- Top of page
- Materials and Methods
The formation of complex subcellular organelles requires the coordinated targeting of multiple components. Melanosome biogenesis in mouse melanocytes is an excellent model system for studying the coordinated function of multiple gene products in intracellular trafficking. To begin to order events in melanosome biogenesis and distribution, we employed the classical coat-color mutants ashen, dilute, and leaden, which affect melanosome distribution, but not melanin synthesis. The loci have been renamed Rab27a, Myo5a, and Mlph for their gene products. While each of the three loci has been shown to be required for melanosome distribution, the point(s) at which each acts is unknown. We have utilized primary melanocytes to examine the interdependencies between rab27a, myosin-Va, and melanophilin. The localization of rab27a to melanosomes did not require the function of either myosin-Va or melanophilin, but leaden function was required for the association of myosin-Va with melanosomes. In leaden melanocytes permeabilized before fixation, myosin-Va immunoreactivity was greatly attenuated, suggesting that myosin-Va is free in the cytoplasm. Finally, we have complemented both the leaden and ashen phenotypes by cell fusion and observed redistribution of mature melanosomes in the absence of both protein and melanin synthesis. Together, our data suggest a model for the initial assembly of the machinery required for melanosome distribution.
A central goal of cell biology is the identification and understanding of the molecular mechanisms that underlie the identity, assembly, and transport of organelles. In mammalian melanocytes, the formation of the melanosome is thought to occur through the convergence of multiple pathways for the delivery of the enzymes required for melanin synthesis. Trafficking of these enzymes involves the endoplasmic reticulum, trans-Golgi network, clathrin- and nonclathrin-coated vesicles, and endosomes (1–7).
Loci other than those that encode melanogenic enzymes also influence the coat colors of mice (8). Mutations at many of these loci affect other systems, including the nervous system and immune system. A subset of coat-color mutants (dilute, ashen and leaden) has been identified that synthesize normal levels of melanin, but inefficiently transfer melanosomes to neighboring cells (9–11). This leads to a clumping of melanin granules in the hair shaft, decreasing the amount of light absorbed. All three loci have been cloned and renamed Myo5a, Rab27a, and Mlph, respectively (12–14). The Myo5a (dilute) locus encodes myosin-Va (12), a processive molecular motor (15–17). Mutations in the human MYO5A gene underlie some cases of Griscelli Syndrome (18). Rab proteins, members of the ras superfamily of small GTPases, are involved in multiple pathways of vesicle trafficking (19,20). Mutations in Rab27a cause the ashen phenotype in mice (13) and a subset of Griscelli syndrome cases in humans (21,22).
In culture, wild-type primary melanocytes have a uniform distribution of melanosomes throughout the cytoplasm. Melanosomes from Myo5ad, Rab27aash, or leaden homozygous mutant mice, however, accumulate around the nucleus (13,23,24). Genetic analysis of compound mutants between the Myo5ad, Rab27aash, and leaden loci showed little synergy in affecting coat color phenotype (25), suggesting that they may act within the same pathway. Furthermore, the dilution of coat color caused by all three mutations is suppressed by a mutation at a fourth locus, dilute suppressor (25).
While the evidence that all three genes are required for melanosome distribution is unequivocal, the evidence that they are components of the actual machinery mediating distribution is circumstantial at best. Initial examinations of mutant melanosomes have suggested that melanosome biogenesis is normal (10,24). Therefore, it has been hypothesized that a complex consisting of myosin-Va, rab27a, and melanophilin is formed and targeted to the melanosome, during or after its genesis, to allow its distribution (26). Others have previously shown (27,28) that Rab27a is required for the interactions between myosin-Va and melanosomes. In this study, we show that leaden function also is required for the interaction of myosin-Va with melanosomes. Others have shown that Rab27aash mutant phenotype can be rescued by transfection (28), but we show that mature melanosomes assembled in the absence of either Rab27a or melanophilin can be redistributed by fusion of mutant with wild-type cells or of the two mutants with each other, even during inhibition of melanin and protein synthesis.
