Intracellular antigen migration in interspecific myoblast heterokaryons

Intracellular migration of species-specific nuclear antigens was studied in chick-rat heterokaryons. These cells were produced by virus-induced or spontaneous fusion of different chick cells with rat myoblasts or myotubes. Chick erythrocyte nuclei introduced into rat myogenic cells increased in volume and were reactivated to synthesize RNA. As the chick erythrocyte nuclei enlarged, they rapidly accumulated rat nuclear antigens. Rat nucleolar and nucleoplasmic antigens assumed a distribution in the chick nuclei corresponding to that in rat nuclei. In hybrid myotubes formed by the spontaneous fusion of chick myoblasts and rat myoblasts antigen exchange was at a much lower level. Some exchange of both rat and chick nuclear antigens could, however, be detected also in this system. Thus chick nuclear envelope and nucleolar antigens migrated into the rat myoblast nuclei and assumed an intranuclear localization analogous to that in chick nuclei. On the basis of these results it appears that antigenic nuclear macromolecules are constantly exchanged between the rat and chick nuclear compartments and the cytoplasm of the heterokaryon. During the rapid nuclear swelling which occurs when chick erythrocyte nuclei are activated in rat myoblast heterokaryons, the inward migration of rat nuclear antigens into the chick erythrocyte nucleus is more impressive than the migration of chick antigens into the rat nuclei.     

Nucleo-cytoplasmic protein migration during the activation of chick erythrocyte nuclei in heterokaryons

The reactivation of the chick erythrocyte nucleus was studied after erythrocytes were induced to fuse with rat epithelial cells in the presence of Sendai virus. The chick nucleus swells, shows an increase in dry mass and protein content and resumes RNA synthesis. Nucleoplasmic antigens characteristic of the rat cell are found to migrate into the erythrocyte nucleus. The rate of uptake of these molecules, which are believed to be proteins, appears to be directly related to increases in nuclear size, ³H-uridine incorporation and RNA polymerase activity. The polymerase activity which increases during the first days after cell fusion is sensitive to α-amanitin but relatively resistant to actinomycin D. At later time points there is an increase in α-amanitin resistant polymerase activity which probably reflects the appearance of ribosomal RNA synthesis.When heterokaryons containing different proportions of rat: chick nuclei are compared, reactivation is found to proceed most rapidly in those containing a high rat: chick nuclear ratio. As the number of erythrocyte nuclei in heterokaryons increases, the rate of reactivation in the individual nuclei is progressively reduced suggesting that the erythrocyte nuclei compete with each other for macromolecules of specific importance for the activation process.     

Ultrastructure of chick erythrocyte nuclei undergoing reactivation in heterokaryons and enucleated cells

The reactivation of chick erythrocyte nuclei after Sendai virus induced fusion of chick erythrocytes with intact or anucleate rat myoblasts or rat epithelial cells was studied by electron microscopy. Both in heterokaryons and in reconstituted cells formed by the fusion of chick red cells with anucleate rat L6 myoblasts the amount of highly condensed chromatin in the chick nuclei decreased with time after fusion at the same time as the proportion of dispersed chromatin increased. Nuclear organelles, typical of active nuclei but absent in the nuclei of unfused erythrocytes, appeared during reactivation. The percentage of chick nuclei containing a nucleolus was low 24 h after fusion but increased so that almost all nuclei contained one or more nucleoli 120 h after fusion. In reconstituted cells the frequency of nucleoli was much lower than in heterokaryons. In other respects, the erythrocyte nuclei introduced into anucleate rat cells underwent a normal reactivation and appeared to be well integrated with the cytoplasm. Thus, the nuclear envelope consisted of two normal leaflets in direct contact with the cytoplasm. Nuclear pores were observed in front of interchromatin channels. A normal cytoplasmic geometry appeared to be re-established since the Golgi apparatus occupied a position close to the poles of the chick nucleus.     

Immunological similarities between microsomal, cytosolic and nuclear progesterone receptors in the chick oviduct

Progesterone receptor of microsomal, cytosolic and nuclear fractions of the chick oviduct was studied by using biochemical, immunochemical and immunohistochemical analyses. In the oviducts of estrogen-treated immature chicks cytosolic, microsomal and nuclear PR were 90, 9.6 and 0.4% of the total binding, respectively, whereas the corresponding values 1 h after progesterone administration were 33, 6 and 61%, respectively. Progesterone decreased the cytosolic and microsomal PR 90 and 88%, respectively. All the receptor forms were similarly recognized by anti-PR-IgG raised against B-subunit of the PR. By using a sensitive immunoelectron microscopy in most cells of the oviduct only nuclear PR antigen was detected both in estrogen-treated and estrogen-progesterone-treated chick oviductal cells. In most cells no PR was found in the cytoplasm nor in the microsomes. Occasionally in very few cells small amounts of PR were found, associated with rough endoplasmic reticulum close to the nucleus containing a high concentration of the PR. This is probably due to a nascent synthesis of the PR. It is concluded that the major part of the cytosolic as well as microsomal PR is due to a homogenization artefact caused by a redistribution of the unoccupied PR located in the nuclei in situ.     

