Niemann-Pick disease type C2 is a lipid storage disorder in which mutations in the NPC2 protein cause accumulation of lipoprotein-derived cholesterol in late endosomes and lysosomes (LE/LYSs). Whether cholesterol delivered by other means to NPC2 deficient cells also accumulates in LE/LYSs is currently unknown. We show that the close cholesterol analog dehydroergosterol (DHE), when delivered to the plasma membrane (PM) accumulates in LE/LYSs of human fibroblasts lacking functional NPC2. We measured two different time scales of sterol diffusion; while DHE rich LE/LYSs moved by slow anomalous diffusion in disease cells (D ∼ 4.6∙10⁻⁴ μm²/sec; α∼0.76), a small pool of sterol could exchange rapidly with D ∼ 3 μm²/s between LE/LYSs, as shown by fluorescence recovery after photobleaching (FRAP). By quantitative lipid mass spectrometry we found that esterification of ¹³C-labeled cholesterol but not of DHE is reduced 10-fold in disease fibroblasts compared to control cells. Internalized NPC2 rescued the sterol storage phenotype and strongly expanded the dynamic sterol pool seen in FRAP experiments. Together, our study shows that cholesterol esterification and trafficking of sterols between the PM and LE/LYSs depends on a functional NPC2 protein. NPC2 likely acts inside LE/LYSs from where it increases non-vesicular sterol exchange with other organelles.

尼曼-匹克病C2型是一种脂质存储疾病，其中NPC2蛋白的突变导致晚期内体和溶酶体（LE / LYSs）中脂蛋白衍生的胆固醇蓄积。目前尚不清楚通过其他方式传递给NPC2缺陷细胞的胆固醇是否还会积聚在LE / LYSs中。我们显示，接近的胆固醇类似物脱氢麦角固醇（DHE），当传递到质膜（PM）时，会在缺乏功能性NPC2的人成纤维细胞的LE / LYS中积聚。我们测量了两种不同的固醇扩散时间尺度。而DHE丰富LE / Lyss的移动通过在疾病的细胞缓慢反常扩散（d〜4.6∙10 ^-4 微米² /秒;α~0.76），甾醇的一小池可与d迅速交换〜3μm的²LE / LYS之间的/ s，如光漂白（FRAP）后的荧光恢复所示。通过定量脂质质谱分析，我们发现，与对照组细胞相比，疾病成纤维细胞中¹³ C标记的胆固醇的酯化作用降低了10倍，而DHE的作用则没有降低。内化的NPC2拯救了固醇表型，并大大扩展了在FRAP实验中看到的动态固醇库。总之，我们的研究表明，胆固醇酯化和PM与LE / LYS之间的固醇转运取决于功能性NPC2蛋白。NPC2可能在LE / LYSs内部起作用，从那里它会增加与其他细胞器的非囊状固醇交换。