Electron Microscopic Evidence for Stimulation of Melanosomal Maturation by Lysosomotropic Agents and Monensin in Cultured B16 Mouse Melanoma Cells

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Abstract
Mouse melanoma cells, B16‐C2M in monolayer culture were treated with either lysosomotropic agent, 10 mM NH4Cl, or 20 μM chloroquine, an ionophore, or 10 μm monensin for 3 h at 37°C, and examined with regard to the site of melanin deposition and numbers of melanized (type 1) and unmelanized (type 2) melanosomes under a transmission electron microscope. The numbers of these two types of melanosomes were counted on electron micrographs of thin sections of 20 to 40 cells for each experimental group and expressed in terms of number per unit area of sectioned cytoplasm. Although most melanosomes were largely swollen in monensin‐treated cells, melanin deposition was apparently confined in melanosomes in all experimental groups. The compound melanosomes were scarcely found. The mean population density (number per unit cytoplasmic area) of type 1 melanosomes was highest in the NH4Cl‐treated cell group followed by monensin‐treated, chloroquine‐treated, and control cell groups. When the relative abundance of type 1 melanosomes was expressed as a fraction of total number of type 1 and 2 melanosomes (melanosomal maturation index, MMI), the differences were much more evident. Type 1 melanosomes were found in every cell (MMI ≠ 0) of the groups treated with NH4Cl and chloroquine only, which suggested the existence of a subpopulation of cells responsive to lysosomotropic agents but not to monensin in regard to melanosome maturation. All these findings indicate that the stimulation of melanogenesis proceeds mainly through maturation of preexisting melanosomes under these conditions.