Isolation of viable reconstituted cells from human karyoplasts fused to mouse cytoplasts.

Long-term survivors of reconstituted human-mouse cells have been isolated and characterized by utilizing nuclear and cytoplasmic genetic markers. Karyoplasts were derived from the human SV40-transformed fetal lung fibroblast strain WI38 VA13, while cytoplasts were obtained from the mouse fibroblast A9 cell line which was both hypoxanthine-aminopterin-thymidine-sensitive (HAT^s; nuclear marker) and chloramphenicol-resistant (CAP^r; cytoplasmic marker). The fusion products were isolated in medium containing HAT and CAP. Clones initially showed a growth pattern different from either human or mouse parental cell, but after repeated subculturing, morphologically resembled the nuclear donor cell. The human and mouse components in these cells were identified from other possible fusion combinations by karyotypic, enzymatic and mitochondrial DNA (mDNA) analyses. The karyotype, using both Q-banding and C-banding revealed only human chromosomes. Electrophoretic mobility of the enzyme malate dehydrogenase, a nuclear controlled enzyme, confirmed the human nucleus. Buoyant density centrifugation of radioactive labelled isolated mitochondrial DNA from the reconstituted cells provided evidence that the cytoplasm was of mouse origin.     

Clonal isolation and characterization of myoblast-like reconstituted cells formed by fusion of karyoplasts from mouse teratocarcinoma cells with rat myoblast cytoplasts

To examine the roles of the cytoplasms of differentiated somatic cells on nuclear gene expression, reconstituted cells (RC-cells) were isolated clonally by fusing karyoplasts (isolated nuclei) from neomycin-resistant mouse teratocarcinoma PCC4-neor cells with cytoplasts (isolated cytoplasms) of chloramphenicol (CAP)-resistant rat myoblasts L6TG.CAPr cells, and after double selection in the medium containing 400 micrograms/ml of neomycin and 100 micrograms/ml of CAP (G418 plus CAP medium). The RC-cells were characterized by the presence of two genetic markers, neomycin- and CAP-resistance, by the absence of latex beads which had incorporated into karyoplast donor PCC4-neor cells as a cytoplasmic physical marker, and by the similar karyotypes as that of parental PCC4-neor cells. In contrast to the teratocarcinoma cybrids previously isolated, all the isolated RC-clones expressed myoblast-like morphologies of three types. The phenotypic expression of these RC-cells was compared with that of PCD-1 cells, a teratocarcinoma-derived myoblast line. RC-cells and PCD-1 cells did not express alkaline phosphatase (ALPase) activity while parental PCC4-neor expressed it strongly. After induction of myogenic differentiation by treatments with excess thymidine and conditioned medium, two clones were capable of forming short multinucleated cells. The protein synthetic patterns of RC-cells analysed by two-dimensional polyacrylamide gel were different from PCC4-neor cells, and quite resembled those of PCD-1 cells. Particularly, multinucleated RC-clones expressed alpha-tropomyosin, and contained 10 nm filaments, characteristic markers of early myogenic cells. These results suggest that the RC-cells are myoblast-like cells, that a few of them maturate to partially differentiated myogenic cells, that the rat myoblast cytoplasm contains regulatory factor(s) able to determine the myogenic cell lineage of the undifferentiated stem cells, and that this factor is continuously expressed in these myoblasts.     

Cloned mice derived from embryonic stem cell karyoplasts and activated cytoplasts prepared by induced enucleation

Our objective was to induce enucleation (IE) of activated mouse oocytes to yield cytoplasts capable of supporting development following nuclear transfer. Fluorescence microscopy for microtubules, microfilaments, and DNA was used to evaluate meiotic resumption after ethanol activation and the effect of subsequent transient treatments with 0.4 micro g/ml of demecolcine. Using oocytes from B6D2F1 (C57BL/6 x DBA/2) donors, the success of IE of chromatin into polar bodies (PBs) was dependent on the duration of demecolcine treatment and the time that such treatment was initiated after activation. Similarly, variations in demecolcine treatment altered the proportions of oocytes exhibiting a reversible compartmentalization of chromatin into PBs. Treatment for 15 min begun immediately after activation yielded an optimized IE rate of 21% (n = 80) when oocytes were evaluated after overnight recovery in culture. With this protocol, 30-50% of oocytes were routinely scored as compartmentalized when assessed 90 min postactivation. No oocytes could be scored as such following overnight recovery, with 66% of treated oocytes cleaving to the 2-cell stage (n = 80). Activated cytoplasts were prepared by mechanical removal of PBs from oocytes whose chromatin had undergone IE or compartmentalization. These cytoplasts were compared with mechanically enucleated, metaphase (M) II cytoplasts whose activation was delayed in nuclear transfer experiments using HM-1 embryonic stem cells. Using oocytes from either B6D2F1 or B6CBAF1 (C57BL/6 x CBA) donors, the in vitro development of cloned embryos using activated cytoplasts was consistently inferior to that observed using MII cytoplasts. Live offspring were derived from both oocyte strains using the latter, whereas a single living mouse was cloned from activated B6CBAF1 cytoplasts.     

