Nuclear-cytoplasmic relationships in human cells in tissue culture

Goldstein, Lester; Micou, Julie; Crocker, T. Timothy

doi:10.1016/0006-3002(60)91428-1

Cited by 51 publications

(2 citation statements)

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“…Enucleated cells have without exception failed to survive for an extended period, though oocytes have been reported to segment irregularly for a while in the absence of nuclei (21). Goldstein (22,23) has made observations on enucleated fragments of HeLa cells and has described behavioral characteristics similar to those observed by the cytoplasts studied here. The degree to which his enucleates contained the central apparatus was not recorded but many of the nucleated cell parts in his experiments seemed to survive.…”

Section: Transmission Microscopy Of Whole Cho-cellsmentioning

confidence: 87%

The Surface Morphology and Fine Structure of CHO (Chinese Hamster Ovary) Cells Following Enucleation

Shay

Porter

Prescott

1974

Proc. Natl. Acad. Sci. U.S.A.

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Chinese hamster ovary cells grown in monolayer culture and exposed to cytochalasin B were enucleated by centrifugation. Thereafter, the karyoplasts (the nucleated parts obtained from the bottoms of the centrifuge tubes) and the cytoplasts (the enucleated cytoplasmic parts attached to the coverslips) were allowed to recover and subsequently were examined by scanning and transmission electron microscopy. Cytochalasin B causes rapid and dramatic changes in the morphology of vertebrate cells growing in monolayer culture (1). In some instances the nucleus is segregated into an outpocketing on the free surface of the cell and may remain attached to the main body of the cell only by a thin stalk of cytoplasm. Occasionally the stalk breaks resulting in a separation of the cell into nuclear and cytoplasmic components (1-3). This process can be made to occur in virtually all cells in a culture by using centrifugal force during treatment with cytochalasin B so that the nucleus is pulled away from the cell (4, 5). Thus, it becomes possible to separate more than 95% of the cells in a culture into nuclear and cytoplasmic parts (6, 7). Both parts recover quickly from the effects of cytochalasin B. The nucleated part, called the karyoplast (8), consists of a nucleus enclosed in a thin shell of cytoplasm with an intact plasma membrane (9). The cytoplasmic part, called the cytoplast (8), contains the bulk of the cytoplasm and has representatives of all cytoplasmic organelles and systems including centrioles and microtubules. The karyoplasts continue to synthesize RNA and protein for at least several hours and remain intact for 48-72 hr. Cytoplasts synthesize protein for at least 12 hr and in this period can support metabolic activities of viruses (4, 6, 7). The cytoplasts survive for up to 48 hr.In this paper we describe observations by scanning and transmission electron microscopy on the structure and behavior of karyoplasts and cytoplasts derived from a Chinese hamster ovary (CHO) cell line. These observations extend those in earlier reports (8,9) and establish a basic background in the experimental use of karyoplasts and cytoplasts for the analysis of nuclear-cytoplasmic interactions. MATERIALS AND METHODSChinese hamster ovary cells were grown to semi-confluency on no. 1 thickness glass (round 18 mm) or plastic (25 mm) coverslips in Ham's F-12 medium containing 10% fetal calf serum. The plastic coverslips were punched out of the bottoms of Falcon plastic tissue culture dishes (Falcon Plastics, Oxnard, Calif.) with a heated piece of stainless steel tubing. The coverslips were then inserted cell-side down into centrifuge tubes containing 10 sg/ml of cytochalasin B (Imperial Chemical Industries, Ltd., Great Britain) in Ham's F-12 and centrifuged in a Sorvall SS-34 prewarmed head as previously described (4). Glass coverslips were spun for 40 min at 3000 to 3400 X g at 370, whereas the more sturdy plastic coverslips were spun at 17,000 X g for 20 min. After one centrifugation in cytochalasin B the enucleation was usually gr...

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Section: Transmission Microscopy Of Whole Cho-cellsmentioning

confidence: 87%

The Surface Morphology and Fine Structure of CHO (Chinese Hamster Ovary) Cells Following Enucleation

Shay

Porter

Prescott

1974

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Goldstein et al (51), using enu cleated pieces of cytoplasm from human amnion cells, could not demonstrate an incorporation of either purines or pyri midines into RNA under conditions which permitted the incorporation of amino acids into proteins. Nevertheless, the authors regard the problem of the site of RNA synthesis as still unre solved, since Harris (52) has provided evidence, based on radioautographic data, which suggests that the cytoplasmic RNA is not derived from nu clear RNA; however, the latter results have been contradicted by Taylor (53).…”

Section: Sites Of Rna Synthesismentioning

confidence: 98%