Cellular localization and trafficking of the human ABCA1 transporter.

ABCA1, the ATP-binding cassette protein mutated in Tangier disease, mediates the efflux of excess cellular sterol to apoA-I and thereby the formation of high density lipoprotein. The intracellular localization and trafficking of ABCA1 was examined in stably and transiently transfected HeLa cells expressing a functional human ABCA1-green fluorescent protein (GFP) fusion protein. The fluorescent chimeric ABCA1 transporter was found to reside on the cell surface and on intracellular vesicles that include a novel subset of early endosomes, as well as late endosomes and lysosomes. Studies of the localization and trafficking of ABCA1-GFP in the presence of brefeldin A or monensin, agents known to block intracellular vesicular trafficking, as well as apoA-I-mediated cellular lipid efflux, showed that: (i) ABCA1 functions in lipid efflux at the cell surface, and (ii) delivery of ABCA1 to lysosomes for degradation may serve as a mechanism to modulate its surface expression. Time-lapse fluorescence microscopy revealed that ABCA1-GFP-containing early endosomes undergo fusion, fission, and tubulation and transiently interact with one another, late endocytic vesicles, and the cell surface. These studies establish a complex intracellular trafficking pathway for human ABCA1 that may play important roles in modulating ABCA1 transporter activity and cellular cholesterol homeostasis.

The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo.

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that the NPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(-) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 degrees C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.

Substantial narrowing of the Niemann-Pick C candidate interval by yeast artificial chromosome complementation.

Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.

Intracellular trafficking of cholesterol monitored with a cyclodextrin.

The sterol binding agent 2-hydroxypropyl-beta-cyclodextrin is shown to be a convenient and useful experimental tool to probe intracellular pathways of cholesterol transport. Biochemical and cytochemical studies reveal that cyclodextrin specifically removes plasma membrane cholesterol. Depletion of plasma membrane sphingomyelin greatly accelerated cyclodextrin-mediated cholesterol removal. Cholesterol arriving at the plasma membrane from lysosomes and the endoplasmic reticulum was also removed by cyclodextrin. Cellular cholesterol esterification linked to the mobilization of cholesterol from lysosomes was strongly attenuated by cyclodextrin, suggesting that the major portion of endocytosed cholesterol is delivered from lysosomes to the endoplasmic reticulum via the plasma membrane. Evidence for translocation of lysosomal cholesterol to the endoplasmic reticulum by a plasma membrane-independent pathway is provided by the finding that cyclodextrin loses its ability to suppress esterification when plasma membrane sphingomyelin is depleted. The Golgi apparatus appears to play an active role in directing the relocation of lysosomal cholesterol to the plasma membrane since brefeldin A also abrogated cyclodextrin-mediated suppression of cholesterol esterification. Using cyclodextrin we further show that attenuated esterification of lysosomal cholesterol in Niemann-Pick C cells reflects defective translocation of cholesterol to the plasma membrane that may be linked to abnormal Golgi trafficking.

Colony inhibition mediated by nonimmune leukocytes in vitro and skin reactivity in vivo as indices of tumorigenicity of guinea pig cultures transformed by chemical carcinogens.

Two short-term quantitative assays that correlate with the tumorigenicity of strain 2 guinea pig fetal cells transformed in culture by chemical carcinogens are described; one measures inhibition of colony growth mediated by nonimmune leukocytes and the other measures skin reactivity in unimmunized syngeneic guinea pigs. Mineral oil-induced peritoneal exudate (PE) cells were obtained from healthy unimmunized syngeneic guinea pigs. The PE cells were cultured for 24 hr and the nonadherent cells with culture medium were incubated with tumorigenic or nontumorigenic target cells in ratios of 1000/1 to 10/1. After 7 to 9 days of incubation in the presence of peritoneal exudate cell culture (PEC), fewer target cell colonies were observed in cultures with tumorigenic than with nontumorigenic cells. The inhibition of tumorigenic cells was dependent on PEC concentration; at 1000/1 PEC/traget cell ratio, a reduction of as much as 80% in the number of colonies and a 100% decrease in colony size relative to controls were noted. Inhibitory activity was present primarily in the supernatant fluid of the culture medium of the PE cells. Phytohemagglutinin stimulation of the PE cells increased PEC and peritoneal exudate cell culture medium supernatant (PES) colony-inhibitory activity as much as twofold. The differential colony inhibition activity of PES with or without phytohemagglutinin was stable during storage of PES for 5 months at -35 degrees. In the 2nd assay, 2 or 5 X 106 tumorigenic or nontumorigenic cells were inoculated intradermally into 12- to 16-seek-old male unimmunized syngeneic guinea pigs; skin reactivity, in terms of the degree and persistence of induration during the next 4 days, was greater to tumorigenic than to nontumorigenic cells. In both assays, tumor-producing cells, morphologically transformed in culture, or tumor-derived cells, were affected more than early-passage-untreated fetal cells, morphologically nontransformed long-term-cultured cells previously exposed to noncarcinogenic chemicals, or chemical carcinogen-transformed but non-tumor-producing cells. The two assays, particularly the nonimmune leukocyte-mediated colony inhibition with its greater degree of discrimination, provide rapid estimations of the tumorigenic potential of cells transformed in in vitro model systems of chemical carcinogenesis.

Tumor-specific and forssman antigens of guinea-pig hepatoma cells: comparison of tumor cells grown in vivo and in vitro.

Cloned tumor-cell lines were derived from two antigenically distinct ascites variants of diethylnitrosamine-induced guinea-pig hepatomas (designated line 1 and line 10). Cell-surface antigens on the ascites and in vitro-grown tumor cells were analyzed with immunofluorescence, C1 fixation and transfer, and antibody-complement-mediated cytotoxity tests. Tumor-specific and Forssman antigens continued to be expressed during 3-6 months in vitro cultivation. Differences between ascites and cultured cells were noted in the degree of antigen expression and sensitivity to antibody-complement-mediated cytotoxicity. Cells grown in vitro exhibited a greater number of Forssman and tumor-specific antigen sites than in vivo grown cells as determined by the quantitative C1 fixation and transfer test. Immunofluorescent staining indicated that some cloned lines were considerably more homogeneous in terms of antigen expression than were the cultured, non-cloned parent cells or ascites-grown cells. Cloned lines were frequently more sensitive to the cytotoxic action of antibody and complement than were the in vivo grown and cultured non-cloned parent tumor cells. Sensitivity to cytotoxicity did not necessarily correlate, however, with the degree of antigen expression. These results suggest that; (1) the expression of Forssman and tumor-specific antigens does not diminish on cells cultivated in vitro and (2) ascites hepatoma cells in vivo are a heterogeneous population of cells differing in their degree of antigen expression and sensitivity to antibody-complement-mediated cytotoxicity.