The distribution of two hnRNP-associated proteins defined by a monoclonal antibody is altered in heat-shocked HeLa cells

A monoclonal antibody obtained after mice were immunized with hnRNP purified from HeLa cells recognizes two polypeptides of Mr 35,000 and 37,000. By immunocytofluorescence, these antigens can be visualized only in cells previously heat shocked at 45 degrees C for 5 or 10 min, although they are present at the same level in unstressed and stressed cells. The signal, which is mostly concentrated in the interchromatin space, where hnRNP fibrils are located, does not accumulate with time and disappears 4 to 5 h after heat shock. Discrimination between the two types of hnRNP substructures, the 30-50 S monoparticles and the nuclear matrix fibrils, based on differential sensitivity to salt or ribonuclease treatment, showed that in unstressed cells the antigens behave as monoparticle proteins. In contrast, in heat-shocked cells, most 35-37K antigens behave as nuclear matrix proteins. Thus, heat shock seems to induce a rapid and reversible switch of these two antigens from hnRNP monoparticles to the nuclear matrix. The data demonstrate that heat shock, which was previously shown not to alter the overall RNA: protein packaging ratio of hnRNP, induces subtle modifications of their substructure. Such modifications might be of importance since heat shock is known for instance to affect pre-mRNA processing.     

Desmin filaments are stably associated with the outer nuclear surface in chick myoblasts

Eukaryotic cells have highly organized, interconnected intracellular compartments. The nuclear surface and cytoplasmic cytoskeletal filaments represent compartments involved in such an association. Intermediate filaments are the major cytoskeletal elements in this association. Desmin is a muscle-specific structural protein and one of the earliest known muscle-specific genes to be expressed during cardiac and skeletal muscle development. Desmin filaments have been shown to be associated with the nuclear surface in the myogenic cell line C2C12. Previous studies have revealed that mice lacking desmin develop imperfect muscle, exhibiting the loss of nuclear shape and positioning. In the present work, we have analyzed the association between desmin filaments and the outer nuclear surface in nuclei isolated from pectoral skeletal muscle of chick embryos and in primary chick myogenic cell cultures by using immunofluorescence microscopy, negative staining, immunogold, and transmission electron microscopy. We show that desmin filaments remain firmly attached to the outer nuclear surface after the isolation of nuclei. Furthermore, positive localization of desmin persists after gentle washing of the nuclei with high ionic strength solutions. These data suggest that desmin intermediate filaments are stably and firmly connected to the outer nuclear surface in skeletal muscles cells in vivo and in vitro.     

Dual location of MAR-binding, filament-like protein 1 in Arabidopsis, tobacco, and tomato

Matrix attachment region-binding filament-like protein 1 (MFP1) is a plant-specific long coiled-coil protein that binds double-stranded DNA. While originally identified as a component of the tobacco nuclear matrix, it was subsequently shown that the majority of MFP1 resides in mature chloroplast where it is located at the stroma side of the thylakoids and is able to bind to nucleoids. On the other hand, a 90 kDa MFP1-like protein from onion has been convincingly shown to be an intrinsic component of the onion meristematic nuclear matrix. Here, we have expanded the analysis of the subcellular location of MFP1 by using high-resolution confocal immunofluorescence microscopy and immunogold electron microscopy. Two different antisera raised against MFP1 from two species were used on isolated nuclei and chloroplasts from tomato, tobacco, and Arabidopsis. Our data show that both antibodies detect a signal in both compartments in all three species. An Arabidopsis MFP1 T-DNA insertional mutation abolishes both nuclear and chloroplast signals, indicating that the nuclear and plastidic antigens are derived from the same gene. We therefore suggest that MFP1 is a protein with a dual location, in both nuclei and chloroplasts, consistent with prior findings in onion and the dicot species investigated here.     

