On the association of centrioles with the interphase nucleus.

Preparations of nuclei from rat liver and bovine spleen purified by centrifugation through dense sucrose solutions are shown to contain centrioles. These centrioles retain their in situ ultrastructure and are surrounded by a network of filaments adjacent to the nucleus and probably attached to it. The number of centrioles in isolated nuclei depends on the conditions of cell homogenization. Under certain conditions of homogenization, the fraction of purified nuclei contains almost all centrioles of the original tissue. The number of centrioles in isolated nuclei sharply decreases if the nuclei are rehomogenized under conditions that do not cause damage to nuclei. The number of nucleus-associated centrioles does not decrease after solubilization of nuclear membranes by Triton X-100. Nuclei retain the associated centrioles after treatmentwith RNase-free DNase I. It is concluded that in interphase the centrioles are associated with the nucleus and that this association which is probably mediated by filaments involves nuclear structures other than nuclear membranes or whole chromatin.     

Reorganization of the system of intermediate filaments and detection of the organization centers after centrifugation of cells attached to a substrate

Cultured pig kidney epithelial cells were centrifuged at 20,000 gav so that the centrifugation force was oriented parallel to the substrate, fixed and processed for indirect immunofluorescent staining with tubulin and vimentin antibodies. After a 2 hour centrifugation vimentin filaments aggregated in the centripetal parts of the cells (probably, because of their association with floating lipid vesicles). Microtubule-organizing centers were found near the centripetal poles of the nuclei, which migrated in the direction of the centrifugal force. The distribution of the cytoplasmic microtubules did not change during centrifugation. The staining of the cultures one hour after centrifugation revealed vimentin-containing spots with radiating intermediate filaments in most of the cells. These spots were localized near the cell nuclei; double immunofluorescent staining with tubulin and vimentin antibodies showed that their position was identical to that of the microtubule-organizing centers. Similar foci of vimentin filaments were seen in the cells after a 3-4 hour centrifugation. Probably, these structures participate in organizing the intermediate filament cytoskeleton in cells.     

INTERCELLULAR MIGRATION OF CENTRIOLES IN THE GERMARIUM OF DROSOPHILA MELANOGASTER : An Electron Microscopic Study

A cluster of centrioles has been found in the early Drosophila oocyte. Since the oocyte is connected to 15 nurse cells by a system of intercellular bridges or ring canals, the possibility that the cluster of centrioles arose in the germarium from an intercellular migration of centrioles from the nurse cells to the oocyte was analyzed in serial sections for the electron microscope. Initially, all of the 16 cells of the future egg chambers possess centrioles, which are located in a juxtanuclear position. At the time the 16 cell cluster becomes arranged in a lens-shaped layer laterally across the germarium, the centrioles lose their juxtanuclear position and move towards the oocyte. By the time the 16 cell cluster of cells is surrounded by follicle cells (Stage 1), between 14 and 17 centrioles are found in the oocyte. Later, these centrioles become located between the oocyte nucleus and the follicle cell border and become aggregated into a cluster less than 1.5 µ in its largest dimension. The fate of these centrioles in the oocyte is not known. The fine structure of the germarium and the early oocyte is also described.     

EFFECT OF CENTRIFUGATION ON THE DEVELOPMENT AND TIMING OF PREMITOTIC POSITIONING OF THE NUCLEUS IN ADIANTUM PROTONEMATA

When single-celled protonemata of Adiantum capillus-veneris L. were centrifuged immediately before transferring to darkness from continuous irradiation with red light, their nuclei were displaced basipetally. Both filamentous and branched protonemata were obtained. The stronger the centrifugal acceleration, the more frequently the branched protonemata were induced.
The effect of centrifugation at 1,300 x g for 15 min on nuclear displacement was different at different stages of the cell cycle. In early G₁ phase, the nucleus was easily displaced by centrifugation, but quickly returned to the original position after centrifugation. In late G₁ phase, the nucleus was displaced, but after centrifugation it never came back to the original position. In late G₂ and M phases, the nucleus was no longer displaced by the centrifugation. Premitotic positioning of the nucleus in cytokinesis took place about 5 hr before cell plate formation in all centrifugal treatments described above.     