The effect of in vitro heating on the distribution of nuclear matrix polypeptides in HeLa cells

The in situ nuclear matrix was obtained from HeLa cells. After permeabilization with nonionic detergent, the resulting structures were incubated for 1h at 37°C to determine whether or not such an incubation might result in the redistribution of nuclear polypetides which resisted extraction with buffers of high-ionic strength (1.6 M NaCl or 0.25 M (NH₄)2SO₄ as well as DNase I digestion. Using indirect immunofluorescence experiments and monoclonal antibodies we show that heating to 37° C changes the distribution of a 160 kDa protein previously shown to be a component of the inner matrix network. On the other hand, a 125 kDa polypeptide was not affected at all by the incubation. Our results clearly indicate that the inclusion of a 37°C incubation (for example during digestion with DNase I) in the protocol to obtain the in situ nuclear matrix can result in the formation of in vitro artifacts.     

Distribution of ferritin receptors and coated pits on giant HeLa cells.

HeLa cells bind horse spleen ferritin when the two are incubated at 0 degrees C. Since the majority of this bound ferritin is located in coated pits, we conclude that the ferritin binds to a specific receptor which takes part in an endocytic cycle. When substrate-attached and well-spread giant HeLa cells are briefly labelled at 0 degrees C with ferritin, ferritin particles are found to be concentrated towards the cell periphery, where they exist largely outside coated pits. This peripheral concentration is a property of circulating (and not just newly synthesized) receptors because it is not affected by prior incubation of giant cells in cycloheximide. However, coated pits are themselves roughly uniformly distributed over the surface of these cells. These results provide evidence that the membrane internalised by coated pits on these cells is returned to the cell surface at the leading edge of the cell. Because of this separation of the sites of endocytosis and exocytosis, a flow of membrane must occur across the cell surface. This flow is composed of lipid plus receptors. The implications of this for capping and for cell spreading are discussed.     

New poliovirus-related polypeptides in the nucleus-associated fraction of HeLa cell cytoplasm

Lysates of poliovirus-infected HeLa cells were fractionated by low speed centrifugation into a sediment (“nuclear fraction”) which contained the nuclei and part of the cytoplasm (nucleus-associated cytoplasm, NAC) and a supernatant (nucleus-unassociated cytoplasm, NUC). Both the nuclear fraction and NUC promoted the conversion of 14 S precursor particles to poliovirus procapsids. The NAC contained numerous species of poliovirus-related particles. Their analysis revealed the presence of two new viral polypeptides, NACP-1 and NACP-2, which were absent from the NUC; their molecular weight was estimated as 37,000 and 54,000, respectively.     

DNA synthesis in permeabilized karyoplasts from cytochalasin B-enucleated mouse L cells

DNA synthesis was examined in karyoplasts permeabilized by hypotonic buffer or lysolecithin. DNA synthesis was not affected by the omission of a single deoxyribonucleated triphosphate but was greatly reduced in the absence of three of the deoxyribonucleotide triphosphates. The rate of DNA synthesis was linear for 40 min and continued for up to 2–3 h. The DNA synthesized in the permeabilized karyoplasts was a continuation of semi-conservative replication occurring in the intact cell.     

Distribution of a platinum anti-tumour drug in HeLa cells by analytical electron microscopy

The distribution of Pt in HeLa cell sections after cell treatment with cis-Pt(NH3)2Cl2 dissolved in dimethylsulphoxide was probed by analytical electron microscopy. Primary targets were the nucleolus and the inner side of the nuclear double membrane. Even after solvolysis in dimethylsulphoxide the drug reached similar sites. It is suggested that cell death may be due to Pt inhibition at the initiation sites of DNA synthesis.     

