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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.

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A ventrally localized melanization-inhibiting factor (MIF) may play an important role in the expression of dorsal-ventral pigment patterns of amphibians. In efforts to purify this putative MIF, ventral skin conditioned medium (VCM) from Rana forreri was partially fractionated and used to immunize mice. A monoclonal antibody that has the ability to block the activity of MIF was isolated, and an immunoaffinity matrix was prepared by cross-linking the antibody to protein G-Sepharose. The fraction of VCM that bound to the affinity matrix decreased the number of melanized cells in the Xenopus laevis neural tube explant assay, but did not reduce significantly the number of cells that emigrated. The monoclonal antibody was used for immunohistochemical studies on R. pipiens skin. Strong staining with the antibody was observed beneath the basement membrane, in mucous glands, and in the subcutaneous tissue of the ventral skin. A weak staining was also observed in the ground substances of both ventral and dorsal skin. These results confirm that a monoclonal antibody has been secured against at least one of the MIF constituents and that it is useful as a probe in detecting the distribution of MIF in tissues. The results of its use in this study support the hypothesis that MIF plays a role in the expression, development, and maintenance of the dorsal-ventral pigmentation patterns of frogs.