Mitochondrial RNA and protein synthesis in enucleated African green monkey cells

The behavior of extramitochondrial protein synthesis and of mitochondrial RNA and protein synthesis was examined in the cytoplasts of African green monkey kidney cells (TC-7 subline) at different times following enucleation by cytochalasin B. The rate of incorporation of [³H]isoleucine into protein of the soluble cytoplasmic fraction decreased in an approximately exponential fashion, with a half-life of about five hours, during the first 26 hours after enucleation. Discrete mitochondrial 16 S, 12 S and 4 S RNA components were identified among the products of cytoplast RNA synthesis. The rates of [³H]uridine incorporation into the 16 and 12 S RNA components as well as into total RNA declined progressively after enucleation to a barely detectable level by the 20th hour. By contrast, the rate of chloramphenicol-sensitive [³H]isoleucine incorporation into protein (due to mitochondrial protein synthesis) did not undergo a substantial decline for at least 20 hours in TC-7 cytoplasts; instead, a reproducible transient stimulation occurred in the first hours following enucleation. The products of mitochondrial protein synthesis pulse-labeled in nucleated cells and in cytoplasts 24 hours after enucleation exhibited similar electrophoretic profiles.     

Modification of a method of cell enucleation using cytochalasin B and the study of cytoplast viability

The modification of Prescott's (Prescott et al., 1972) method of enucleation in vitro was described. A special teflon chamber faciliatating the enucleation of monolayer cultured cells to produce cytoplasts and karyoplasts was constructed. Mouse L-cells were enucleated by exposing to cytochalasine B (10 gamma/ml) followed by centrifugation. The fraction of cells enucleated in the chamber was about 98%. The life time of cytoplasts in cultural medium after their enucleation was 48 hours (sometimes 56-72 hours) as tested by vital neutral red staining. The cytoplasts that survived were shown to accumulate large lysosomes, and the evidence of appearing ring-like fibrillar structures was provided using a simple technique of cytoskeleton observation under light microscope.     

The ultrastructure of the centrosome in cytoplasts and its alteration under the action of ouabain]

It has been shown that after enucleation of the PE cells with cytochalasin D the centrioles remain in approximately 80% of cytoplasts. Some cytoplasts contain only single centriole, either a mother (active) of a daughter (inactive) one. 20% cytoplasts have no centrioles. 2h after enucleation the centrosome structure in the cytoplasts did not differ from that in normal cells. 14-16 h after enucleation in many cytoplasts large secondary lysosomes and lipid droplets appeared around the centrosome. At this time in some cytoplasts in the centrosome we observed free microtubule convergence foci. 14-16 h after the enucleation, some cytoplasts have doubling centrioles. Under the influence of ouabain (30 min), the number of active centrioles oriented perpendicularly to the substrate plane in the cytoplasts increased. We suggest that the preferentially perpendicular orientation of centrioles to the substrate plane does not depend on the nuclear activity.     

Use of cell enucleation in studying the stability of cytoplasmic organelles and cytoplasm organization

A study was made of the viability and ultrastructure of cytoplasts produced by enucleation of cytochalasin-induced A9 cells in suspension. These cytoplasts are in general as viable as cells enucleated in the monolayer. The organization of the cytoplasm, i.e. a specific distribution of cytoplasmic organelles, is conserved for at least 24 hours in the absence of the nucleus. This fact may reflect a high degree of autonomy of the cytoskeleton.     

Assessment of mitochondrial function in the surviving cytoplasts of cultured cells of pig embryonic kidney by using ethylrhodamine

A study was made of the functional state of chondriome in cytoplasts previously cultivated for a long time under vital staining with fluorescent dye ethylrhodamine (10 micrograms/ml) which is known to accumulate preferentially in mitochondria. The energization was estimated by the intensity of mitochondrial fluorescence. It was realized that 30 minutes after enucleation cytoplasts retained the same fluorescence as did the untreated cells, and that the mitochondrial distribution within the cell was very similar. Such a high intensity of fluorescence seen within one day after enucleation gives a strong evidence on the high degree of independence on the cell nucleus of organelles that provide the energy to metabolic processes. In the course of survival in the cultivation medium for 1-4 days the intensity of fluorescence is shown to fall, especially on the second day after enucleation. All these changes coincide with the changes in cytoplast shape: originally well spread bodies transform into squeezed, ball-shaped or strongly deformed ones. The adhesive ability is going down, and in result only single units of cytoplast can hardly be found on the cover slips by the 4th day after enucleation. These changes give evidence on the enhanced degeneration of cultured cytoplasts.     

