Preparation and characterization of chick erythrocyte nuclei from heterokaryons

A method for the isolation of reactivated chick erythrocyte nuclei from heterokaryons was developed. The heterokaryons were produced by fusing chick erythrocytes with HeLa or L cells in the presence of inactivated Sendai virus. At various time intervals after fusion nuclei were isolated directly from the monolayer by treatment with an acidic detergent solution. Chick erythrocyte nuclei were then separated from other nuclei (HeLa or L cell) by centrifugation on sucrose gradients. The purified preparation of reactivated chick erythrocyte nuclei was shown to be free from other nuclei and cytoplasmic contamination. By using L cells which had been labelled with ³H-leucine before fusion or heterokaryons labelled after fusion it was demonstrated that labelled mouse proteins migrate from the cytoplasm of the heterokaryons into the reactivating chick erythrocyte nuclei. ³H-uridine labelling of heterokaryons made by fusing UV-irradiated chick erythrocytes with L cells failed to reveal any significant migration of mouse RNA into the chick erythrocyte nuclei.     

Dynamics of Nuclear Pore Distribution in Nucleoporin Mutant Yeast Cells

To follow the dynamics of nuclear pore distribution in living yeast cells, we have generated fusion proteins between the green fluorescent protein (GFP) and the yeast nucleoporins Nup49p and Nup133p. In nup133⁻ dividing cells that display a constitutive nuclear pore clustering, in vivo analysis of GFP-Nup49p localization revealed changes in the distribution of nuclear pore complex (NPC) clusters. Furthermore, upon induction of Nup133p expression in a GAL-nup133 strain, a progressive fragmentation of the NPC aggregates was observed that in turn led to a wild-type nuclear pore distribution. To try to uncouple Nup133p- induced NPC redistribution from successive nuclear divisions and nuclear pore biogenesis, we devised an assay based on the formation of heterokaryons between nup133⁻ mutants and cells either expressing or overexpressing Nup133p. Under these conditions, the use of GFP-Nup133p and GFP-Nup49p fusion proteins revealed that Nup133p can be rapidly targeted to the clustered nuclear pores, where its amino-terminal domain is required to promote the redistribution of preexisting NPCs.     

ACQUISITION OF CHICK CYTOSOL THYMIDINE KINASE ACTIVITY BY THYMIDINE KINASE-DEFICIENT MOUSE FIBROBLAST CELLS AFTER FUSION WITH CHICK ERYTHROCYTES

Chick-mouse heterokaryons were obtained by UV-Sendai virus-induced fusion of chick erythrocytes with thymidine (dT) kinase-deficient mouse fibroblast [LM(TK^-)] cells. Autoradiographic studies demonstrated that 1 day after fusion, [³H]dT was incorporated into both red blood cell and LM(TK^-) nuclei of 23% of the heterokaryons. Self-fused LM(TK^-) cells failed to incorporate [³H]dT into nuclear DNA. 15 clonal lines of chick-mouse somatic cell hybrids [LM(TK^-)/CRB] were isolated from the heterokaryons by cultivating them in selective hypoxanthine-aminopterin-thymidine-glycine medium. LM(TK^-) and chick erythrocytes exhibited little, if any, cytosol dT kinase activity. In contrast, all 15 LM(TK^-)/CRB lines contained levels of cytosol dT kinase activity comparable to that found in chick embryo cells. Disk polyacrylamide gel electrophoresis and isoelectric focusing analyses demonstrated that the LM(TK^-)/CRB cells contained chick cytosol, but not mouse cytosol dT kinase. The LM(TK^-)/CRB cells also contained mouse mitochondrial, but not chick mitochondrial dT kinase. Hence, the clonal lines were somatic cell hybrids and not LM(TK^-) cell revertants. The experiments demonstrate that chick erythrocyte cytosol dT kinase can be activated in heterokaryons and in hybrid cells, most likely as a result of functions supplied by mouse fibroblast cells.     