The centriolar cycle in polykaryons containing prematurely condensed chromosomes or telophase-like nuclei]

In fused interphase-mitotic cells, either interphase nuclei are induced to premature chromosome condensation (PCC) or mitotic chromosomes are induced to telophase-like nuclei (TLN) formation. This study concerns structural and functional changes in centrioles of fused cells in which PCC or TLN are induced. Embryonic pig kidney cells were fused using a modified PEG-DMSO-serum method. Cell cycle period of the nuclei was determined before cell fusion using double-labeling autoradiography. Polykaryons containing desirable type of PCC or interphase nuclear combination in TLN were selected on the basis of isotope labeling after being embedded in epon. Selected cells were cut into serial sections and studied under electron microscope. The data obtained showed that centrioles at every interphase period undergo mitotic activation when their nuclei are induced to PCC. They acquire fibrillar halo and form half-spindles. Daughter centrioles at G1, S and G2 periods are also capable of mitotic activation when separated from their mother centriole. Inert centrioles were found in some cells with G1-PCC. When mitotic nuclei are induced to TLN formation, their centrioles also become inactivated. They lose fibrillar halo and mitotic spindles break down. Some mitotic centrioles develop features characteristic of interphase period such as satellites and vacuoles. Induced nuclear and centriolar changes are simultaneous and may be controlled by the same factor. Mitotic factor of mitotic cell partner which induces PCC may also induce interphase centrioles to mitotic activation. Degradation of the mitotic factor leading to TLN formation may also cause the loss of the mitotic activity of centrioles and disorganization of mitotic spindles.     

Effect of colchicine on rat mast cells

In the mast cell, a well-developed array of microtubules is centered around the centrioles. Complete loss of microtubules is observed when mast cells are treated with 10(-5) M colchicine for 4 h at 37 degrees C. The loss of ultrastructurally evident microtubules is associated with a marked change in the shape of mast cells from spheroids to highly irregular, frequently elongated forms with eccentric nuclei. In colchicine-treated cells the association of nucleus, Golgi apparatus, and centrioles is also lost. Mast cells exposed to 10(-5) M colchicine for 4 h at 37 degrees C retain 80% of their capacity to release histamine when stimulated by polymyxin B. Exocytosis is evident in stimulated cells pretreated with colchicine and lacking identifiable microtubules. When the conditions of exposure of mast cells to colchicine are varied with respect to the concentration of colchicine, the length of exposure, and the temperature of exposure, dissociation between deformation of cell shape and inhibition of histamine secretion is observed. These observations indicate that microtubules are not essential for mast cell histamine release and bring into question the assumption that the inhibitory effect of colchicine on mast cell secretion depends on interference with microtubule integrity.     

MITOSIS IN THE AQUATIC FUNGUS RHIZOPHYDIUM SPHEROTHECA (CHYTRIDIALES)

The structure of centric, intranuclear mitosis and of organelles associated with nuclei are described in developing zoosporangia of the chytrid Rhizophydium spherotheca. Frequently dictyosomes partially encompass the sides of diplosomes (paired centrioles). A single, incomplete layer of endoplasmic reticulum with tubular connections to the nuclear envelope is found around dividing nuclei. The nuclear envelope remains intact during mitosis except for polar fenestrae which appear during spindle incursion. During prophase, when diplosomes first define the nuclear poles, secondary centrioles occur adjacent and at right angles to the sides of primary centrioles. By late metaphase the centrioles in a diplosome are positioned at a 40° angle to each other and are joined by an electron-dense band; by telophase the centrioles lie almost parallel to each other. Astral microtubules radiate into the cytoplasm from centrioles during interphase, but by metaphase few cytoplasmic microtubules are found. Cytoplasmic microtubules increase during late anaphase and telophase as spindle microtubules gradually disappear. The mitotic spindle, which contains chromosomal and interzonal microtubules, converges at the base of the primary centriole. Throughout mitosis the semipersistent nucleolus is adjacent to the nuclear envelope and remains in the interzonal region of the nucleus as chromosomes separate and the nucleus elongates. During telophase the nuclear envelope constricts around the chromosomal mass, and the daughter nuclei separate from each end of the interzonal region of the nucleus. The envelope of the interzonal region is relatively intact and encircles the nucleolus, but later the membranes of the interzonal region scatter and the nucleolus disperses. The structure of the mitotic apparatus is similar to that of the chytrid Phlyctochytrium irregulare.     