Immunocytochemical localization of glucocorticoid receptors in cells, cytoplasts, and nucleoplasts

A monoclonal antibody has been used to assess the intracellular localization of the glucocorticoid receptor in rodent L-929 fibroblasts and GH3 pituitary tumor cells. Whole cells from both cell lines showed immunoreactivity in the cytoplasm and nucleus. However, when cytoplasts and nucleoplasts of these cells were examined, only L-cells showed strong antibody binding in both fractions; in contrast, GH3 cells exhibited nuclear staining and slight cytoplasmic staining. These results are discussed in terms of the current findings regarding the intracellular location of steroid hormone receptors.     

Budded karyoplasts from multinucleated fibroblast cells contain centrosomes and change their morphology to mitotic cells

Cell changes to accelerated rates of growth are determined by random, new gene mutations that have their origin in vitro in a morphologically visible process of cell alterations. It is a continuous process that, step by step, transforms the normal cell into extended life (EL) cells. These latter cells with limited life spans can further transform to immortalized cells (i.e., cell lines) by the same sequence of morphological cell changes. In contrast to human epithelial cells, fibroblasts in culture have not given rise spontaneously to EL cells. Therefore, it was assumed that some of the cell changes (i.e., cell indicators of the process of transformation) might not be present in near senescent fibroblast cell cultures. As a positive control to normal fibroblast expansion to senescence, the same cells were stressed by inadequate nutrition and confluence. Another positive control was cell indicators induced by SV40 infections. The consecutive sequence of the cell indicators in the transformation process reported previously were: (1) large polyploid cells with nuclei that contained more than two genomes, (2) fragmentation/amitosis of the polyploid nuclei to bi- and multinucleated cells (MNCs), and (3) nuclear budding (i.e., karyoplasts) from MNCs that gave rise to EL cell colonies with various longevities beyond the senescent phase. The present study shows that MNCs and karyoplasts were present in the near-senescent fibroblast cell cultures. Furthermore, new data on the following aspect of the cell transformation process are presented: (1) association of the nuclear fragmentation process with death of cells, (2) cytological markers that distinguish between fragmentation-MNCs versus MNCs from cell fusion, (3) cytological changes of karyoplasts that go through mitotic division to produce daughter cells, and (4) presence of two centrosomes (spindle polar bodies) in the budded karyoplasts. These new findings are discussed in regard to the nuclear fragmentation process in polyploid cells which gives rise to smaller, viable nuclei in MNCs with reduced numbers of whole genomes.     

The preparation and properties of cytoplasts from Ehrlich ascites tumor cells

A method is described in which cytochalasin B is used to fractionate Ehrlich ascites tumor cells into cytoplasts and (nucleated) karyoplasts. The plasma membrane and cytoplasm are selectively removed from these cells by this method such that the cytoplasts rarely contain membranous organelles (e.g., mitochondria) which are retained in the karyoplast during fractionation. ATP concentrations similar to those found in whole cells and glycolytic activity were measured in cytoplasts in the presence but not the absence of glycose. Cytoplasts also actively transport Na⁺, K⁺, and α-aminoisobutyric acid to steadystate distribution ratios similar to those found in whole cells. It was concluded that these cytoplasts are a simplified model system for the study of active transport in Ehrlich cells.     

Separation of cytoplasts and whole cells using density gradients of renografin.

Cytoplasts prepared from L929 or Hepa-2 cells were separated from whole cells using density gradients of renografin. Using this technique, cytoplasts can be isolated from cell lines which cannot be routinely enucleated with an efficiency of 100%. The purified cytoplasts excluded the vital dye trypan blue and were utilized in nuclear transplantation experiments to reconstruct whole viable cells capable of division. In addition, the renografin gradient technique was used to separate the newly reconstructed cells from any contaminating "non-renucleated" cytoplasts. This will permit immediate biochemical characterization of cytoplasmic-nuclear hybrid cells without interference from contaminating cytoplasts.     