Comparison of the coverslip and the discontinuous Percoll density gradient methods of enucleation of mouse cells

Two methods of enucleation of LB 10 cells, a subline of mouse L cells, were used: the method of enucleation of cells growing in monolayers, and the newly improved method of enucleation in discontinuous Percoll gradients. The second method was more effective and, as shown by incorporation of 3H-lysine, protein synthesis in cytoplasts was prolonged twice when compared with that in cytoplasts obtained by the coverslip method.     

Non-selective isolation of fibroblast-cybrids by flow sorting

Red- and green-fluorescing polystyrene beads were used to label different populations of cultured human fibroblasts. After enucleation of the green-fluorescing population, the cytoplasts were fused with red-fluorescing cells. Twenty-four hours after cell fusion the population of red-green heterofluorescent cells was isolated with a FACS II cell sorter. When Lesch-Nyhan fibroblasts (HPRT⁻) were fused with cytoplasts from control fibroblasts (HPRT⁺) more than 95% of the sorted cells were heterofluorescent and 90% of the sorted cells showed HPRT⁺ activity. Therefore almost all sorted heterofluorescent cells are true cybrids. With this procedure for cybrid isolation, earlier complementation studies using cybrids from different variants of β-galactosidase deficiency could be confirmed.     

An improved cellular enucleation method with extracellular matrix and colchicine facilitates the study of nucleocytoplasmic interaction

Enucleated mammalian cells (cytoplasts) have been widely used for studying differential roles of the cytoplasm and nucleus in various cellular processes. Here, we reported an improved enucleation protocol, in which cells were seeded in extracellular matrix (ECM)-coated 24-wells and spun at 4600 g and 35 °C for 60 min in the presence of cytochalasin B and colchicine. When glass-bottom wells were used, cellular structures and organelles in cytoplasts could be examined directly by confocal microscopy. Nuclear envelope rupture did not occur probably due to mild centrifugation conditions used in this study. Addition of paclitaxel or doxorubicin completely blocked proliferation of residual nucleated cells; however, to our surprise, paclitaxel dramatically prolonged the survival of cytoplasts. Results from Annexin V and Propidium Iodide staining showed that cytoplasts died predominantly by apoptosis, which was partially inhibited by ECM and further by paclitaxel. Mitochondria were mostly rod-shaped and formed a connected network in paclitaxel-treated cytoplasts, indicating lack of fusion and fission dynamics. Moreover, paclitaxel increased mitochondrial membrane potential, suggesting that perturbation of mitochondria might be critical to the survival of cytoplasts. In conclusion, we had established an efficient and fast procedure for enucleation of adherent animal cells, which could facilitate the investigation of nucleocytoplasmic interaction.     

Reproduction of murine encephalomyocarditis virus in enucleated Krebs 2 ascitic carcinoma cells]

Enucleated cells of ascites carcinoma Krebs 2 (cytoplasts) were prepared. The cells were centrifugated for this purpose in a discontinuous Ficoll density gradient containing cytochalasine B. The cytoplasts formed turbid bands in Phycol solution at the density of 1.037--1.053 g/ml. The cytoplasts failed to synthesize RNA, but proteins were synthesized for several hours. Encephalomyocarditis virus replicated in the ascites carcinoma cytoplasts, but its yield was 10 to 100-fold lower as compared to that in the intact cells. Apparently the reduction of the virus yield was not due to the low efficiency of early stages of the virus-cytoplast interaction. Viral infection resulted in inhibition of the synthesis of cellular proteins in the cytoplasts as well as in the intact cells.     

Degradation of short-lived proteins is decreased by centrifugation

We have examined the effects of enucleation and of inhibitors of mRNA synthesis (actinomycin D and cordycepin) on protein turnover of HeLa cells. Enucleation markedly inhibited the rate of protein degradation for short-lived proteins. However, cells centrifuged in the absence of cytochalasin B at the speed required to obtain cytoplasts showed protein degradation rates identical to those of cytoplasts, while inhibitors of mRNA synthesis did not affect the process. Although enucleation may affect degradation of specific proteins, these results suggest that centrifugation is largely responsible for the inhibition of protein degradation in cytoplasts.     