The centriolar cycle in polykaryons formed in the fusion of heterophasic cells]

In this work we studied the centriolar cycle in fused cells containing heterophasic nuclei. Embryonic pig kidney cells were double-labeled with 3H- and 14C-thymidine and fused using a PEG-DMSO-serum method (Manandkhar et al., 1991). Fused cells containing nuclei at various cell cycle periods were selected after embedding in epon on the basis of isotope marking. Ultrastructure of centrioles was studied in serial ultrathin sections of selected cells. Centrioles of cells fused at different interphase periods showed a tendency to become synchronized in a manner similar to that of the nuclei. The G1-cell partners suppressed replication of S-centrioles or induced disorientation of centrioles of G2-diplosomes. In G1-S fused dikaryons, procentriole formation in G1-centrioles was not observed. This indicates the absence in the S-cell partner of an excess of a factor which could induce replication of the G1-centriole. However, G2-cell partner stimulated procentriole formation in G1- or unreplicated early S-centrioles. Asynchronous replication of G1-, S- and G2- centrioles was observed in some G1-S, G1-G2 and S-G2 fused cells. Heterophasic cellular environment containing mixed cell cycle factors appears to be responsible for the opposite effects on the structure of centrioles. In oocytes or early embryonic cells, due to the presence of a large amount of centriolar precursor material, centriolar replication cycle can proceed independently of the synthetic activity controlled by the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)     

A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation

We have isolated a nucleus-basal body complex from Chlamydomonas reinhardtii. The complex is strongly immunoreactive to an antibody generated against a major protein constituent of isolated Tetraselmis striata flagellar roots (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, J. Cell Biol., 99:962-970). Electrophoretic and immunoelectrophoretic analysis indicates that, like the Tetraselmis protein, the Chlamydomonas antigen consists of two acidic isoforms of approximately 20 kD. Indirect immunofluorescent staining of nucleus- basal body complexes reveals two major fibers in the connector region, one between each basal body and the nucleus. The nucleus is also strongly immunoreactive, with staining radiating around much of the nucleus from a region of greatest concentration at the connector pole. Calcium treatment causes shortening of the connector fibers and also movement of nuclear DNA towards the connector pole. Electron microscopic observation of negatively stained nucleus-basal body complexes reveals a cluster of approximately 6-nm filaments, suspected to represent the connector, between the basal bodies and nuclei. A mutant with a variable number of flagella, vfl-2-220, is defective with respect to the nucleus-basal body association. This observation encourages us to speculate that the nucleus-basal body union is important for accurate basal body localization within the cell and/or for accurate segregation of parental and daughter basal bodies at cell division. A physical association between nuclei and basal bodies or centrioles has been observed in a variety of algal, protozoan, and metazoan cells, although the nature of the association, in terms of both structure and function, has been obscure. We believe it likely that fibrous connectors homologous to those described here for Chlamydomonas are general features of centriole-bearing eucaryotic cells.     