The cytoplasmic appearance of three functions expressed during the G0→G1→S transition is nucleus-dependent

tsAF8, ts13, tsHJ-4, and TK^?ts13 cells are G₁-specific temperature-sensitive (ts) mutants of BHK cells that do not enter S phase when serumstimulated from quiescence at nonpermissive temperature (39.6°-40.6°). TK^?ts13 are, in addition, defective in thymidine kinase. Different G₁ functions must be involved in these cells, since the first three cell lines complement each other when forming heterokaryons. We have used these cells to study the role of the nucleus in the cytoplasmic expression of these G₁ functions during the transition of cells from the non-proliferating to the proliferating state. We fused cytoplasts from either serumstarved (G₀) or serum-stimulated (S) tsAF8 cells with G₀-ts13, G₀-tsHJ-4, and G₀-TK^?ts13 recipient cells and determined, after serum stimulation of the fusion products, which type of cytoplasts could complement the defective G₁ functions. Cytoplasts from S-tsAF8 cells complemented all three functions, i.e., cybridoids between S phase cytoplasts and ts13 or tsHJ-4 recipient cells entered S at the nonpermissive temperature, and TK^?ts13 recipient cells incorporated exogenous thymidine. Cytoplasts isolated from G₀-tsAF8 cells (3 days of serum starvation) complemented ts13 cells but not tsHJ-4 and TK^?ts13 cells. Cytoplasts from 6-day starved tsAF8 cells lost the complementing capacity for ts13 cells. However, when the 6-day starved tsAF8 cells were fused with G₀-ts13 cells, the heterokaryons entered S phase at the nonpermissive temperature. Also, cytoplasts isolated from the 6-day starved cells that were serum stimulated for 40 hr before enucleation regained the capacity to complement ts13 cells. These results demonstrate that three functions required in G₁ cannot be detected in the cytoplasm of serum-starved cells, although they are present in the cytoplasm of S-phase cells. These results suggest that a functional nucleus is required for the cytoplasmic appearance of certain G₁ functions in serumstimulated cells.     

Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana

The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5·10⁵ protoplasts (0.5 ml of cells at 10⁶/ml) can be fused in 5–7 min with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.Abbreviations AC alternating current - DC direct current - PEG polyethylene glycol     

An ultrastructural study of the fusion of cultured amphibian cells with higher plant protoplasts

Summary CulturedXenopus cells have been induced to fuse with carrot suspension cell protoplasts using PEG at high pH in the presence of high Ca²⁺. Ultrastructural observations confirm unambiguously that the fusion bodies seen by light microscopy are animal/plant cell heterokaryons. The csytoplasmic events occurring in theseXenopus/carrot fusion products during the first 48 hours of culture provide evidence for their viability. Some of the factors influencing the formation and subsequent survivalin vitro of interkingdom heterokaryons are discussed.     

Species specificity of interferon action: maintenance and establishment of the antiviral state in the presence of a heterospecific nucleus.

The expression of the interferon-induced antiviral state was studied in heterokaryons and cytoplasmic hybrids (cybrids). An autoradiographic assay for the antiviral state, in which the percentage of cells containing vaccinia viral DNA factories was determined, was used. The expression of the antiviral state was dominant in homokaryons and heterokaryons formed by fusion of interferon-treated cells with untreated cells. Cytoplasts derived from treated cells conferred resistance to virus growth on cybrids formed by fusing such cytoplasts with untreated cells. Treatment of L cell x HeLa cell heterokaryons with human interferon or mouse interferon was much less effective in inducing a detectable antiviral state than was similar treatment of parental cells with homospecific interferon. The antiviral state was fully induced when heterokaryons were treated simultaneously with both types of interferon. Cybrids formed by fusing L cell cytoplasts with HeLa cells or HeLa cytoplasts with L cells did not enter a detectable antiviral state after treatment with interferon specific for the cell type of the enucleated parent. However, treatment of cybrids with interferon specific for the cell type of the nucleated parent was effective in inducing a detectable antiviral state.     

