Purification and characterization of regenerating mouse L929 karyoplasts

Zorn, Glenn A.; Lucas, Joseph J.; Kates, Joseph R.

doi:10.1016/0092-8674(79)90121-1

Cell

1979

DOI: 10.1016/0092-8674(79)90121-1

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Purification and characterization of regenerating mouse L929 karyoplasts

Glenn A. Zorn¹,

Joseph J. Lucas

Joseph R. Kates³

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1979

1992

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Cited by 43 publications

(27 citation statements)

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“…An examination of the organelle content of karyoplasts at various times throughout regeneration showed that not all cytoplasmic structures are re-formed at the same rate (31). This suggested that various cellular proteins might be synthesized differentially, perhaps at discrete times during regeneration.…”

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confidence: 99%

“…Karyoplasts initially contain approximately 10% of the cytoplasmic volume of whole cells and approximately 10% of certain organelles, such as mitochondria (5,13,21,31). However, not all cytoplasmic structures are randomly partitioned between cytoplasts and karyoplasts.…”

mentioning

confidence: 99%

“…Thus, karyoplasts contain only a small portion (ca. 2 to 3c) of the lysosomes and Golgi apparatus of the cells and only fragments of major cytoskeletal structures, such as microtubules; in fact, they completely lack centrioles (21,31).…”

mentioning

confidence: 99%

“…1, which summarizes methods described in detail elsewhere (13,31). In brief, the crude karyoplast pellet was freed from contaminating cytoplasmic fragments by sedimentation through a gradient of Ficoll; from whole cells by two 90-minute incubations in tissue culture dishes; and from dead karyoplasts by sedimentation through Ficoll-paque.…”

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confidence: 99%

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Programmed macromolecular synthesis in regenerating karyoplasts.

White¹,

Bruno²,

Lucas³

1983

Mol. Cell. Biol.

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Conditions for the preparation, purification, and maintenance of karyoplasts which could regenerate to reform whole viable cells were defined. Results of biochemical analyses of such karyoplasts at various times during regeneration indicated that a reproducible biosynthetic program was followed. Thus, an examination of the polypeptides made during regeneration by two-dimensional gel electrophoresis showed that the pattern of radiolabeled polypeptides synthesized at each time studied was specific and was significantly different from that observed at other times during regeneration. Polypeptides associated with three major cellular fractions-nuclear, cytoskeletal-microtrabecular, and solublewere among the most dramatically regulated molecules. Other polypeptides, such as the major components of microfilaments and intermediate filaments, were synthesized at relatively constant rates and were assembled into structures throughout regeneration. Likewise, microtubules appeared to be reformed throughout regeneration, even in the absence of identifiable centriole-associated organizing centers. Finally, analysis of DNA synthesis by autoradiography showed that, even when prepared from whole cells synchronized at the Gl/S interface, karyoplasts could not begin making DNA until they had regenerated an almost complete complement of cytoplasm.Cytochalasin-induced enucleation of mammalian cells produces primarily two types of cellular fragments: cytoplasts, or enucleated cells, and karyoplasts, or nuclei surrounded by thin shells of cytoplasm enclosed within outer cell membranes (5,18,21

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confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Programmed macromolecular synthesis in regenerating karyoplasts.

White¹,

Bruno²,

Lucas³

1983

Mol. Cell. Biol.

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“…By judicious choice of the parental cell types and the use of a variety of staining techniques, such reconstituted cells can be produced in large numbers and definitively identified immediately after fusion (8)(9)(10)(11). These cells, having a minimal contribution of cytoplasm from the nuclear donor cell type (12)(13)(14)(15), would appear to be excellent systems for investigating the interaction of cytoplasm and nucleus in determining a variety of cellular properties and events, including cell shape and cytoarchitecture and genome replication and expression. However, analyses of both reconstructed and cybrid cells performed so far have indicated that, except for a few instances (16)(17)(18)(19)(20)(21), exposure to a foreign cytoplasm has little long-term effect on a transplanted nucleus.…”

mentioning

confidence: 99%

Identification of key regulated events early in the life of hybrid animal cells constructed by nuclear transplantation.

