the latter process, Slp2-a anchors melanosomes to the plasma membrane through simultaneous interaction with Rab27a on the melanosomes and phosphatidylserine in the plasma membrane. The data above also suggests that Rab27a uses two Rab27a effectors consecutively, starting with Slac2-a followed by Slp2-a, to transport melanosomes from the perinucleus to the cell periphery (Kuroda and Fukuda, 2004). Further functional studies of the protein’s SHD domains (known to bind Rab27a) revealed that Slac2-a is a low affinity Rab27a effector involved in the early stage of melanosome transport, and Slp2-a is a high affinity Rab27a effector involved in a subsequent stage (Fukuda, 2006). These in vitro data might suggest that a temporal hierarchy of Rab27a effectors is important in the regulation of melanosome transport in melanocytes.
Functional redundancy of RAB27 proteins: clues to the pathogenesis of GS
Besides RAB27A, other Rabs are also present on melanosomes of which the exact function is not yet defined. A homolog of RAB27A, namely RAB27B, has been cloned whereby subsequent studies have revealed in great detail much of its structure. RAB27B proteins are found to be mainly expressed in platelets, unlike melanocytes, where they are very low expressed or not expressed at all (Chen et al., 1997). Transient expression of EGFP-Rab27b constructs in mouse melanocytes depicted the colocalization of Rab27b and Myo5a on melanosomes (Barral et al., 2002; Chen et al., 2002; Ramalho et al., 2001). Overexpression of dominant negative mutants of Rab27b accomplished the relocalization of melanosomes from the cell periphery to the perinuclear region, suggesting Rab27b could serve to be at least partial functionally redundant with Rab27a (Chen et al., 2002). These observations were further confirmed in another study performed by Barral et al. (2002). The authors demonstrated that transgenic expression of Rab27b in ashen mice rescued their coat color phenotype. These data presume the involvement of Rab27b in melanosome transport. Interestingly, Westbroek et al. (2004) described a GS patient in whom RAB27A protein expression was lost due to deletion of exons 3 and 4 at the genomic level. This defect results in the perinuclear aggregation of melanosomes in the patient’s melanocytes. Nevertheless, a normal melanosomal distribution was still observed in about 10% of these cultured melanocytes. Expression analysis of RAB27B at both the mRNA and protein level revealed a significant upregulation of RAB27B in melanocytes obtained from this GS patient compared with that in normal melanocytes. Immunofluorescence analysis and yeast two-hybrid screening studies have confirmed that RAB27B associates with the melanosomal membrane and is part of a RAB27B-MLPH-MYO5A-exon F tripartite complex in melanocytes. These results conclude that upregulated RAB27B had partially taken over the function of RAB27A in this particular GS patient. This case study provided new insights into the pathogenesis of GS and could stimulate others to perform additional screenings for RAB27B upregulation in GS type 2 patients.
Melanosome motility in RPE: the Rab27a-Myrip-Myosin VIIa tripartite complex
In mammals, melanosomes are found not only in epidermal melanocytes, but also in choroidal melanocytes of the eye and retinal pigment epithelium (RPE) cells. The latter cells also provide an excellent model system for the study of organelle transport because they contain numerous melanosomes that are essential in maintaining photoreceptor vitality (reviewed in Coudrier, 2007; Futter, 2006). The molecular regulation of melanosome distribution, as a response to light changes, in mammalian RPE cells has been recently examined. Liu et al. (1998) demonstrated that shaker-1 mice, which carry a mutation in the gene encoding myosin VIIa (Myo7a), have an abnormal perinuclear distribution of the melanosomes found in their RPE cells. Parallel with Myo5a in skin melanocytes, the melanosomes are excluded from the apical processes of RPE cells indicating that Myo7a is required for movement of melanosomes into, or retention of melanosomes within, the actin-rich apical region. In addition, Myo7a has been illustrated to be associated with retinal melanosomes by immunoelectron microscopy (El-Amraoui et al., 2002; Liu et al., 1998).