Expression of embryonic and MHC antigens in heterokaryons of murine embryonal carcinoma and human melanoma cells

Mouse embryonal carcinoma cells were fused with human melanoma cells or with cytoplasts of these cells. The expression of embryonic and major histocompatibility complex (MHC) antigens was studied in single heterokaryons and cybrids in the population after fusion. Recognition of heterokaryons by differential staining of mouse and human nuclei was combined with indirect immunofluorescent staining of specific membrane antigens. Complete suppression of embryonic antigen expression was found in heterokaryons within 2 days after fusion. Cybrids, formed by fusion of embryonal carcinoma cells with melanoma cytoplasts, showed a transient decrease in the expression of embryonic antigens. The expression of human MHC antigens, both class I (HLA-A, B, C) and class II (HLA-DR), was only slightly influenced in heterokaryons. No activation of mouse MHC antigens was found. The results indicate that melanoma cells contain trans-acting factors exerting a negative control on the expression of embryonic antigens. In contrast the continued expression of human MHC antigens in heterokaryons suggests that embryonal carcinoma cells either are devoid of or contain only a very limited amount of trans-acting factors controlling the expression of MHC antigens.     

Arginine-rich histones do not exchange between human and mouse chromosomes in hybrid cells

Following division of HeLa-3T3 heterokaryons, human and mouse chromosomes occupy distinct regions within the resulting hybrid nuclei. This favorable orientation of genomes has allowed us to determine whether histones exchange between chromosomes in vivo. Acrylamide gel electrophoresis of the proteins from HeLa cells labeled with ³H-arginine during S phase showed that the core histones were labeled preferentially, constituting 30% of the total cellular tritium and 50% of the label in a crude nuclear fraction. Autoradiographic analysis of cells formed by fusion of ³H-arginine-labeled HeLa cells and 3T3-4E cells showed that ³H-arginine-labeled proteins did not migrate between nuclei in heterokaryons; hybrid cells formed from such heterokaryons contained nuclei in which ³H proteins occupied a sector within the nucleus; “sectored nuclei” could persist for at least 4 days; and the unequal distribution of ³H proteins did not change during DNA synthesis. Electron microscopic examination of hybrid nuclei failed to reveal a physical partition between human and mouse chromosome sets. Sectored nuclei were also observed in synkaryons derived from ³H-arginine-labeled HeLa and unlabeled HeLa cells, indicating that the unequal distribution of ³H-arginine-labeled proteins in HeLa-3T3 hybrid cells did not result from species-specific binding of proteins and DNA. The persistent unequal distribution of ³H-arginine-labeled proteins within hybrid nuclei in the apparent absence of a barrier between mouse and human chromosomes indicates that histones, the principal ³H-arginine-labeled proteins, do not dissociate from DNA in vivo.     

Expression and distribution of vimentin and keratin filaments in heterokaryons of human fibroblasts and amnion epithelial cells

The expression of intermediate filaments of the keratin- and the vimentin-type was studied in heterokaryons of human fibroblasts and amnion epithelial cells by immunofluorescence microscopy. Fibroblasts and their homokaryons showed a fibrillar, vimentin-specific fluorescence throughout the cytoplasm but were negative when stained for keratin. Amnion epithelial cells and their homokaryons, on the other hand, showed a keratin-specific fibrillar staining, and only some of them contained also detectable vimentin. When suspended epithelial cells were fused with adherent fibroblasts, keratin fibrils spread within 3 h into the fibroblasts, intermixing with the vimentin fibrils. 1-3 d after fusion, both vimentin and keratin filaments were expressed as typical fibrillar cytoplasmic arrays, and the distribution of keratin in heterokaryons resembled closely that of vimentin. A typical cell-to-cell arrangement of keratin fibrils, seen in cultures of amnion epithelial cells, could also be found between heterokaryons. Treatment of the cultures with vinblastine sulphate induced coiling of the vimentin filaments in both homo- and heterokaryons, whereas the keratin organization was only slightly affected. Our results show that both vimentin and keratin filaments are incorporated into the cytoskeleton of heterokaryons formed between fibroblasts and epithelial cells, and that they behave in the same way as in their parental cells. Both epithelial and fibroblastic characteristics thus appear to the coexpressed in such heterokaryons.     

Two methods of heterokaryon formation to discover HCV restriction factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers^1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle ^6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected naïve cells in a receptor-dependent fashion.To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells ⁹) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection ¹⁰ . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV¹¹) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.     