The Two Nuclei in the Dikaryon of the Homobasidiomycete Coprinus cinereus Change Position after Each Conjugate Division

We constructed a common-AB diploid strain of Coprinus cinereus and mated this to a compatible haploid strain to construct a diploid-haploid dikaryon. We examined the positions of the diploid and haploid nuclei in the apical and subapical cells of the dikaryon by fluorescence microscopy and microfluorometry. In 60% of apical cells the leading nucleus (the nucleus proximal to the hyphal apex) was diploid and the second nucleus (the nucleus distal to the apex) was haploid, whereas in the remaining 40% of apical cells the order of the two nuclei was reversed. It was also observed that in 97% of hyphae examined the order of the diploid and haploid nuclei was reversed between the apical cell and the subapical cell. Based on these observations, we conclude that the two nuclei alternate in taking the leading and second positions in the apical cell at almost every conjugate division in the dikaryon. Copyright 1998 Academic Press.     

The differentiation of monocytes into macrophages,epithelioid cells,and multinucleated giant cells in subcutaneous granulomas

The morphological changes occurring in monocytes during their differentiation into macrophages, epithelioid cells, Langhans-type giant cells, and foreign-body-type giant cells were investigated in foreign-body granulomas induced by subcutaneous implantation of pieces of Melinex plastic. Analysis based on Adams's (1974) criteria for discrimination between the several types of cell of the monocyte line, showed that each type has a characteristic type of granule. Primary and secondary granules, numerous in the Golgi area of monocytes were generally found close to the cell membrane and decreased in number in maturing macrophages. This was accompanied by an increase in the number of microtubules. Mature macrophages show numerous characteristic macrophage granules, which are round (average diameter: 280 nm) and have a halo between the limiting membrane and granular matrix. Mature epithelioid cells have characteristic epithelioid cell granules, and multinucleated giant cells a heterogenous population of granules. Fusing macrophages generally have their Golgi areas facing each other, and also show a reduced thickness of the cell coat. The morphology of the multinucleated giant cell is closely related to the number of nuclei present. In Langhans-type giant cells, which generally have two to ten nuclei, a giant centrosphere with numerous aggregated centrioles is found. In transition forms between Langhans-type and foreign-body-type giant cells, which generally contain 10--30 nuclei, the centrioles show less aggregation. In the foreign-body-type giant cells, which generally have more than 30 nuclei, centrioles are virtually absent and never aggregated. These differences between the Langhans-type giant cells, the foreign-body-type giant cells, and the transition forms, support our previous finding that Langhans-type giant cells are the precursors of foreign-body-type giant cells.     

NUCLEAR MEMBRANE FUSION IN FERTILIZED LYTECHINUS VARIEGATUS EGGS

Fusion of apposed nuclear envelopes is frequently seen at telophase during postmitotic reorganization of the nucleus, but only rarely at other times in the cell cycle. We attempted to define an experimental system for studying changes in the nuclear envelope related to the cell cycle by varying the time of pronuclear apposition in fertilized Lytechinus variegatus eggs. This approach was based on the assumption that the period from fertilization to metaphase of the first cleavage division corresponds to the period from telophase to metaphase in the generalized cell cycle. The experimental approach used was to block the movement of the pronuclei with Colcemid and then to release this block at varying times after insemination by photochemically inactivating the Colcemid. The results show that apposed pronuclear envelopes can fuse from soon after insemination until the anticipated time of prometaphase. Fusion occurred in about 3 min as scored by light microscopy and this time did not vary significantly with the time after insemination. The potential for nuclear fusion is not restricted to pronuclei alone since diploid nuclei in binucleate cells could be fused using centrifugation in solutions of Colcemid to bring the nuclei into apposition. It is suggested that the potential for nuclear fusion is not necessarily related to the cell cycle and that modification of the nuclear envelope, possibly by association with chromatin or other fibrous material restricts nuclear fusion in most multinucleated cells.     

