Department for Experimental Vitreoretinal Surgery, University Eye Hospital Tübingen, Germany.
Cell Tissue Res. 2010 Mar;339(3):551-60. doi: 10.1007/s00441-009-0920-9. Epub 2010 Feb 6.
Premelanosomes are presumed to be essential for melanogenesis in melanocytes and pre-natal retinal pigment epithelium (RPE) cells. We analysed melanin synthesis in adenoviral-transduced tyrosinase-gene-expressing amelanotic RPE (ARPE) 19 cells to determine whether premelanosome formation is needed for post-natal melanogenesis. The synthesis of melanogenic proteins and melanin granules was investigated by immunocytochemistry and light and electron microscopy. The occurrence of tyrosinase was analysed ultrastructurally by dihydroxyphenylalanine histochemistry. The viability of transduced cell cultures was examined via MTT assay. We found active tyrosinase in small granule-like vesicles throughout the cytoplasm and in the endoplasmic reticulum and nuclear membrane. Tyrosinase was also associated with multi-vesicular and multi-lamellar organelles. Typical premelanosomes, structural protein PMEL17, tyrosinase-related protein 1 and classic melanosomal stages I-IV were not detected. Instead, melanogenesis took place inside multi-vesicular and multi-lamellar bodies of unknown origin. Viability was not affected up to 10 days after transduction. We thus demonstrate a pathway of melanin formation lacking typical hallmarks of melanogenesis.
前黑素小体被认为是黑素细胞和产前视网膜色素上皮（RPE）细胞中黑素生成所必需的。我们分析了转导的酪氨酸酶基因表达的无色素 RPE（ARPE）19 细胞中的黑色素合成，以确定前黑素小体的形成是否是产后黑素生成所必需的。通过免疫细胞化学和光镜及电子显微镜研究了黑素生成蛋白和黑素颗粒的合成。通过二羟苯丙氨酸组织化学法超微结构分析了酪氨酸酶的发生。通过 MTT 测定法检查了转导细胞培养物的活力。我们发现小颗粒状囊泡整个细胞质中都有活性的酪氨酸酶，在内质网和核膜中也有。酪氨酸酶也与多泡体和多层体细胞器有关。未检测到典型的前黑素小体、结构蛋白 PMEL17、酪氨酸酶相关蛋白 1 和经典黑素小体阶段 I-IV。相反，黑色素的形成发生在未知来源的多泡体和多层体内部。转导后 10 天内活力不受影响。因此，我们证明了一种缺乏典型黑素生成特征的黑色素形成途径。