Vaccinia Virus Replication I. Requirement for the Host-Cell Nucleus

Using cytochalasin B-induced enucleation techniques, we examined the ability of vaccinia virus to replicate in the absence of the host-cell nucleus in several mammalian cell lines. It was found that virus-infected enucleated cells (cytoplasts) prepared from BSC-40, CVC, and L cells were incapable of producing infectious progeny virus. The nature of this apparent nuclear involvement was studied in detail in BSC-40 cells. Modulations designed to maximize cytoplast integrity and longevity, such as reduction of the growth temperature and initial multiplicity of infection, did not improve virus growth in cytoplasts. Sodium dodecyl sulfate-polyacrylamide gel analysis of the [(35)S]methionine pulse-labeled proteins synthesized in vaccinia virus-infected cytoplasts demonstrated that both early and late viral gene products were being expressed at high levels and with the proper temporal sequence. Vaccinia virus cytoplasmic DNA synthesis, as measured by [(3)H]thymidine incorporation, peaked at 3 h postinfection and was 70 to 90% of control levels in cytoplasts. However, in the cytoplasts this DNA was not converted to a DNase-resistant form late in infection, which was consistent with the failure to isolate physical particles from infected cytoplasts. Treatment of vaccinia virus-infected cells with 100 mug of rifampin/ml from 0 to 8 h to increase the pools of viral precursors, followed by subsequent removal of the drug, resulted in a threefold increase virus yield. This treatment had no effect on virus-infected cytoplasts. Finally, vaccinia virus morphogenesis was studied under an electron microscope in thin sections of virus-infected cells and cytoplasts which had been prepared at various times during a single-step growth cycle. It was apparent that, although early virus morphogenetic forms appeared, there was no subsequent DNA condensation or particle maturation in the cytoplasts. These results suggest that vaccinia virus requires some factor or function from the host-cell nucleus in order to mature properly and produce infectious progeny virus.     

De novo formation of cytokeratin filaments in calf lens cells and cytoplasts after transfection with cDNAs or microinjection with mRNAs encoding human cytokeratins.

We have studied the formation of new intermediate-sized filaments (IFs) by human cytokeratins (CKs) 8 and/or 19 in cultured bovine lens cells stably transfected with the corresponding cDNAs under SV40 promoter control. In the transfected cells, polypeptides of both type I and type II CKs were synthesized to near-equimolar amounts, formed heterotypic complexes and assembled into IFs with a peculiar tendency to accumulate into variously sized, often roundish aggregates in the juxtanuclear region, usually one per cell. Electron microscopy of these large CK IF aggregates revealed typical 7 to 12-nm IFs, tightly packed together in an apparently haphazard mode. By immunoelectron microscopy, the CK IFs could be readily distinguished from the vimentin IFs which were abundant in these cells. Electron microscopy also showed that many of the CK IF aggregates were located in the vicinity of the nucleus but did not have direct contact with the nuclear envelope; moreover, their location did not regularly correspond to those of the centrosomes and the Golgi apparatus. During enucleation of transfected cells in the presence of cytochalasin B, the CK aggregates were often retained in the cytoplast. After microinjection of CK 8 and 19 mRNAs, synthesized in vitro from cDNA molecules, into enucleated cytoplasts prepared from untransfected cells, CK IFs similar to those observed in microinjected whole cells were formed but often showed a wider cytoplasmic distribution. Our observations indicate that typical CK IFs can form, in vivo, in the absence of any nuclear structures. We discuss possible reasons for the tendency of the CK IFs to accumulate, in this cell line, into a juxtanuclear aggregate, in relation to similar CK-IF aggregates formed in certain normal cell types and upon toxic damages.     