Isolation and characterization of cytoplasts and miniprotoplasts derived from protoplasts of cultured cells

We have isolated and partially characterized subprotoplasts containing nuclei, i.e. miniprotoplasts, and enucleated subprotoplasts, i.e. cytoplasts, from freshly isolated protoplasts of cultured cells from Hyoscyamus muticus, Nicotiana tabacum and especially Zea mays . Protoplasts were fragmentated by centrifugation through discontinuous iso-osmotic density gradients containing colloidal silaca gel (Percoll), calcium chloride and mannitol. Using this method metabolically active miniprotoplasts and highly purified cytoplast fractions with less than 4% contamination with nucleated protoplasts were obtained. The cytoplasts prepared by our method are suitable for use in fusion experiments aimed at transferring nuclear and cytoplasmic genetic information separately.     

Architecture and polypeptide composition of HeLa cytoskeletons: Modification of cytoarchitectural polypeptides during mitosis

Substrate-attached asynchronous HeLa cells were extracted with Triton X-100 and analysed by electron microscopy and two-dimensional gel electrophoresis. Such Triton cytoskeletons showed actin filament bundles, microtubules, intermediate filaments, and actin networks in the substrate-associated lamellae, and contained around 90 polypeptides (48 basic, 42 acidic; 52% of total actin, 99% of vimentin, 41% of α-actinin and 30% of β-tubulin).Cytoskeletons produced by further extraction in high and low salt buffers (L-H-L) showed only intermediate filaments, the nucleus and residual actin, and contained a total of 19 polypeptides (13 acidic, 6 basic). Of these, 12 corresponded to abundant acidic proteins in the 47,000 to 70,000 M_r region as determined by staining with Coomassie blue and labelling with a mixture of ¹⁴C-labelled amino acids. Using L-H-L extracted cytoplasts, and employing an actin depolymerising protein from slime moulds, seven abundant acidic IEF³ polypeptides were shown to be present in these intermediate filament-enriched, substrate-attached cytoplast cytoskeletons. These polypeptides (L-H-L cytoplast polypeptides) corresponded to vimentin (IEF 26, 54,000 M_rmr) and six polypeptides (IEF 12, 68,000 M_r; IEF 24, 56,000 M_r; IEF 31, 50,000 M_r; IEF 35, 49,000 M_r; IEF 36, 48,500 M_r and IEF 46, 43,500 M_r) not previously reported as present in cytoskeletons. Peptide analysis showed that these were not related as products of modification or proteolysis.Labelling of mitotic and interphase cells with [³⁵S]methionine followed by one-dimensional peptide map analysis showed that IEF 24, 26 (vimentin), 31 and 36 are preferentially modified during mitosis. These modifications correspond to phosphorylations of IEF 26 (vimentin) and 31, and to an unknown type for IEF 24. IEF 36 is phosphorylated in interphase to yield IEF 37, and the latter is further phosphorylated in mitosis. These results suggest that modification of the L-H-L cytoplast polypeptides may be important in the reorganization of cytoskeletal elements that takes place during cell division.     

Cryoinjury in human granulocytes and cytoplasts.

Although most isolated cells can be successfully cryopreserved, human granulocytes have little functional recovery after cryopreservation, even under optimized conditions. Cytoplasts, which are vesicles created from human granulocytes by depletion of organelles including granules and the nucleus, can carry out some of the complex functions of the parent granulocyte such as phagocytosis of bacteria, even after cryopreservation. Human granulocytes and cytoplasts were used in this comparative study of low-temperature responses to assess the relative importance of the plasma membrane and the granules in cryoinjury to human granulocytes. Boyle-van't Hoff plots of cell volume as a function of the reciprocal of osmolality showed that granulocytes and cytoplasts have similar osmometric behavior and equivalent osmotically inactive fractions. The hydraulic conductivities were also similar, indicating that the osmotic properties of the plasma membrane and cytoplasm were retained during preparation of the cytoplasts. Assessment of membrane integrity using fluorescein diacetate after graded freezing stresses showed that the low-temperature responses of cytoplasts were similar to those of human lymphocytes and hamster fibroblasts, with recoveries much higher than those of human granulocytes, particularly after post-thaw incubation at 37 degrees C. The results indicate that the plasma membrane is not the primary site of injury to granulocytes during freezing and thawing, and suggest that activation of cytoplasmic elements, such as granules, may constitute the early events in cryoinjury to human granulocytes. These studies have significance in approaches to the cryopreservation of granulocytes and other types of cells, such as platelets, with increased sensitivity to the conditions encountered during freezing and thawing.     

Programmed macromolecular synthesis in regenerating karyoplasts.

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 soluble--were 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 G1/S interface, karyoplasts could not begin making DNA until they had regenerated an almost complete complement of cytoplasm.