Hightower¹,

Bruno²,

Lucas³

1983

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

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Reconstituted cells were constructed by fusion of cytoplasts from the human diploid fibroblast cell strain Detroit 532 and karyoplasts from the mouse fibroblast cell line A9. Several cellular properties were examined during the first 48 hr after nuclear transplantation. (i) The overall morphology of the cells originally resembled that of the cytoplasmic donor, Detroit 532, but rapidly changed to approximate that of the nuclear donor, A9. However, definitive changes in the microfilament structure of the reconstituted cells were not seen until 24-48 hr after fusion. These observations support the idea that the presence or absence of an ordered array of microfilament bundles is not the sole determinant of cell shape. (ii) Although cytoplasts and karyoplasts were prepared from cultures of randomly growing cells, the first division of reconstituted cells occurred in a synchronous manner. However, the initiation of DNA synthesis was not synchronized. It thus appeared that, in their first cell cycle, the cells had a G2 period of variable length. The results further suggest that the cytoplasm of interphase fibroblasts contains the material necessary to initiate or support DNA synthesis in a transplanted nucleus but not entry into mitosis. (iii) A two-dimensional gel electrophoretic analysis of polypeptide synthesis in reconstituted cell cultures showed that synthesis directed by transplanted mouse nuclei could be detected as early as 3-6 hr after fusion. Some of the mouse polypeptides detected at the earliest time points studied were not among the major polypeptides synthesized by the parental A9 cells. By about 48 hr after fusion, the pattern of polypeptides produced by reconstituted cells was almost indistinguishable from that of the nuclear donor parent cells.Cytoplasts and karyoplasts prepared from animal cells by cytochalasin-induced enucleation (1, 2) can be fused together to reconstitute whole viable cells (3)(4)(5). If the fragments are from two different cell types, cells containing single nuclei within foreign cytoplasm can be constructed (6, 7). By judicious choice of the parental cell types and the use of a variety of staining techniques, such reconstituted cells can be produced in large numbers and definitively identified immediately after fusion (8-11). These cells, having a minimal contribution of cytoplasm from the nuclear donor cell type (12-15), would appear to be excellent systems for investigating the interaction of cytoplasm and nucleus in determining a variety of cellular properties and events, including cell shape and cytoarchitecture and genome replication and expression. However, analyses of both reconstructed and cybrid cells performed so far have indicated that, except for a few instances (16-21), exposure to a foreign cytoplasm has little long-term effect on a transplanted nucleus. In this report it is shown that, in most respects, cells constructed by the method of nuclear transplantation are indistinguishable from the nuclear donor parental cell type as early as 48 hr after fusion....

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Partitioning of the Golgi apparatus in rat primary and secondary spermatocytes during meiosis

Oke

Suárez‐Quian

1992

Anat. Rec.

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We examined the disassembly and reformation of the Golgi apparatus as a function of the cycle of the seminiferous epithelium in adult rats during stages XIII and XIV, i.e., just prior to and during meiosis I and II. Serial section analysis of primary spermatocytes at metaphase I demonstrated the presence of two Golgi complexes. At the ultrastructural level, these Golgi complexes were shown to be composed of stacks of cisternae and vesicles, with each stack having a varying number of saccules. Although Golgi complex intermediates resulting from the process of organelle disassembly were not clearly identified in diplotene spermatocytes immediately prior to nuclear envelope vesiculation, we did observe clusters of vesicles resembling the "nuage," with each cluster varying in size and number of vesicles. Meiosis I results in the formation of secondary spermatocytes that exhibit a well-formed spherical Golgi complex approximately half the size of the diplotene spermatocyte Golgi. Next, secondary spermatocytes enter meiosis II. In contrast to metaphase I, during metaphase II reformation of the Golgi apparatus into stacks was not observed and only small clusters of vesicles at two poles of dividing cells were detected. In addition, "nuage"-like structures were not identified during meiosis II. Our results begin to characterize the process by which Golgi apparatus partitioning is accomplished during meiosis, presumably resulting in the delivery of equal complements of this organelle to four round spermatids. We suggest that partitioning of the Golgi apparatus takes place prior to metaphase I and that the two steps of meiosis may exhibit subtle differences with respect to Golgi partitioning.

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Purification and characterization of regenerating mouse L929 karyoplasts

Cited by 43 publications

References 17 publications

Programmed macromolecular synthesis in regenerating karyoplasts.

Programmed macromolecular synthesis in regenerating karyoplasts.

Identification of key regulated events early in the life of hybrid animal cells constructed by nuclear transplantation.

Partitioning of the Golgi apparatus in rat primary and secondary spermatocytes during meiosis

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