Tight association of DNA polymerase alpha with granular structures in the nuclear matrix of chick embryo cell: immunocytochemical detection with monoclonal antibody against DNA polymerase alpha

Immunofluorescent methods using a monoclonal antibody against chick DNA polymerase alpha and a rabbit antibody against chick DNA polymerase beta demonstrated that both DNA polymerases alpha and beta are present mainly in nuclei of cultured chick embryo cells. Fluorescence produced by anti-DNA polymerase alpha was more intense in the small granules than in other parts of the nucleus but, fluorescence produced by anti-DNA polymerase beta was distributed evenly in the nucleus. Cells first were treated with Nonidet P-40, followed by treatment with 50 micrograms/ml pancreatic DNase and 2 M NaCl in order to prepare the nuclear matrix. Fluorescence produced by anti-DNA polymerase alpha was still detectable in the granules after these treatments, but most of the fluorescence produced by anti-DNA polymerase beta disappeared. Our results indicate that a part of DNA polymerase alpha is tightly bound to a special structure present in the nuclear matrix which presumably is the DNA replication machinery.     

Immunofluorescent localization of the proteins of nuclear ribonucleoprotein complexes

Antibodies were raised in chickens against heterogeneous nuclear RNA (hnRNA)-binding proteins from 30S ribonucleoprotein (RNP) complexes of mouse Taper hepatoma ascites cell nuclei. The antibody preparations were characterized for immunological specificity and purity by double- diffusion gels, binding to specific bands in SDS polyacrylamide gels, and crossed immunoelectrophoresis. Antibodies raised against either whole 30S RNP complexes or purified RNP core proteins had a strong selective affinity for the four 34,000- to 40,000-dalton polypeptides which comprise the major structural proteins of hnRNP. The intracellular distribution of 30S RNP antigens in mouse ascites cells was determined by indirect immunofluorescence microsacopy. In interphase cells immunofluorescent sites were restricted to the nucleus, and nucleoli were free of fluorescence. The chicken anti-mouse- RNP antibodies were also able to react with cells from many different vertebrate species, showing a similar nucleus-restricted localization of the reacting sites. The antibodies also bound chick 30S RNP-proteins and reacted with the nuclei of chick cells. An exception to this was the failure of the antibody to bind to adult chick erythrocytes, suggesting that these major hnRNA binding proteins may be found only in nuclei capable of RNA synthesis.     

Phenotypic expression in chick erythrocyte X rat myoblast hybrids and in chick myoblast X rat myoblast hybrids

Attempts were made to reprogram chick erythrocyte nuclei to specify the synthesis of chick myosin. Chick erythrocytes were fused with rat myogenic cells with the aid of UV-inactivated Sendai virus. In the heterokaryons and hybrid myotubes which resulted from this fusion, the erythrocyte nuclei resumed RNA synthesis and formed nucleoli. Although some new chick antigens developed in those myotubes which contained fully reactivated chick erythrocyte nuclei, accumulation of chick myosin could not be detected by immunological methods. Neither small heterokaryons nor large hybrid myotubes which were actively synthesizing rat myosin reacted with antibodies directed against chick myosin. A small number of mononucleated cells, believed to be synkaryons formed by mitotic division of heterokaryons, did, however, react strongly with antibodies directed against chick myosin and showed a cross striation typical of skeletal muscle. The frequency of such cells was too low, however, to permit karyological analysis or further characterization of the antigen. Hybrids between chick myoblasts and rat myoblasts produced both chick and rat myosin thus indicating that simultaneous translation of chick and rat mRNA for myosin in a common cytoplasm was possible. In summary the evidence obtained suggested that reprogramming of chick erythrocyte nuclei, if it did occur in the present system, was a rare phenomenon.The possibility that hybrids between chick erythrocytes and rat myoblasts expressed markers typical of an erythroid phenotype was examined by immune staining with antibodies directed against chick haemoglobin. The results suggested that haemoglobin was introduced into hybrid cells by erythrocytes which failed to lyse before fusion. The intensity of this immune fluorescence decreased with increasing time after fusion. The rate at which this decrease occurred was not affected by inhibition of RNA synthesis. Thus, there was no evidence for the accumulation of haemoglobin in the hybrid cells.     

The effect of sodium tetrathionate stabilization on the distribution of three nuclear matrix proteins in human K562 erythroleukemia cells

Using both conventional fluorescence and confocal laser scanning microscopy we have investigated wheter or not stabilization of isolated human erythroleukemic nuclei with sodium tetrathionate can maintain in the nuclear matrix the same spatial distribution of three polypeptides (M_r 160 kDa and 125 kDa, previously shown to be components of the internal nuclear matrix plus the 180-kDa nucleolar isoform of DNA topoisomerase II) as seen in permeabilized cells. The incubation of isolated nuclei in the presence of 2 mM sodium tetrathionate was performed at 0° C or 37° C. The matrix fraction retained 20–40% of nuclear protein, depending on the temperature at which the chemical stabilization was executed. Western blot analysis revealed that the proteins studied were completely retained in the high-salt resistant matrix. Indirect immunofluorescence experiments showed that the distribution of the three antigens in the final matrix closely resembled that detected in permeabilized cells, particularly when the stabilization was performed at 37° C. This conclusion was also strengthened by analysis of cells, isolated nuclei and the nuclear matrix by means of confocal laser scanning microscopy. We conclude that sodium tetrathionate stabilization of isolated nuclei does not alter the spatial distribution of some nuclear matrix proteins.     