- Top of page
- Materials and Methods
While significant progress in understanding the delivery of melanogenic enzymes to melanosomes has been made (1–7), the identification of components functioning in the motility, distribution, and transfer of melanosomes to other cells is important as well (33,34) and has been relatively neglected until a spate of studies published in the last 6 years. The Rab27aash, Myo5ad, and leaden mutants are ideal for studying distribution, since the levels of melanin in mutant mice are not different from wild-type levels; only melanosome distribution is abnormal and accounts for the dilution of coat color (8,10,13,24,35). The apparent maturity of melanosomes in these mutants suggests separate pathways for the acquisition of melanogenic and motile components by melanosomes (8,10,24). Using these mutants and others, we should be able to derive the temporal sequence of events required for melanosome assembly and distribution. Melanophilin, rab27a, and myosin-Va have been hypothesized to function in a complex that mediates the movement or capture of mature melanosomes (13,14). We tested this model using Rab27aash, Myo5ad, and leaden (now Mlph) homozygous mutant melanocytes. In all cases, our results do not exclude the possibility that rab27a, myosin-Va, and/or melanophilin are targeted to (and function to localize) a precursor organelle that subsequently fuses with melanosomes, providing them with other components of the motility machinery.
If independent pathways deliver melanophilin, rab27a, and myosin-Va, delivery of rab27a to melanosomes should not be dependent on myosin-Va function. This prediction was confirmed by the melanosomal localization of rab27a in dilute (Myo5ad/Myo5ad) mutant melanocytes (Figure 3C,G). In addition, rab27a partially colocalized with melanosomes in leaden mutant melanocytes, indicating that it is not necessary for rab27a to interact with either myosin-Va or melanophilin for its targeting to melanosomes (Figure 3B,F). The reciprocal experiments, however, suggested dependency, since both melanophilin and rab27a were required for myosin-Va binding to melanosomes. Two other groups (27,28) have previously published the latter observation.
Our observation that myosin-Va targeting requires melanophilin suggested that assembly of any motility/capturing complex on melanosomes might require melanophilin early in melanosome biogenesis. The result of our cell fusion experiment was not consistent with this hypothesis, since the introduction of melanophilin to melanosomes that were formed in its absence resulted in a uniform distribution of mature melanosomes (Figure 4); the same result was obtained for Rab27aash/Rab27aash mutants rescued by fusion with melan-c melanocytes (Figure 5), and the mutant melanocytes fused with each other (Figure 6). These data indicate that neither melanophilin nor rab27a function is required until after all the components necessary for pigmentation have been delivered and are functional. It further suggests that melanosomes have a mechanism for melanophilin and rab27a delivery that diverges from that of lysosomes. This is an important consideration since the melanosome has been widely considered to be a derivative of the lysosome (36–39), but recent data suggest significant differences (7). The results presented here appear to conflict with our previously published immunofluorescence results (24); however, those experiments were performed with an antibody against a peptide. The antibody used in this study has been tested more extensively, and is more specific in both Western blotting and immunofluorescence (29) (data not shown). Also, the earlier experiments were performed using only methanol fixation, which may have allowed the differential loss of myosin-Va immunoreactivity during subsequent steps, given the data presented in Figure 2.
Melanophilin was recently identified as the product of the leaden gene (14) and is related to Slp3-a (40), the granuphilins (41), and rabphilin (42). These sequence similarities suggest that melanophilin functions in the same manner as rabphilin (14), the best-characterized member of the family, and the data presented here strongly support that hypothesis, consistent with rab27a and melanophilin as ‘co-keystones’ for the assembly of distribution components. Our cell fusion experiments suggest that this assembly occurs independently of, and possibly after, the delivery of components involved in melanogenesis. It must be emphasized that our experiments cannot directly address the existence of any hypothesized motility complex consisting of myosin-Va, rab27a, and melanophilin. However, they do suggest that if such a complex exists, rab27a and melanophilin, either simultaneously or sequentially, provide a foundation for the interaction of myosin-Va with melanosomes. The recent cloning of the leaden gene (14) should allow the further dissection of these pathways.