ISOLATION OF A TEMPERATURE-SENSITIVE VARIANT CHINESE-HAMSTER OVARY CELL-LINE WITH A MORPHOLOGICALLY ALTERED ENDOCYTIC RECYCLING COMPARTMENT

We have enriched a mutagenized population of Chinese hamster ovary (CH O) cells for those defective in endocytosis by selection for survival to treatment with transferrin (Tf)-ricin and Tf-diphtheria toxin conju gates. Surviving cells were screened with a fluorescently labeled Tf u ptake assay to identify cells with morphologically aberrant endocytic phenotypes. One of the cell lines identified, B104-5, has a striking t emperature-induced alteration in the morphology of its endocytic recep tor recycling compartment. In parental cells the tightly clustered end ocytic recycling compartment is located near the Golgi complex. In the mutant cells, following incubation at 40-degrees-C, this compartment appears fragmented and widely dispersed. Surprisingly, this alteration in the morphology of the recycling compartment has no effect on the k inetics of Tf internationalization and recycling. The wild-type endocy tic compartment is closely aligned with the microtubule-organizing cen ter and the Golgi apparatus, and like the Golgi, its clustered appeara nce is dependent upon intact microtubules. Although the disruption of the B104-5 receptor recycling compartment morphology can be phenocopie d in wild-type cells by microtubule depolymerizing drugs, the microtub ule cytoskeleton in B104-5 cells appears normal in immunofluorescent s taining. B104-5 cells, unlike the parental cells, do not proliferate a t 40-degrees-C. The mutation in B104-5 cells is recessive, as fusion w ith wild-type cells results in a reversion of the B104-5 phenotype. Th e finding that the morphology of the recycling compartment in CHO cell s can be altered without affecting recycling of endocytosed Tf is cons istent 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 determinin g the endocytic compartment morphologies observed in different cell li nes.