Wüstner Daniel, Dupont Juhl Alice, Egebjerg Jacob Marcus, Werner Stephan, McNally James, Schneider Gerd
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Department of X-Ray Microscopy, Helmholtz-Zentrum Berlin, Berlin, Germany.
Front Cell Dev Biol. 2023 Oct 31;11:1144936. doi: 10.3389/fcell.2023.1144936. eCollection 2023.
Niemann Pick type C1 and C2 (NPC1 and NPC2) are two sterol-binding proteins which, together, orchestrate cholesterol transport through late endosomes and lysosomes (LE/LYSs). NPC2 can facilitate sterol exchange between model membranes severalfold, but how this is connected to its function in cells is poorly understood. Using fluorescent analogs of cholesterol and quantitative fluorescence microscopy, we have recently measured the transport kinetics of sterol between plasma membrane (PM), recycling endosomes (REs) and LE/LYSs in control and NPC2 deficient fibroblasts. Here, we use kinetic modeling of this data to determine rate constants for sterol transport between intracellular compartments. Our model predicts that sterol is trapped in intraluminal vesicles (ILVs) of LE/LYSs in the absence of NPC2, causing delayed sterol export from LE/LYSs in NPC2 deficient fibroblasts. Using soft X-ray tomography, we confirm, that LE/LYSs of NPC2 deficient cells but not of control cells contain enlarged, carbon-rich intraluminal vesicular structures, supporting our model prediction of lipid accumulation in ILVs. By including sterol export via exocytosis of ILVs as exosomes and by release of vesicles-ectosomes-from the PM, we can reconcile measured sterol efflux kinetics and show that both pathways can be reciprocally regulated by the intraluminal sterol transfer activity of NPC2 inside LE/LYSs. Our results thereby connect the function of NPC2 as sterol transfer protein between membranes with its function.
尼曼-皮克C1型和C2型（NPC1和NPC2）是两种固醇结合蛋白，它们共同协调胆固醇通过晚期内体和溶酶体（LE/LYSs）的转运。NPC2可以使模型膜之间的固醇交换加快几倍，但人们对其在细胞中的功能如何与之相关却知之甚少。我们最近使用胆固醇的荧光类似物和定量荧光显微镜，测量了对照和成纤维细胞中NPC2缺陷型细胞的质膜（PM）、循环内体（REs）和LE/LYSs之间固醇的转运动力学。在这里，我们使用这些数据的动力学模型来确定细胞内区室之间固醇转运的速率常数。我们的模型预测，在没有NPC2的情况下，固醇被困在LE/LYSs的腔内小泡（ILVs）中（导致NPC2缺陷型成纤维细胞中固醇从LE/LYSs的输出延迟）。使用软X射线断层扫描，我们证实NPC2缺陷型细胞而非对照细胞的LE/LYSs含有扩大的、富含碳的腔内小泡结构，支持了我们关于ILVs中脂质积累的模型预测。通过将ILVs作为外泌体的胞吐作用以及从PM释放囊泡-外小体纳入固醇输出过程，我们可以协调测量的固醇流出动力学，并表明这两种途径都可以由LE/LYSs内部NPC2的腔内固醇转移活性进行相互调节。因此，我们的结果将NPC2作为膜间固醇转移蛋白的功能与其功能联系起来。
