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Titolo:
An arrested late endosome-lysosome intermediate aggregate observed in a Chinese hamster ovary cell mutant isolated by novel three-step screening
Autore:
Ohashi, M; Miwako, I; Nakamura, K; Yamamoto, A; Murata, M; Ohnishi, S; Nagayama, K;
Indirizzi:
Natl Inst Physiol Sci, Dept Mol Physiol, Okazaki, Aichi 4448585, Japan Natl Inst Physiol Sci Okazaki Aichi Japan 4448585 i, Aichi 4448585, Japan Kyoto Univ, Fac Sci, Dept Biophys, Kyoto 6068501, Japan Kyoto Univ KyotoJapan 6068501 c Sci, Dept Biophys, Kyoto 6068501, Japan Kansai Med Univ, Dept Physiol, Osaka 5708506, Japan Kansai Med Univ Osaka Japan 5708506 , Dept Physiol, Osaka 5708506, Japan
Titolo Testata:
JOURNAL OF CELL SCIENCE
fascicolo: 8, volume: 112, anno: 1999,
pagine: 1125 - 1138
SICI:
0021-9533(199904)112:8<1125:AALEIA>2.0.ZU;2-W
Fonte:
ISI
Lingua:
ENG
Soggetto:
LOW-DENSITY-LIPOPROTEIN; RECEPTOR-MEDIATED ENDOCYTOSIS; MANNOSE 6-PHOSPHATE RECEPTOR; MULTIVESICULAR ENDOSOMES; MEMBRANE-GLYCOPROTEINS; HUMAN-FIBROBLASTS; COMPARTMENT; FUSION; PROTEIN; GTPASE;
Keywords:
lysosome; endosome; mutant; microtubule; rab7;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
53
Recensione:
Indirizzi per estratti:
Indirizzo: Ohashi, M Natl Inst Physiol Sci, Dept Mol Physiol, Okazaki, Aichi 4448585,Japan Natl Inst Physiol Sci Okazaki Aichi Japan 4448585 448585, Japan
Citazione:
M. Ohashi et al., "An arrested late endosome-lysosome intermediate aggregate observed in a Chinese hamster ovary cell mutant isolated by novel three-step screening", J CELL SCI, 112(8), 1999, pp. 1125-1138

Abstract

Chinese hamster ovary cell mutants defective in the post-uptake degradation of low-density lipoprotein (LDL) in lysosomes were selected from mutagenized cells by novel three-step screening. First, in the presence of LDL, clones sensitive to an inhibitor of the rate-limiting enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3methylglutaryl-CoA reductase, were isolated. Second, from the selected clones, those lacking in the degradation of a constituent of a fluorescent LDL were qualitatively screened by microscopy, Third, the clones were further screened by previously established quantitative analytical flow cytometry that detects the early-phase disintegration of LDL by lysosomal acid hydrolases, One of the isolated mutant clones, LEX1 (Lysosome-Endosome X 1), was a recessive mutant, and exhibited a specific disorder in the late endocytic pathway, LEX1 cells showed an unusual perinuclear aggregate of vesicles, heterogeneously positive for lysosomal glycoprotein-B/cathepsin D and rab7, yet negative for the cation-independent mannose 6-phosphate receptor. The aggregate was formed around the microtubuleorganizing center, and was disrupted by nocodazole treatment. Internalizedoctadecyl rhodamine B-labeled LDL (R18-LDL) was accumulated in the perinuclear rab7-positive vesicles. In a Percoll density gradient, neither internalized R18-LDL nor internalized horseradish peroxidase was efficiently chased into heavy lysosomal fractions positive for P-hexosaminidase, LEX1 cells showed differences in the activity and subcellular distribution of lysosomal enzymes, These characteristics of LEX1 cells are consistent with the ideas that the perinuclear vesicle aggregate is an arrested intermediate of direct fusion or divergence between lysosomes and rab7-positive, cation-independent mannose 6-phosphate receptor-negative late endosomes, and that equilibrium between the lysosomes and the late endosomes is shifted towards the late endosomes in LEX1 cells, Such fusion or divergence between the late endosomes and the lysosomes would determine an appropriate equilibrium betweenthem, and might thereby play an important role for proper lysosomal digestive functions. LEX1 mutant cells would be helpful for the dissection of theas yet unrevealed details of the late endocytic membrane dynamics and for the identification of factors involved in the process arrested by the mutation.

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Documento generato il 07/08/20 alle ore 07:04:29