Electron microscopic study of preleptotene-stage oocyte nuclei in the prophase of meiosis in the hen

The morphology of oocyte nuclei at preleptotene and early leptotene stages of meiotic prophase I in the chick embryo was examined by electron microscopy and light cytochemistry. The intrenuclear fibrillar body (IFB) of proteinaceous nature is described. The IFB has a spherical or irregular form and consists of the disoriented fibrils ranging from 3 to 18 nm in diameter and of globules lying along the course of fibrils. The condensed chromatin in the oocyte nuclei at the early--late preleptotene stages and the thread-like chromosomes in the oocyte nuclei at the middle preleptotene--early leptotene stages either closely adjoin to IFB, localized in the centre of the nucleus, or are situated at some distance from it, and then the fibrils or fibrillar filaments are seen between chromosome material and IFB. The chromosomes are attached to IFV by the telomer or interstitial segment. The chromosomes lose the connection with IFB after the attachment of both the telomeres to the nuclear envelope (middle--late leptotene), and IFB removes from the centre of the nucleus to its periphery. It is supposed that IFB represents a nuclear skeleton element and takes part in the spatial organization of the chromosomes in the oocyte nuclei at the early stages of meiotic prophase I.     

The development of branched silk gland nuclei

Nuclei in the giant polyploid silk gland cells of Calpodes ethlius grow by endomitosis and can develop hundreds of branches during larval life. The shape of the these nuclei is characteristic for each region of the gland. We have found shape to be correlated with arrangement of the nuclear matrix. Scanning electron microscopy showed nuclear matrices with shapes similar to those of feulgen stained nuclei. Profiles of isolated matrices seen by transmission electron microscopy had filaments aligned parallel to the long axis of nuclear branches. DNA stained by Hoechst had a similar parallel alignment within the branches. Nuclear shape may be maintained by a small number of components, since electrophoretic analysis showed only a few abundant polypeptides in the matrix fraction. Silk gland nuclei have some of the same nuclear matrix antigens found in smaller, more regularly shaped, eukaryote nuclei.     

The influence of Triton X-100 on the nuclear envelope of the isolated liver cell nuclei.

In order to obtain more precise information on an eventual presence of extra-membranous lipids in the interior of the nucleus, the effects of Triton X-100 on the lipid content and ultrastructure of isolated rat liver nuclei was investigated. Enzyme markers (a.o. glucose-6-phosphatase) were used to control impurities of the nuclear fractions biochemically along with transmission electron microscopy and qualitative and quantitative light microscopy to check the condition of the nuclei obtained. Treatment of the nuclear fraction with increasing concentrations of Triton X-100 resulted in a decrease of the phospholipid content down to 25% at a Triton X-100/protein ratio of 0.4. A further decrease to 8% was measured at a ratio of 1.5. Electron microscopy of nuclei of the latter group showed nuclei containing outer membrane fragments in 2.5% of their surfaces. The composition of lipids extracted from a nuclear fraction appeared to be markedly changed after treatment with Triton X-100 with an increase of the percentage of neutral lipids and the phospholipids diphosphatidyl-glycerol and spingomyelin. From the chemical and morphological data obtained, the conclusion was drawn that a substantial part of the lipids remaining in the isolated nuclei after treatment with Triton X-100 is localized in both membranes of the nuclear envelope. It cannot however, be excluded that a small portion would be present in the interior of the nuclei.     

Nuclear transport as an ultimate step of multidrug resistance

Adriamycin (ADM) incorporation into nuclei of whole multidrug resistant (MDR) CEM cells is lower than into sensitive ones (S), that is mostly thought to be the consequence of a decrease of drug related to the activity of the multidrug resistance plasma membrane protein P 170. Isolated nuclei of the lymphoblastic tumor cell line CEM, which structures were controlled by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy, where incubated with 10(-6) mole/l of ADM. Incorporation into DNA was quantified by spectrofluorimetry. It was lower and slower into MDR nuclei than into S ones. Different modulators of active transport influence drug transfer into S nuclei and had no effect in MDR nuclei. The nuclear transfer into S nuclei appeared divided into two components: one was decreased by WGA, increased by cytosolic factors and an other part was purely passive in an identical intensity to MDR nuclei. Resistance of MDR nuclei seemed indebt to a defect, in these cells, of factors that mediate and/or activate nuclear transport of drug.