B16-F1黑色素瘤细胞经常被用作研究黑素生成的模型,但在正常黑素细胞和B16-F1细胞中,黑素体生物发生和运输通过相同机制发生的证据是不够的。在这项研究中,我们建立了敲除B16-F1细胞的黑素生成中的几个关键因素,即,酪氨酸酶(Tyr),Hps4,Rab27A,和Rab32·Rab38(Rab32/38),然后将它们的表型与相应的突变小鼠黑素细胞系的表型进行比较,即,melan-c,melan-le,melan-ash,和缺乏Rab32的melan-cht细胞,分别。结果表明,Tyr和Rab27A也是黑色素合成和外周黑素小体分布不可或缺的,分别,在B16-F1细胞中,但是Hps4或其下游目标Rab32/38对于B16-F1细胞中的Tyr运输不是必需的,表明B16-F1细胞中存在Rab32/38独立的Tyr转运机制。然后,我们对Rab小GTP酶进行了全面的敲低筛选,并鉴定了Rab10和Rab24,这是黑素细胞中以前未表征的Rabs,在Rab32/38-null条件下参与Tyr运输。我们的发现表明黑素细胞和B16-F1细胞中Tyr转运机制在Rab32/38依赖性方面存在差异,并且在使用黑色素瘤细胞作为黑素细胞模型方面存在局限性。特别是在研究内体Tyr转运机制时。
B16-F1 melanoma cells have often been used as a model to investigate melanogenesis, but the evidence that melanosome biogenesis and transport occur by the same mechanisms in normal melanocytes and B16-F1 cells is insufficient. In this study, we established knockout B16-F1 cells for each of several key factors in melanogenesis, i.e., tyrosinase (Tyr), Hps4, Rab27A, and Rab32·Rab38 (Rab32/38), and then compared their phenotypes with the phenotypes of corresponding mutant mouse melanocyte cell lines, i.e., melan-c, melan-le, melan-ash, and Rab32-deficient melan-cht cells, respectively. The results showed that Tyr and Rab27A are also indispensable for melanin synthesis and peripheral melanosome distribution, respectively, in B16-F1 cells, but that Hps4 or its downstream targets Rab32/38 are not essential for Tyr transport in B16-F1 cells, suggesting the existence of a Rab32/38-independent Tyr transport mechanism in B16-F1 cells. We then performed comprehensive knockdown screening of Rab small GTPases and identified Rab10 and Rab24, previously uncharacterized Rabs in melanocytes, as being involved in Tyr transport under Rab32/38-null conditions. Our findings indicate a difference between the Tyr transport mechanism in melanocytes and B16-F1 cells in terms of Rab32/38-dependency and a limitation in regard to using melanoma cells as a model for melanocytes, especially when investigating the mechanism of endosomal Tyr transport.