Isolation of a temperature-sensitive variant Chinese hamster ovary cell line with a morphologically altered endocytic recycling compartment

Timothy E. McGraw, Kenneth Dunn, Frederick R. Maxfield

Research output: Contribution to journalArticle

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Abstract

We have enriched a mutagenized population of Chinese hamster ovary (CHO) cells for those defective in endocytosis by selection for survival to treatment with transferrin (Tf)-ricin and Tf-diphtheria toxin conjugates. Surviving cells were screened with a fluorescently labeled Tf uptake assay to identify cells with morphologically aberrant endocytic phenotypes. One of the cell lines identified, B104-5, has a striking temperature-induced alteration in the morphology of its endocytic receptor recycling compartment. In parental cells the tightly clustered endocytic recycling compartment is located near the Golgi complex. In the mutant cells, following incubation at 40°C, this compartment appears fragmented and widely dispersed. Surprisingly, this alteration in the morphology of the recycling compartment has no effect on the kinetics of Tf internationalization and recycling. The wild-type endocytic compartment is closely aligned with the microtubule-organizing center and the Golgi apparatus, and like the Golgi, its clustered appearance is dependent upon intact microtubules. Although the disruption of the B104-5 receptor recycling compartment morphology can be phenocopied in wild-type cells by microtubule depolymerizing drugs, the microtubule cytoskeleton in B104-5 cells appears normal in immunofluorescent staining. B104-5 cells, unlike the parental cells, do not proliferate at 40°C. The mutation in B104-5 cells is recessive, as fusion with wild-type cells results in a reversion of the B104-5 phenotype. The finding that the morphology of the recycling compartment in CHO cells can be altered without affecting recycling of endocytosed Tf is consistent with the variety of recycling compartment morphologies observed among different cell lines. An interpretation of this result is that the lesion in B104-5 cells is in a gene that is involved in determining the endocytic compartment morphologies observed in different cell lines.

Original languageEnglish (US)
Pages (from-to)579-594
Number of pages16
JournalJournal of Cellular Physiology
Volume155
Issue number3
StatePublished - Jun 1993
Externally publishedYes

Fingerprint

Cricetulus
Recycling
Ovary
Cells
Cell Line
Temperature
Transferrin
Microtubules
Golgi Apparatus
Ricin
Endocytosis
Microtubule-Organizing Center
Assays
Phenotype
Fusion reactions
Genes
Cytoskeleton
Kinetics
Pharmaceutical Preparations
Staining and Labeling

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry
  • Physiology

Cite this

Isolation of a temperature-sensitive variant Chinese hamster ovary cell line with a morphologically altered endocytic recycling compartment. / McGraw, Timothy E.; Dunn, Kenneth; Maxfield, Frederick R.

In: Journal of Cellular Physiology, Vol. 155, No. 3, 06.1993, p. 579-594.

Research output: Contribution to journalArticle

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