According to the exocytosis theory, melanin transfer is accomplished by fusion of the melanosomal membrane with the melanocyte plasma membrane, resulting in extracellular melanin and ensuing phagocytosis of released melanin by a neighbouring keratinocyte (Figure 1B). This hypothesis has been suggested by electron microscopic observations of human skin and hair follicles, depicting ‘naked melanin’ in the intercellular space and the enfolding of these pigment granules by keratinocyte pseudopods or clathrin-coated pits (12,13) (Figure 2A). Melanocytes discharge melanin in the extracellular space in vitro and do so increasingly when stimulated with α-melanocyte-stimulating hormone or with the soluble domain of the β-amyloid precursor protein (14,15). The process involved is considered to be exocytosis, as on electron microscopy discharged melanin is not surrounded by membrane and moreover, melanocytes express molecules with a well-known role in regulated exocytosis as SNAREs and Rab GTPases. SNAREs comprise three conserved families of membrane-associated proteins (the synaptobrevin/VAMP, syntaxin and SNAP-25 families) that act late in the events of membrane fusion. They associate into core complexes, and usually SNAP25 and syntaxin on the plasma membrane bind VAMP on the vesicle membrane. Different SNAREs have been identified in melanosome-enriched fractions: SNAP23, SNAP25, VAMP2, syntaxin4 and syntaxin6 (16,17). Immunoprecipitation shows association of VAMP2 and SNAP23, but not syntaxin4. Possibly, VAMP2 on melanosomes interacts with SNAP23 and an as yet to be identified syntaxin on the melanocyte plasma membrane to achieve fusion. Another family of proteins that acts in membrane fusion, particularly in tethering and docking of membranes before actual fusion, is Rab GTPases. The Rab3 proteins consisting of Rab3a-d are the central Rabs in regulated exocytosis. Rab3a is expressed in melanocytes and downregulation of its expression is effected by UV irradiation (16,18). Interestingly, downregulation of Rab3 in other cell types has proved to stimulate regulated exocytosis (19,20). Rab27a is also involved in regulated exocytosis of various types of organelles (21). As mentioned before, this molecule has an established role in melanosome transport at the cell periphery before actual transfer occurs. Synaptotagmin-like protein2-a (Slp2-a) is a new Rab27a effector which has been discovered in melanocytes (22). It links Rab27a with phosphatidylserine, thereby attaching melanosomes to the plasma membrane. This suggests that Rab27a has a role in docking melanosomes at the plasma membrane, which is an essential step in exocytosis. Interestingly, Slp2-a is structurally homologous to synaptotagmin, the putative regulator of exocytosis at the neuronal synapse.
Further support for the exocytosis hypothesis was provided by the finding that melanin in the keratinocyte cytoplasm presents in the same way, be it exogenously administered melanin or melanin supplied by melanocytes (14,23–26). Small melanin granules form aggregates after ingestion, while larger granules are dispersed singly in the keratinocyte cytoplasm. The distribution pattern seems to depend on size: phagocytosis of small or large latex beads also results in aggregates and singly dispersed beads, respectively. Aggregates of melanin granules are considered as indicators of cytophagocytosis of a whole dendrite tip with dispersion occurring in a later stage (see below). These studies demonstrate that melanin granules aggregate after being ingested as single granules and suggest that aggregates represent a final stage in the life cycle of melanin in keratinocytes.
Lastly, melanocytes are closely related to both neuronal and haemopoietic cells: to the former because of their neural crest origin and to the latter because melanosomes belong to the family of secretory lysosomes (specialized organelles that display substantial homology to lysosomes) (27). The relation between secretory lysosomes shows in genetic diseases affecting the biogenesis, transport and/or secretion of these organelles. For example, mutation of Rab27a in Griscelli syndrome impairs the exocytosis of secretory granules in cytotoxic T lymphocytes, resulting in immunodeficiency (28). As both synaptic vesicles and secretory lysosomes undergo regulated exocytosis on stimulation, it would be tempting to speculate that melanin transfer occurs by similar mechanisms.