Protoplast fusion of Schizosaccharomyces pombe auxotrophic mutants of identical mating-type

Summary Protoplasts of methionine-and lysine-requiring h^- mutants isolated from the L972 h^- strain of Schizosaccharomyces pombe were fused. The protoplasts were obtained from the cells with enzymes produced by Trichoderma viride. When a mixture of the protoplasts was treated with 30% PEG 4000 solution containing 10 mM CaCl₂, cell fusion and complementation was attained with a frequency of 0.17%. Both fusion partners were recovered among the spores after crossing of the fusion products with the strain M210 ade6 h⁺. Cytological and haploidization examinations showed that the fusion cells are not heterokaryons, and that the increased amount of genetic material is situated in one nucleus.     

Complementation of murine cells for human immunodeficiency virus envelope/CD4-mediated fusion in human/murine heterokaryons.

Murine cell lines expressing human CD4 are resistant to the fusogenic effect of the human immunodeficiency virus (HIV) envelope. Consequently, they cannot be infected by HIV or form syncytia with HIV envelope-expressing cells. Murine cells could either lack human-specific cofactors necessary for the CD4/envelope-mediated membrane fusion or express inhibitors of this process. To address this question, we have tested the ability of heterokaryons made from CD4-expressing murine cells and human cells to undergo HIV envelope-mediated fusion. We have devised a rapid and specific assay based on the induction of lacZ expression, in which membrane fusion events with HIV-infected cells can be detected by a simple histochemical technique. CD4-positive murine/human heterokaryons, but not murine/simian heterokaryons, were found able to fuse with HIV envelope-expressing cells. In these experiments, the fusion resistant phenotype of murine-CD4 cells could be complemented by human cellular factors.     

Reactivation of chick erythrocyte nuclei in young and senescent WI38 cells

The chromatin of the dormant chick nucleus is dispersed in the heterokaryons made by Sendai virus fusion of phase II WI38 cells with chick erythrocyte nuclei. The erythrocyte nucleus resumes RNA synthesis and enters into DNA synthesis with the host nucleus. In the heterokaryons of phase III WI38 cells and chick erythrocytes, the nuclear chromatin is not dispersed and RNA synthesis occurs at a reduced rate. The differences in the physiological state of the young and senescent cells measured by [³H]uridine incorporation into nuclear RNA is reflected in the extent of reactivation of the chick erythrocyte nuclei in the cytoplasm of these cells. The reactivation of the chick nucleus in enucleated fibroblasts parallels the nucleated cells. The results of these studies are interpreted as evidence that there is a specific loss of nuclear function in the senescent cells.     

DNA synthesis and division in interkingdom heterokaryons

Summary Drosophila cells and plant protoplasts were fused by polyethylene glycoldimethylsulfoxide treatment. DNA synthesis in plant and animal cells and in their heterokaryons were monitored by [³H]thymidine autoradiography. In appropriate media both the parental cells and the heterokaryons preserved their DNA synthetic activity. Division of the heterokaryons were also observed.     

Restoration of metabolic cooperation in heterokaryons between HGPRT-deficient mouse A9 fibroblasts and chick embryo erythrocytes

Genetic determinants of metabolic cooperation were studied by fusing chick erythrocytes to HGPRT- mammalian cells. Heterokaryons were then tested for their ability to incorporate [3H]hypoxanthine and to transfer radioactive material to HGPRT- recipient cells. Chick erythrocytes (CE) have nuclei which are inactive but contain the HGPRT gene and some cytoplasmic HGPRT enzyme activity. They are unable, however, to cooperate with HGPRT- cells. Of the two mammalian cell lines used, the human GM29 line is HGPRT- and capable of functioning as a receptor cell in cooperation experiments with HGPRT+ cells. The HGPRT- mouse A9 line on the other hand is unable to cooperate. Immediately after fusion, both types of heterokaryons incorporated [3H]hypoxanthine, indicating the presence of some chick HGPRT enzyme contributed by the erythrocyte partner at the time of fusion. While the CE-GM29 heterokaryons participated in metabolic cooperation shortly after fusion, the CE-A9 heterokaryons did not. However, four days after fusion, i.e., at a time when the erythrocyte nucleus had been reactivated, the CE-A9 heterokaryons did cooperate. This suggests that in CE-A9 heterokaryons the genes required for metabolic cooperation are expressed by the previously dormant chick erythrocyte nucleus.     

