Faculty Bibliography

The movement of melanosomes from post-Golgi compartments to the periphery of melanocytes is known to be regulated by factors including myosin Va and at least one Rab protein, Rab27a. Mutations in the genes encoding either protein in the mouse result in a hypopigmented phenotype mimicking the human disease Griscelli syndrome. Rab27b and Rab27a share 72% identity and they belong to the same melanocyte/platelet subfamily of Rab proteins. Rab27a orchestrates the transport of melanosomes by recruitment of the actin motor, myosin Va, onto melanosomes. By contrast, the function of Rab27b has remained elusive. In this study, we found that Rab27b mRNA is present in melanocytes and demonstrated the intrinsic GTPase activity of Rab27b protein. We explored the function of Rab27b by overexpression of two dominant negative mutants as well as the wild-type Rab27b in melan-a melanocytes. Green-fluorescent-protein-tagged Rab27b colocalizes with the melanosome marker tyrosinase-related protein 1 and with myosin Va at the cell periphery, whereas Rab27b mutants do not decorate melanosomes, and melanosomes in these mutant transfected cells redistribute from cell periphery to the perinuclear region. Furthermore, transient overexpression of the dominant negative forms of Rab27b caused diminution in both numbers and length of dendrites of melan-a cells. Our results suggest that Rab27b may regulate the outward movement of melanosomes and the formation or maintenance of dendritic extensions in melanocytes

The processing of tyrosinase, which catalyzes the limiting reaction in melanin synthesis, was investigated in melan-p1 melanocytes, which are null at the p locus. Endoglycosidase H digestion showed that a significant fraction of tyrosinase was retained in the endoplasmic reticulum. This retention could be rescued either by transfection of melan-p1 cells with an epitope-tagged wild-type p transcript or by treatment with either bafilomycin A1 or ammonium chloride. We found that the endoplasmic reticulum contains a significant amount of p protein, thus supporting a role for p within this compartment. Using immunofluoresence, we showed that most mature full-length tyrosinase in melan-p1 cells was located in the perinuclear area near the Golgi, in contrast to its punctate melanosomal pattern in wild-type melanocytes. Expression of p in melan-p1 cells restored tyrosinase to melanosomes. Triton X-114 phase separation revealed that an increased amount of tyrosinase was proteolyzed in melan-p1 cells compared with wild-type melanocytes. The proteolyzed tyrosinase was no longer membrane bound, but remained enzymatically active and a large proportion was secreted into the culture medium of melan-p1 cells. We conclude that p regulates posttranslational processing of tyrosinase, and hypopigmentation in melan-p1 cells is the result of altered tyrosinase processing and trafficking

BACKGROUND: The Ink4a-Arf tumor suppressor locus encodes two growth inhibitors, p16 and Arf, both of which are also implicated as effectors in cellular senescence. Because human germline defects in the INK4A-ARF locus are associated with familial melanoma, melanocytes may have unusual INK4A-ARF functions or controls of cell senescence. Because senescence is believed to be an anticancer mechanism, we investigated the role of Ink4a-Arf and its individual components in melanocyte senescence. METHODS: Melanocytes were cultured from littermate mice with zero, one, or two functional copies of the Ink4a-Arf locus. Senescence was evaluated by cumulative population doubling curves and by the assessment of acidic beta-galactosidase (an indicator of senescence) expression. Pigmentation and cell size were evaluated by spectrophotometry and microscopy. p16 and Arf expression in primary and spontaneously immortalized melanocyte or melanocyte precursor cell lines were evaluated by immunoblotting. Retroviral vectors containing normal p16 and Arf complementary DNAs were used to restore expression of these genes in Ink4a-Arf(-/-) melanocytes. RESULTS: Wild-type melanocytes (i.e., Ink4a-Arf(+/+)) senesced within 4-5 weeks of culture. Ink4a-Arf(-/-) melanocytes did not senesce and readily became immortal. Ink4a-Arf(+/-) melanocytes showed defective senescence. Senescent Ink4a-Arf(+/+) melanocytes were heavily pigmented, but Ink4a-Arf(+/-) and Ink4a-Arf(-/-) melanocytes were less pigmented. All of six spontaneously immortalized melanocyte or melanocyte precursor lines from Ink4a-Arf(+/+) mice lacked p16 protein expression, although most retained Arf protein expression. After restoration of p16 but not Arf expression, Ink4a-Arf(-/-) melanocytes stopped growing, became highly melanized, and expressed acidic beta-galactosidase. By contrast, restoration of Arf but not p16 expression led to cell death without evidence of senescence. CONCLUSION: Normal mouse melanocyte senescence and associated pigmentation require both copies of Ink4a-Arf and appear to depend more on p16 than on Arf function. Mutations of the INK4A-ARF locus may favor tumorigenesis from melanocytes by impairing senescence, cell differentiation, and (where ARF is disrupted) cell death

The ocular albinism type 1 (OA1) gene product is a membrane glycoprotein that may play a role in controlling melanosome growth and maturation. A number of mutations in the OA1 gene lead to ocular albinism due at least in part to retention of the aberrant protein in the endoplasmic reticulum. To examine whether N-glycosylation plays a role in the post-translational trafficking of the Oa1 protein, we constructed a series of mutant mouse Oa1 cDNAs encoding an Oa1-green fluorescent protein fusion in which some or all of the potential glycosylation sites were eliminated by site-directed mutagenesis. Biochemical studies in transfected cells treated with tunicamycin and peptide:N-glycosidase F suggest that asparagine at amino acid 106 is essential for N-glycosylation of the protein. Mutation at amino acid 106 that eliminated glycosylation did not affect the endo/lysosomal distribution of the Oa1 protein in either COS cells or cultured murine melanocytes

The pink-eyed dilution protein (p) plays a pivotal role in the synthesis of eumelanin. In its absence, critical melanosomal proteins fail to traffic to the melanosome. Pink-eyed dilution gene (P) mutations are the most common cause of tyrosinase-positive oculocutaneous albinism worldwide. Thus, reports that bafilomycin A1 was able to induce synthesis of melanin in tyrosinase-positive melanomas led us to test the drug on p-null murine melanocytes. We found that in melanocytes lacking p, bafilomycin A1 was able to induce melanin synthesis. These cells, once transfected with an expression vector encoding an epitope-tagged p transcript, failed to respond to the drug. The increase in melanin synthesis is accompanied by a reduction in tyrosinase protein cleavage and secretion with subsequent accumulation within the melanocyte. Bafilomycin A1 has also been reported to induce pigmentation of normal Caucasian melanocytes. Based on these data we hypothesize that p may serve as a key control point at which ethnic skin color variation is determined

Ocular albinism type 1 (OA1) is an X-linked recessive disorder characterized by a severe reduction of visual acuity, and hypopigmentation of the retina that leads to nystagmus, strabismus, and photophobia/photodysphoria. Microscopic examination of both retinal pigment epithelium and skin melanocytes in OA1 reveals the presence of macrome-lanosomes, suggesting that the OA1 gene product plays a role in melanosome biogenesis. Studies of mutations identified from OA1 patients and an Oa1 knock-out mouse model further implicate OA1 protein function in the late stage of melanosome development. Because its effects are primarily limited to the eye, OA1 represents an ideal model system to study the relationship between pigmentation and visual development. Based upon sequence homology and biochemical studies, OA1 may represent a novel intracellular G-protein coupled receptor. Understanding the function of OA1 will contribute greatly to our understanding of melanosome biogenesis and the role of pigmentation in visual development