Involvement of Microtubules on Nuclear Positioning during Apical Growth in Adiantum protonemata

The position of the nucleus during apical growth of a single-celledprotonema in Adiantum capillus-veneris under continuous redlight was observed to find whether any cytoskeletons were involvedin determining its location. The nucleus migrated through thefilamentous cell keeping a constant distance of ca. 55 µmfrom the tip, but was not able to maintain this position inthe presence of colchicine. The nuclei in most cells could bedisplaced by centrifugation at 110?g for 15 min in the presenceof anti-micro-tubule drugs such as colchicine, ethyl N-phenylcarbamateand griseofulvin, but not when these drugs were absent. Similartreatment with cytochalasin B did not cause the displacing effect.These results suggest that microtubules have a role determiningthe position of the nucleus when it migrates during apical growth. ¹ Present address: Department of Developmental Biology, ResearchSchool of Biological Sciences, The Australian National University,Canberra City, A.C.T. 2601, Australia. (Received November 27, 1984; Accepted February 18, 1985)     

MICROTUBULAR ORGANIZATION IN ELONGATING MYOGENIC CELLS

Microtubule organization has been studied in serially sectioned myogenic cells in the tail muscle regeneration blastema of Rana pipiens tadpoles. In mesenchymal cells and in some premyoblasts, microtubules radiate from centriolar satellites in a cell center, while in more mature myoblasts and myotubes the centrioles no longer appear to serve as organizing centers for microtubules. In all elongate, fusiform myogenic cells, the microtubules are predominately oriented in the longitudinal axis of the cell. Counts of microtubules in transverse sections spaced at regular intervals along the cells show that the absolute number of microtubules is greatest in the thickened midregions of the cells and decreases relatively smoothly toward the tapered ends of the cells. Close paraxial association of microtubules (within 40 nm surface-to-surface) occurs along the entire lengths of cells but appears with greatest frequency in their tapered ends. In two myoblasts, serial sections were used to trace all microtubules in 8-µm long segments of the cells located about midway between the nucleus and one end of the cell. Since tracings show that as many as 50% of the microtubules terminate within the 8-µm long segment, it seems unlikely that any microtubules extend the entire length of the cell. It is proposed that lateral interactions between paraxial microtubules stabilize the overall microtubular apparatus and contribute to maintenance of the bipolar form of the cells. A three-dimensional model of the complete microtubular array in one of the 8-µm long segments of a myoblast has been constructed. The model reveals that a few microtubules within the segment are bent into smooth curves and loops that could be generated by sliding interaction between paraxial microtubules.     

A study of the glycerol phosphate acyltransferase and dihydroxyacetone phosphate acyltransferase activities in rat liver mitochondrial and microsomal fractions. Relative distribution in parenchymal and non-parenchymal cells, effects of N-ethylmaleimide, palmitoyl-coenzyme A concentration, starvation, adrenalectomy and anti-insulin serum treatment.

1. GPAT (glycerol phosphate acyltransferase) and DHAPAT (dihydroxyacetone phosphate acyltransferase) activities were measured both in subcellular fractions prepared from fed rat liver and in whole homogenates prepared from freeze-stopped pieces of liver. 2. GPAT activity in mitochondria differed from the microsomal activity in that it was insensitive to N-ethylmaleimide, had a higher affinity towards the palmitoyl-CoA substrate and showed a different response to changes in hormonal and dietary status. 3. Starvation (48 h) significantly decreased mitochondrial GPAT activity. The ratio of mitochondrial to microsomal activities was also significantly decreased. The microsomal activity was unaffected by starvation, except after adrenalectomy, when it was significantly decreased. Mitochondrial GPAT activity was decreased by adrenalectomy in both fed and starved animals. 4. Acute administration of anti-insulin serum significantly decreased mitochondrial GPAT activity after 60 min without affecting the microsomal activity. 5. A new assay is described for DHAPAT. The subcellular distribution of this enzyme differed from that of GPAT. The highest specific activity of DHAPAT was found in a 23 000 gav. pellet obtained by centrifugation of a post-mitochondrial supernatant. This fraction also contained the highest specific activity of the peroxisomal marker uricase. DHAPAT activity in mitochondrial fractions or in the 23 000 gav. pellet was stimulated by N-ethylmaleimide, whereas that in microsomal fractions was slightly inhibited by this reagent. The GPAT and DHAPAT activities in mitochondrial fractions had a considerably higher affinity for the palmitoyl-CoA substrate. 6. Total liver DHAPAT activity was significantly decreased by starvation (48 h), but was unaffected by administration of anti-insulin serum. 7. The specific activities of GPAT and DHAPAT were lower in non-parenchymal cells compared with parenchymal cells, but the GPAT/DHAPAT ratio was 5--6-fold higher in the parenchymal cells.     

