Novel method for isolating mammalian cells defective in fluid-phase endocytosis

Wang, Ru Hung; Colbaugh, Penelope A.; Kuo, Peter T.; Bau, Mu Yeh; Poppe, Lisa M.; Draper, Rockford K.

doi:10.1007/bf01232650

Cited by 4 publications

(2 citation statements)

References 25 publications

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“…Trf1 cells were not resistant but were significantly more sensitive to such toxins [19]. This contrasts with the CHO cell endocytosis (acidification) mutants, which are all resistant to Diphtheria toxin [37]. The acid nature of the endosomal compartments in Trf2-Trf7 is indicated by the sensitivity to Diphtheria toxin, which requires the low pH of the endosome to trigger a conformational change in the toxin, permitting exposure and interaction of previously sheltered tryptophan residues with the endosomal membrane [38].…”

Section: Discussionmentioning

confidence: 89%

New liver cell mutants defective in the endocytic pathway

Stockert

Potvin

Nath

et al. 2007

Biochimica et Biophysica Acta (BBA) - Biomembranes

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To isolate mutant liver cells defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was utilized. Rare survivors termed trafficking mutants (Trf2-Trf7) were stable and more resistant than the parental HuH-7 cells to both toxin conjugates. They differed from the previously isolated Trf1 HuH-7 mutant as they expressed casein kinase 2 alpha'' (CK2alpha'') which is missing from Trf1 cells and which corrects the Trf1 trafficking phenotype. Binding of (125)I-asialoorosomucoid (ASOR) and cell surface expression of asialoglycoprotein receptor (ASGPR) were reduced approximately 20%-60% in Trf2-Trf7 cells compared to parental HuH-7, without a reduction in total cellular ASGPR. Based on (125)I-transferrin binding, cell surface transferrin receptor activity was reduced between 13% and 88% in the various mutant cell lines. Distinctive phenotypic traits were identified in the differential resistance of Trf2-Trf7 to a panel of lectins and toxins and to UV light-induced cell death. By following the endocytic uptake and trafficking of Alexa(488)-ASOR, significant differences in endosomal fusion between parental HuH-7 and the Trf mutants became apparent. Unlike parental HuH-7 cells in which the fusion of endosomes into larger vesicles was evident as early as 20 min, ASOR endocytosed into the Trf mutants remained within small vesicles for up to 60 min. Identifying the biochemical and genetic mechanisms underlying these phenotypes should uncover novel and unpredicted protein-protein or protein-lipid interactions that orchestrate specific steps in membrane protein trafficking.

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Section: Discussionmentioning

confidence: 89%

New liver cell mutants defective in the endocytic pathway

Stockert

Potvin

Nath

et al. 2007

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…The other half were kept at 34'C. Protein synthesis and accumulation of HRp were measured as described previously (Wang et al, 1992). The kinetics of HA transport were determined following influenza infection as described previously (Lazarovits et al, 1990).…”

Section: Isolation Of Cho Cells With Defects In Secretionmentioning

confidence: 99%

A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

Ktistakis

Kao

Wang

et al. 1995

MBoC

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The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescenceactivated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10-6. Thus, the fluorescent lipid C5-DMBceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.

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Ricin intoxicates End4 mutants that have an aberrant Golgi complex.

Bau

Draper

1993

Journal of Biological Chemistry

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Novel method for isolating mammalian cells defective in fluid-phase endocytosis

Cited by 4 publications

References 25 publications

New liver cell mutants defective in the endocytic pathway

New liver cell mutants defective in the endocytic pathway

A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

Ricin intoxicates End4 mutants that have an aberrant Golgi complex.

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