Fusion between human and Drosophila melanogaster cells induced by polyethylene glycol

Polyethylene glycol was used to induce interspecific somatic cell fusion between human fibroblasts (stock F6) and Drosophila melanogaster cells from established cell lines (C1 82 and 11 P102), characterized by different ploidy levels. The present investigation defines some parameters for Drosophila cell fusion and interspecific fusion between Drosophila and human cells. The cytological analysis provided evidence of spontaneous as well as induced human-Drosophila heterokaryon formation. The presence in the same cell of two types of nuclei, distinguishable because of their different size and morphology, was confirmed autoradiographically by ³H-thymidine pre-labelling of Drosophila cells. Furthermore, the retained DNA synthetic activity and some examples of mitotic figures of both types of nuclei in the heterokaryons indicate the viability of the fused cells.     

Interaction of human and chick DNA repair functions in UV-irradiated xeroderma pigmentosum-chick erythrocyte heterokaryons

Fusion of chick erythrocytes with human primary fibroblasts results in the formation of heterokaryons in which the inactive chick nuclei become reactivated. The expression of chick DNA repair functions was investigated by the analysis of the DNA repair capacity after exposure to ultraviolet (UV) irradiation of such heterokaryons obtained after fusion of chick erythrocytes with normal human or xeroderma pigmentosum (XP) cells of complementation groups A, B, C and D. Unscheduled DNA synthesis (UDS) in normal human nuclei in these heterokaryons is suppressed during the first 2–4 days after fusion. The extent and duration of this suppression is positively correlated with the number of chick nuclei in the heterokaryons. Suppression is absent in heterokaryons obtained after fusion of chicken embryonic fibroblasts with XP cells (complementation group A and C).Restoration of DNA repair synthesis is found after fusion in XP nuclei of all complementation groups studied. It occurs rapidly in XP group A nuclei, starting one day after fusion and reaching near normal human levels after 5–8 days. In nuclei of the B, C and D group increased levels of UDS are found 5 days after fusion. At 8 days after fusion the UDS level is about 50% of that found in normal human nuclei. The pattern of UDS observed in the chick nuclei parallels that of the human counterpart in the fusion. A fast complementation pattern is also observed in chick fibroblast-XP group A heterokaryons resulting within 24 h in a UDS level comparable with that in chick fibroblast-normal human heterokaryons. In heterokaryons obtained after fusion of chick fibroblasts with XP group C cells UDS remains at the level of chick cells. These data suggest that reactivation of chick erythrocyte nuclei results in expression of repair functions which are able to complement the defects in the XP complementation groups A, B, C and D.     

Intergenic complementation in chick melanocyte heterokaryons

Melanocytes from chick embryos of the pinkeye (pk/pk) and recessive white (c/c) genotypes do not produce melanin in cell culture. However, aberrant melanogenic organelles are evident when these cells are examined with the electron microscope. Melanocytes of each genotype, previously grown for 5 days in cell culture, were co-cultured for 24 h and then fused with inactivated Sendai virus. Twenty-four hours after fusion faintly pigmented cells could be seen in the culture dishes. These cells were invariably multinucleated. At 48 h post-fusion many darkly pigmented, multinucleated cells could be seen. Pigment-producing cells were found in four separate experiments and occurred at a frequency of approx. 1 per 40 000 cells treated. Co-culturing of the melanocytes without virus treatment failed to elicit pigment production. When one genotype was labeled with [³H]thymidine prior to fusion, autoradiograms showed that the pigmented cells contained at least one labeled and one unlabeled nucleus. Electron micrographs of the pigmented cells confirmed that cell fusion was complete and showed normal pigment granules with welldefined matrices and deposited melanin. The results show that recessive white and pinkeye can complement as heterokaryons. This indicates that each mutation affects a different melanogenic function and that the expression of the normal function of each does not require nuclear integration. The simplest hypothesis is that the two mutations affect structural genes and that the complementing cytoplasms contain functional gene products. The hypothesis that one or both mutants have altered control functions cannot be ruled out, however.