Behavior of the cell center during epithelial cell spreading

In the epithelial cells of mouse embryo renal channels, centrioles are located near the plasma membrane of the apical part of the cell. In most of the cells an active centriole carries a cilium, which comes out into the channel lumen. In the epithelial cells, suspended after trypsinisation and in single cells adhering to the substrate, the centrioles are located near the nucleus, and the outcoming cilia are not observed. In the spread cells of epithelial islets, the centrioles are also found near the nucleus, and in most cases an active centriole carries a cilium, which comes out of the cytoplasm at the upper side of the cell. In the peripheral cells of the islet, centrioles are positioned between the nucleus and the active edge of the cell. In the epithelial cells in situ, a relatively small number of microtubules radiate from the active centrioles. In the suspended cells, the activation of microtubule formation is observed in the cell center. In the spread cells of the epithelial islets there occurs a further increase in the number of microtubules radiating from the active centrioles. In the peripheral cells which cause translocation of the epithelial islet in the culture, the number of microtubules, radiating from the centrioles does not differ significantly from that of the inner cells of the islet. The cell center of the epithelial cells does not seem to be actively involved in the locomotion of the epithelial cells in the culture.     

Subcellular localization of gamma-aminobutyrate transaminase and glutamate dehydrogenase in adult rat brain. Evidence for at least two small glutamate compartments in brain.

The subcellular localizations of gamma-aminobutyrate transaminase (EC 2.6.1.19) and glutamate dehydrogenase (EC 1.4.1.2) in brain tissue of adult rats were compared with each other and with those of NAD+-isocitrate dehydrogenase (EC 1.1.41) and monoamine oxidase (EC 1.4.3.4; kynuramine as substrate). Crude mitochondrial fractions from brain tissue were centrifuged in continuous sucrose density gradients. gamma-Aminobutyrate transaminase and glutamate dehydrogenase were always found at a higher density than NAD+-isocitrate dehydrogenase and monoamine oxidase. When centrifuged for 1 h at 53 000gav., there was a slight difference between the distribution profiles of glutamate dehydrogenase and gamma-aminobutyrate transaminase. This difference was larger when the centrifugation time was only 15 min. It is concluded that there are subpopulations of brain mitochondria with differing proportions of gamma-aminobutyrate transaminase and glutamate dehydrogenase. The results are discussed in relation to evidence obtained with labelled precursors in vivo that there are at least two small glutamate compartments in adult brain.     

Physical and Functional Interactions between the Herpes Simplex Virus UL15 and UL28 DNA Cleavage and Packaging Proteins

Herpes simplex virus (HSV) DNA is cleaved from concatemers and packaged into capsids in infected cell nuclei. This process requires seven viral proteins, including UL15 and UL28. UL15 expressed alone displays a nuclear localization, while UL28 remains cytoplasmic. Coexpression with UL15 enables UL28 to enter nuclei, suggesting an interaction between the two proteins. Additionally, UL28 copurified with UL15 from HSV-infected cells after ion-exchange and DNA affinity chromatography, and the complex sedimented as a 1:1 heterodimer upon sucrose gradient centrifugation. These findings are evidence of a physical interaction of UL15 and UL28 and a functional role for UL15 in directing UL28 to the nucleus.     

Effect of cytosol on DNA synthesis in isolated HeLa cell nuclei

Cytosol obtained by centrifugation of cytoplasm from synchronized S-phase HeLa cells at 200 000 × g for 30 min had a stimulatory effect on the rate and extent of DNA synthesis in isolated nuclei. The cytosol preserved the ability of isolated nuclei to initiate early nascent intermediates (primary DNA pieces). The stimulatory activity was partially separated from the DNA polymerase activity present in the cytosol.