Karyoplast-cytoplast volume ratio in bovine nuclear transfer embryos: Effect on developmental potential

To evaluate the effect of karyoplast-cytoplast ratio on the development of nuclear transfer embryos, karyoplasts from day 4, day 5, and day 6 embryos were transferred to oocytes enucleated with different volumes of cytoplasm: Type 1, removal of a small volume of cytoplasm equivalent to the first polar body, Type 2, removal of a volume of cytoplasm approximately equal to the volume of the respective karyoplast, and Type 3, removal of half of the oocyte volume. In addition, the effect of experimental reduction of karyoplast cytoplasm was investigated in day 4 and day 5 karyoplasts. Intact day 4 karyoplasts fused to Type 3 cytoplasts did not support development to blastocysts, whereas these karyoplasts yielded blastocysts in combination with Type 1 (7%) and Type 2 cytoplasts (12%). After experimental reduction of cytoplasmic volume in day 4 karyoplasts, blastocysts (10%) were also obtained after fusion with Type 3 cytoplasts, probably due to reduction of cytoplasmic chimerism. With day 5 karyoplasts, blastocyst rate was higher in combination with Type 2 (34%) than with Type 1 (19%) and Type 3 cytoplasts (16%; P < 0.05). The use of day 6 intact karyoplasts resulted in a significantly (P < 0.05) higher proportion of blastocysts when fused with Type 2 (38%) or Type 1 cytoplasts (34%) than with Type 3 cytoplasts (16%). These results suggest that enucleation of oocytes with a volume similar to that of the respective karyoplast creates better conditions for cell cycle interactions with all types of karyoplasts than enucleation with minimal or large volume of cytoplasm. Mol. Reprod. Dev. 48:332–338, 1997. © 1997 Wiley-Liss, Inc.     

Intracellular location of unoccupied 1,25-dihydroxyvitamin D receptors: a nuclear-cytoplasmic equilibrium

In order to investigate the subcellular distribution of unoccupied 1,25-dihydroxyvitamin D3 receptors, highly purified cytoplasts and nucleoplasts were prepared from two kidney cell lines (PK1 and MDBK). This was accomplished utilizing the technique of enucleation by cytochalasin B and density gradient centrifugation. Unoccupied 1,25-dihydroxyvitamin D3 receptors were found in both the nuclear and cytosolic compartments, with approximately 70% of the receptors localized in the cytoplasm. When cells were pretreated with 1,25-[3H]dihydroxyvitamin D, prior to enucleation, it was found that 90% of the receptor-hormone complex was associated with nucleoplasts, thus demonstrating that cytochalasin B treatment does not alter the high-affinity association of the receptor-hormone complex with the nucleus. The ratio of unoccupied receptor/protein was found to be the same in whole cells, cytoplasts, and nucleoplasts for both cell types. The ratio of unoccupied receptor/DNA was highest in cytoplasts and lowest in nucleoplasts. Taken together, these data indicate that the unoccupied 1,25-dihydroxyvitamin D receptor is generally associated with cell proteins and not specifically associated with cell DNA. We therefore propose, at least for these cells, that the unoccupied 1,25-dihydroxyvitamin D receptor exists in equilibrium between the nuclear and cytosolic compartments of the whole cell, and receptor-hormone binding shifts this equilibrium to favor nuclear localization.     

Demecolcine- and nocodazole-induced enucleation in mouse and goat oocytes for the preparation of recipient cytoplasts in somatic cell nuclear transfer procedures

Treatment of pre-activated oocytes with demecolcine (DEM) has been shown to induce the extrusion of all oocyte chromosomes within the second polar body (PB2). However, induced enucleation (IE) rates are generally low and the competence of these cytoplasts to support embryonic development following somatic cell nuclear transfer (SCNT) is impaired. Here, we explored whether short treatments with DEM or another antimitotic, nocodazole (NOC), improve IE efficiency, and determined the most appropriate timing for nuclear transfer in the cytoplasts produced. We show, for the first time, that IE can be accomplished in mouse and goat oocytes using NOC and that short treatments with DEM or NOC result in similar IE rates, which proved to be strain- and species-specific. Because enucleation induced by both antimitotic drugs is reversible, the IE protocol was combined with the mechanical aspiration of PB2s to increase permanent enucleation rates in mouse oocytes. None of the cloned mouse embryos produced from the resultant cytoplasts developed to the blastocyst stage. However, when they were reconstructed prior to the activation and antimitotic treatment, their in vitro embryonic development was similar to that of cloned embryos produced from mechanically-enucleated oocytes.     