Previous studies have already shown that Rab27a is a marker of mature secretory organelles and that this GTPase can bind several classes of myosin motors to their appropriate organelle surface (Seabra and Wasmeier, 2004). Rab27a is also present on the melanosomes in RPE cells. In the RPE cells of the ashen mouse, the melanosomes remain in the cell body, thus showing a similar phenotype with respect to melanosome distribution as observed in the RPE cells of the shaker-1 mouse (Futter et al., 2004; Gibbs et al., 2004). Interestingly, a novel Rab effector, Myrip (also know as Slac2-c, an Mlph homolog), was able to bind to both Rab27a and Myo7a (El-Amraoui et al., 2002; Fukuda and Kuroda, 2002). Due to its observed expression in RPE cells, one may assume that Myrip is implicated in RPE melanosome motility (El-Amraoui et al., 2002). Based on these obtained data, it was hypothesized that a tripartite complex of Rab27a-Myrip-Myo7a regulates melanosome distribution in RPE cells, in a comparable way as the Rab27a-Mlph-Myo5a complex, which is involved in intracellular melanosome transport in melanocytes. Additionally it was demonstrated that in both in vitro and in intact cells, Myrip directly binds actin, indicating a possible role for Myrip in capturing Rab27a-containing organelles around the actin-enriched cell periphery (Fukuda and Kuroda, 2002). As it was also demonstrated that Myrip/Slac2-c could weakly interact with Myo5a, Kuroda and Fukuda (2005) decided to investigate the function of Slac2-c in skin melanosome transport. Ectopic expression of Slac2-c-Myo7a in melanocytes lacking Mlph restored the normal distribution of melanocytes from perinuclear aggregation, whereas the expression of Slac2-c alone did not. This indicates that the Slac2-c-Myo7a complex can substitute for the Slac2-a-Myo5a complex in melanosome transport, or in other words, Myrip binds myosin VIIa in intact cells and operates as a linker between Rab27a on melanosomes and the actin cytoskeleton, confirming the hypothesis stated above. Conclusive evidence that the formation of a functional Rab27a-Myrip-Myo7a tripartite complex is required for RPE melanosome motility came from RNAi studies in cultured primary RPE cells. Loss of any of the three components of the tripartite complex resulted in a redistribution of melanosomes from the F-actin-rich apical region and processes to the MT-rich cell body; the same phenotype was also observed in ashen (Rab27a defective) and shaker-1 (Myo7a mutant)-derived RPE cells (Lopes et al., 2007). Interestingly, Lopes et al. (2007) also demonstrated that nocodazole treatment (to disrupt MTs) led to an almost complete loss of melanosome movement in mammalian RPE cells. Previous studies involving isolated fish RPE cells suggested that MTs and MT-motors were not important for melanosome movement (King-Smith et al., 1997). The afore-mentioned data indicate on the contrary or at least in mammals that melanosome movement in RPE cells requires motor proteins such as kinesins and dyneins. However, the above data state that the Rab27a-Myrip-Myo7a complex is involved in the switch from MT- to actin-based motility as put forward in the skin melanocytes for the Rab27a-Mlph-Myo5a tripartite complex. Nonetheless, the importance of melanosome movements in retinal physiology is not yet completely understood. Mislocalization of melanosomes is probably not the main reason for photoreceptor degeneration found in patients suffering from Usher 1B syndrome (OMIM #276900), caused by mutations in MYO7A. This disease is most likely triggered by a lack of photoreceptor-specific functions of MYO7A and/or related to the role of RPE phagocytosis leading to a delay in degradation of phagosomes (Gibbs et al., 2003). Further research is necessary to resolve these issues.
Knockdown of MYO5A melanocyte-specific isoforms by RNAi: a new strategy to treat hyperpigmentation?
Hyperpigmentation is a frequently seen dyschromatosis of the skin (reviewed in Halder and Nootheti, 2003; Rigopoulos et al., 2007). Most of the time this kind of pigmentation disappears spontaneously, but in some cases the course of the disease may be so prolonged that the condition develops into a chronic problem. The current treatment methods, such as hydroquinone or kojic acid treatment, are insufficient and/or can cause side effects including risk of post-inflammatory hypo- or hyperpigmentation, ochronosis, and scarring, producing carcinogenic effects (reviewed in Solano et al., 2006). Increased melanosome transport from the melanocytes to the keratinocytes is one of the underlying biological mechanisms causing hyperpigmentation (reviewed in Briganti et al., 2003). Modulating melanosome capturing in the periphery of the melanocytic dendrites through RNAi could ultimately reduce pigment transfer and holds perspectives to the development of new topical therapeutics, aiding in the treatment of (hyper)pigmentation.
