Abstract
The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.
Highlights
I(Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Marylnnd 20892, the $$Laboratoire de Bwchimie, Institut National de la Sante et de la Recherche Medicale U 189, Faculte de Medecine Lyon-Sud, BP 12, F-69921 Oullins Cedex, Frame, and the **NeurologicalService, Veterans Administration Medical Center, Newington, Connecticut 061 11
Perinucleafrluo- reported for Type C Niemann-Pick disease [6]
Perinuclear filipin-cholesterol flu-cells leads to an excessive intracellular accumulation and orescence of both normal and mutant cells was co- storage of cholesterol primarily as unesterified sterol [8].The localized with lysosomes by indirectimmunocytochem- inability of internalized cholesterol to initiate timely regulaical stainingof lysosomal membrane protein
Summary
Cells were harvested by washing monolayers three times with phosphate-buffered saline (PBS) and subsequent treatment with 0.05% trypsin (Sigma) for 5 min a t 37"C.Specific experimental culture manipulations and conditions are described in the appropriate legends. In Vitro Esterification of CeUular Cholesterol-Freshly harvested and washed cell pellets (5 mgof protein) were suspended in 1 ml of 250 mM sucrose and 10 mM Tris-HC1,pH 7.4. It has previously been shown that such controlled disruption allows cell-free extracts to essentially retain intact subcellular organelles [18].Aliquots (0.010 ml and 50 pg of protein) of the respective total cell-free extracts were incubated in 0.19mlof250 mM sucrose, 2 mM dithiothreitol, 5 mM KF, and 10 mM Tris-HC1, pH 7.4, containing 6 mM ATP, 0.6 mM CoA, 15 mM MgCI, and 0.40 mM [3H]oleate(370dmp/pmol in 14%fatty acid-free bovine serum albumin). The level of [3H]oleateincorporated into cellular cholesterol to form chole~teryl-[~H]oleawteas measured by thin layer chromatography as described previously [8]
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