Protein synthesis in enuleated fertilized and unfertilized mouse eggs

Summary Fertilized and unfertilized C57BL/6J eggs were microsurgically enucleated and then analyzed for their capacity to synthesize proteins using 2-dimensional polyacrylamide gel electrophoresis. In both types of enucleated eggs (cytoplasts), protein synthesis continued and was still detected up to three days in culture. Shortly after enucleation, the pattern of polypeptides remained similar to the respective non-operated control eggs but it later became gradually reduced in intensity and complexity. After two days of culture the appearance of some new proteins typical for 2-cell embryos was observed in enucleated fertilized eggs only. Our findings suggest that maternal mRNA stored during oogenesis is utilized during the preimplantation period.     

Translation of RNAs Synthesized In Vivo and In Vitro from Bacteriophage SP82 DNA

The synthesis of 69 phage-specific polypeptides during the infection of Bacillus subtilis with bacteriophage SP82 was detected by pulse-labeling, one-dimensional electrophoresis, and autoradiography. SP82 virions were found to contain approximately 22 polypeptides, most of which were synthesized late in infection; evidence was obtained for the processing of the major virion protein. RNAs extracted at different times during infection were translated by using an Escherichia coli cell-free extract. Only smaller-molecular-weight peptides were produced efficiently in vitro; in the 9,000- to 60,000-molecular-weight range, 50 to 60% of the peptides synthesized in vivo were produced by translation of RNAs extracted from infected cells. Eight of the virion peptides were produced by in vitro translation of RNAs extracted from infected cells. RNAs were synthesized under defined conditions by RNA polymerase extracted from uninfected B. subtilis and by polymerases isolated from cells 8 and 20 min after infection with SP82. Translation of these RNAs yielded characteristic and different patterns of polypeptides. Nine of the 12 polypeptides produced by translation of RNAs synthesized by the host polymerase corresponded in mobility to peptides appearing in vivo in the 0 to 3 and 3 to 6 min intervals of pulse-labeling after infection; 12 of the 25 peptides synthesized from RNAs produced by polymerase extracted 8 min after infection corresponded in mobility to peptides detected in vivo 8 min after infection, and 15 of the 22 peptides directed by RNAs made by the polymerase isolated 20 min after infection corresponded to peptides present in vivo late in infection. Five of the peptides produced in vitro from the latter RNA corresponded to virion peptides.     

Purification and characterization of regenerating mouse L929 karyoplasts

Within 72–96 hr after preparation, about 10% of the karyoplasts made from mouse L929 cells regenerated to reform whole viable cells. As soon as 30 hr after preparation, however, nearly all of the remaining 90% of karyoplasts were dead. By separating living and dead karyoplasts at 30 hr, therefore, that fraction destined to complete regeneration was effectively purified. Complete separation was accomplished by sedimentation through Ficoll-paque (Pharmacia), a patented preparation originally developed for the separation of monocytes from whole blood. With the addition of this technique to the previously reported purification scheme for karyoplasts, various biochemical and morphological studies were attempted. Of particular importance are results indicating that karyoplasts that regenerate do not initially contain any more cytoplasm than the average karyoplast in a preparation—that is, about 10% of the cytoplasm within a whole cell. Electron microscopy of karyoplasts immediately after preparation indicated an unequal partitioning of cytoplasmic organelles at the time of enucleation. For example, karyoplasts initially contained about 11.4% of the mitochondrial volume of whole cells, but only 2.9% of the Golgi apparatus. The size of the karyoplasts and the volume occupied by a variety of organelles was followed throughout the process of regeneration. Although there was an approximately linear increase in the diameter of regenerating karyoplasts, there appeared not to be a simple concordant increase in the volume occupied by all cellular organelles. An extensive investigation was performed to determine whether or not karyoplasts contained centrioles. Immediately after enucleation, 15,000 random thin sections through karyoplasts, which represented about 100 complete bodies, were examined for the presence or absence of centrioles. No centrioles were observed. Examination of the cytoplasts revealed that they contained a sufficient number of centrioles to account for all of the centrioles that were present in the whole cells before enucleation. Centrioles were first detected in karyoplasts at 24 hr after preparation, about the same time that karyoplasts regained the ability to adhere to the surface of tissue culture dishes. At this time, however, the average karyoplast had less than one centriole. By 72 hr, the regenerated karyoplasts had approximately the same number of centrioles as whole cells.     

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.