Others, along with our group, have previously shown that the Myo5a-exon F-containing isoforms are abundantly expressed in epidermal melanocytes (Lambert et al., 1998a; Wu et al., 2002b). Furthermore, they are absolutely required for the capturing of melanosomes in the actin-rich regions of the dendrite tips via the formation of the Rab27a-Mlph-Myo5a-exon F tripartite protein complex (Westbroek et al., 2003; Wu et al., 2002a). Recently, we were successful in developing an exon-specific RNAi method to inhibit the expression of the melanocyte-specific exon F isoforms of MYO5A. Downregulation of MYO5A-exon F in primary human melanocytes resulted in a perinuclear melanosome aggregation when using either synthetically synthesized siRNAs (transient gene silencing) or lentiviruses expressing a short hairpin RNA against the exon F sequence (stable gene silencing) (Van Gele et al., 2008). Loss of MYO5A-exon F isoforms was by these means sufficient to inhibit intracellular melanosome transport, due to the disruption of the RAB27A-MLPH-MYO5A-exon F tripartite complex. Interestingly, the observed biological effect (that is, accumulation of melanosomes around the cell nucleus) depicts what is seen in melanocytes from patients with human GS, explaining the pigmentary dilution of the skin. Presently, only one GS patient bearing a deletion of exon F has been reported. The phenotype of this patient was restricted to hypopigmentation, similar to GS type 3 (Ménasché et al., 2003a). The occurrence of this single effect is probably explained by the fact that MYO5A-exon F isoforms are predominantly expressed in melanocytes and not in neurons, for example. The data summarized above implicate that abrogation of the MYO5A-exon F-containing isoforms by siRNA-mediated gene silencing could diminish (hyper) pigmentation. Expanding this siRNA-based methodology toward in vivo studies will be of great importance to obtain a proof-of-principle, which also embraces the exclusion of possible side effects in other cell types of the skin.
Another challenge in the development of topical siRNA-based therapeutics is the delivery of siRNA to the target cells located in the skin. Although the skin is a large and easily accessible organ, it is well-known that the stratum corneum serves as a very efficient barrier preventing the penetration of foreign molecules, including topical applied drugs. Recently, we developed ultradeformable cationic liposomes, based on DOTAP and sodium cholate, which could very efficiently deliver MYO5A-exon F siRNA into human primary melanocytes cultured in vitro (Geusens et al., 2009). The addition of sodium cholate, an edge-activator, increased the flexibility and deformability of the cationic DOTAP liposomes, suggesting that these carrier systems could penetrate the skin easier, compared with conventional cationic liposomes. These results show great promise for future in vivo experiments and could open ways for new topical applications to treat some hyperpigmentary disorders and other skin diseases, such as psoriasis and atopic dermatitis.
The topics in this review demonstrate clearly that GS is an excellent model system to study the function of key molecules involved in intracellular melanosome transport. We pointed out that each member of the RAB27A-MLPH-MYO5A tripartite complex has a specific role in the peripheral distribution of melanosomes, a necessary step in skin pigmentation. Melanosome transport in pigment cells is considered as a paradigm for the further understanding of vesicular trafficking in other cell types. In this context, we refer to the recent discovery of a new Slp2-a isoform (hem-Slp2-a) which is able to associate with RAB27A in cytotoxic T cells and is probably involved in cytotoxic granule secrection (Ménasché et al., 2008). In addition, identifying the exact role of the brain-specific MYO5A isoforms together with the search for a tripartite complex, if any, involved in axonal and dendritic transport in neurons, are other interesting issues to be addressed in the future. Results from these studies, together with existing data, will eventually lead to a better insight into the pathogenesis and disease mechanism of GS and will open new perspectives in the treatment of GS patients.
Mireille Van Gele is a postdoctoral research fellow of the Fund for Scientific Research-Flanders (Belgium). Peter Dynoodt received financial support from the Pierre Fabre Institute (Centre d’Etudes et de Recherche sur la Peau et les Epithéliums de Revêtements, Toulouse, France).