Coxey R A, Pentchev P G, Campbell G, Blanchette-Mackie E J
Endocrinology Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
J Lipid Res. 1993 Jul;34(7):1165-76.
Cholesterol accumulation in the Golgi of normal and Niemann-Pick Type C (NP-C) fibroblasts was shown by freeze-fracture electron microscopy using filipin as a probe for unesterified cholesterol. The specific distribution of cholesterol within individual Golgi compartments could be examined because membrane cholesterol forms complexes with filipin that are visible as membrane deformations (pits and protuberances) in freeze-fracture replicas. The density of filipin-cholesterol deformations, quantitated for cis, medial, and trans Golgi cisternae and trans Golgi vacuoles, was shown to increase in a cis to trans direction. After addition of low density lipoproteins (LDL) to cultured fibroblasts for 24 h, the cholesterol content increased within specific compartments of the Golgi. Normal cells showed an increase in filipin-cholesterol deformations in membranes of cis/medial cisternae and trans Golgi vacuoles, whereas NP-C cells showed only an increase in membranes of trans Golgi cisternae. LDL uptake by cells appears to induce a disparate cholesterol enrichment of Golgi compartments of normal and mutant cells. The ability of cells to process endocytosed cholesterol may in part depend on modulation of cholesterol-enriched membrane transport through the Golgi, a function which appears to be defective in NP-C cells.
利用制霉菌素作为游离胆固醇的探针，通过冷冻蚀刻电子显微镜观察到正常成纤维细胞和尼曼-匹克C型（NP-C）成纤维细胞的高尔基体中存在胆固醇积累。由于膜胆固醇与制霉菌素形成复合物，在冷冻蚀刻复制品中表现为膜变形（凹陷和突起），因此可以检测单个高尔基体区室中胆固醇的特定分布。对顺式、中间和反式高尔基体潴泡以及反式高尔基体液泡的制霉菌素-胆固醇变形密度进行定量分析，结果显示其密度从顺式到反式方向增加。向培养的成纤维细胞中添加低密度脂蛋白（LDL）24小时后，高尔基体特定区室中的胆固醇含量增加。正常细胞顺式/中间潴泡膜和反式高尔基体液泡膜中的制霉菌素-胆固醇变形增加，而NP-C细胞仅反式高尔基体潴泡膜中的变形增加。细胞摄取LDL似乎会导致正常细胞和突变细胞的高尔基体区室出现不同的胆固醇富集。细胞处理内吞胆固醇的能力可能部分取决于对通过高尔基体的富含胆固醇的膜转运的调节，而这一功能在NP-C细胞中似乎存在缺陷。
