TPC2 controls pigmentation by regulating melanosome pH and size
TPC2 controls pigmentation by regulating melanosome pH and size
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DOI：
10.1073/pnas.1600108113
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发表时间：
2016-05-17
影响因子：
11.1
通讯作者：
Di Pietro, Santiago M.
中科院分区：
文献类型：
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作者：
Ambrosio, Andrea L.;Boyle, Judith A.;Di Pietro, Santiago M.
黑色素负责皮肤和毛发的色素沉着，并在黑素细胞中的专门细胞器黑素体中合成。一项全基因组关联研究显示，双孔段通道2（TPCN 2）基因与色素沉着变化密切相关。TPCN 2编码双孔通道2（TPC 2）蛋白，一种阳离子通道。然而，TPC 2如何调节色素沉着仍然未知。在这里，我们表明，TPC 2在黑素细胞中表达，并定位于黑素小体限制膜，并在较小程度上，通过共聚焦荧光和免疫金电子显微镜的内溶酶体室。免疫磁性分离的TPC 2含有细胞器证实了其与黑素标记的共存。通过成簇的规则间隔的短回文重复序列/CRISPR相关的9基因编辑敲除TPCN 2引起MNT-1黑素细胞中色素含量的急剧增加。该效应通过瞬时表达TPC 2-GFP来挽救。同样，siRNA介导的TPC 2敲低也导致MNT-1细胞和原代人黑素细胞中黑素含量的显著增加。使用新开发的针对黑素体的遗传编码的pH传感器，我们确定了TPC 2-KO MNT-1细胞和经受TPC 2敲低的原代黑素细胞中的黑素体腔的酸性比对照细胞中的酸性小。荧光和电子显微镜分析显示，TPC 2-KO MNT-1细胞具有比对照细胞显著更大的黑素体，但细胞器的数量不变。TPC 2可能通过介导Ca 2+从细胞器释放来调节黑素体pH和大小，如用Ca 2+传感器酪氨酸酶-GCaMP 6所确定的，这在TPC 2-KO MNT-1细胞中减少。因此，我们的数据表明，TPC 2通过黑素体功能的两个基本决定因素调节色素沉着：pH和大小。
Melanin is responsible for pigmentation of skin and hair and is synthesized in a specialized organelle, the melanosome, in melanocytes. A genome-wide association study revealed that the two pore segment channel 2 (TPCN2) gene is strongly linked to pigmentation variations. TPCN2 encodes the two-pore channel 2 (TPC2) protein, a cation channel. Nevertheless, how TPC2 regulates pigmentation remains unknown. Here, we show that TPC2 is expressed in melanocytes and localizes to the melanosome-limiting membrane and, to a lesser extent, to endolysosomal compartments by confocal fluorescence and immunogold electron microscopy. Immunomagnetic isolation of TPC2-containing organelles confirmed its coresidence with melanosomal markers. TPCN2 knockout by means of clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 gene editing elicited a dramatic increase in pigment content in MNT-1 melanocytic cells. This effect was rescued by transient expression of TPC2-GFP. Consistently, siRNA-mediated knockdown of TPC2 also caused a substantial increase in melanin content in both MNT-1 cells and primary human melanocytes. Using a newly developed genetically encoded pH sensor targeted to melanosomes, we determined that the melanosome lumen in TPC2-KO MNT-1 cells and primary melanocytes subjected to TPC2 knockdown is less acidic than in control cells. Fluorescence and electron microscopy analysis revealed that TPC2-KO MNT-1 cells have significantly larger melanosomes than control cells, but the number of organelles is unchanged. TPC2 likely regulates melanosomes pH and size by mediating Ca2+ release from the organelle, which is decreased in TPC2-KO MNT-1 cells, as determined with the Ca2+ sensor tyrosinase-GCaMP6. Thus, our data show that TPC2 regulates pigmentation through two fundamental determinants of melanosome function: pH and size.