Plasma membrane heterogeneity in ascites tumor cells. Isolation of a light and a heavy membrane fraction of the glycogen-free Ehrlich-Lettré substrain.

In this work we report on the isolation of two plasma membrane fractions of a glycogen-free substrain of Ehrlich-Lettré ascites cells, a light fraction sedimenting in a sucrose gradient at 1.10 g/ml, and a heavy fraction sedimenting at nuclei by a combination of short-term swelling and mild Dounce homogenization. A 12 000 X g postnuclear pellet (PII) containing major portions of the plasma membrane marker enymes, 5'-nucleotidase, ouabain-sensitive (Na+ + K+)-ATPase and the alkaline phosphatase, was prepared by differential centrifugation. The two plasma membrane fractions were obtained by centrifugation on a discontinuous sucrose gradient, from which they were further purified on a linear sucrose gradient applying sedimentation velocity conditions only. Enrichment factors for the three marker enzymes were between 5- and 14-fold for the light fraction and between 3- and 7-fold for the heavy fraction with an overall yield of 1--4% and 0.5--1.7%, respectively, of cellular protein. Contamination of both fractions with nuclear material was minor. Mitochondrial contamination was about 8% for the light material and somewhat higher for the heavy material. In the light fraction, co-sedimentation of lysosomal and Golgi marker enzymes was detected. The presence of membrane structures of these organelles could not be confirmed definitely by electron microscopy. Differences in sialic acid content and phospholipid composition within the two fractions, especially in the relative proportion of lecithin to sphingomyelin, suggests differences in membrane fluidity. The light material showed mostly unit membrane vesicles in thin-section and freeze-etch electron microscopy, whereas the heavy fraction mainly consisted of sheet-like membrane fragments.

Distinctive subcellular alterations induced by hypertonic stress in sea urchin eggs.

Sea urchin eggs continuously exposed to a hypertonic solution were ultrastructurally examined for osmotic-stress induced alterations. No fertilization membranes formed during the treatment and the surface-cortex complexes remained unaltered from the unfertilized state. However, the osmotic stress did induce a number of subcellular changes. During the first 30 minutes of the treatment the eggs formed many endoplasmic reticulum whorls and compacted Golgi body aggregations. Both of these new formations can be correlated with rapid changes in intracellular calcium, known to occur in hypertonic stressed eggs. Aggregations of mitochondria could be observed at later stages; these aggregations can also be related to subcellular stress and possible changes in internal calcium concentrations. The various morphological transitions within the cytoplasm, along with the lack of a cortical reaction in these eggs, not only supports the idea that calcium is released during parthenogenetic activation, but also suggests that this free calcium originates from stores other than the stores that are involved during fertilization or simple artificial activation.

Membranes of the mitotic apparatus of mammalian cells.

HeLa cells in mitosis are fixed by KMnO4 to obtain a good preservation of membranes. Inside and around the mitotic apparatus a vesicular, a tubular and a cisternal type of membranes are demonstrated. Small spherical or ellipsoidal vesicles are distributed at random in the spindle area. Zones around the poles and in the midbody of telophase cells are almost free from vesicles. Elements of the tubular ER are often aggregated in parallel arrays at the periphery of the spindle region, thus resembling dictyosomes. During telophase tubular ER is most prominent at the cleavage furrow. Layers of concentrically arranged flat cisternae surround the spindle region during the middle stages of mitosis. Radiating from the poles such membranes form large asters, thereby interrupting the concentric arrangement. This pattern might act as a barrier against larger cytoplasmic organelles or as a cisternal system to bring about an oriented transport of molecules from or to the poles. Fragments of the two large indentations of the nuclear envelops in prophase are aggregated in the spindle region and later on contribute to form new envelopes around the daughter nuclei.

Fine structure of the mitotic cycle of unfertilized sea urchin eggs activated by ammoniacal sea water.

Unfertilized sea urchin eggs enter a mitotic chromosome cycle after treatment with sea water containing ammonia. Centrioles cannot be found but microtubules are formed in the later stages of the cycle. The microtubules are displayed in an astral arrangement centered on clusters of osmiophilic bodies. In early stages, distinct kinetochores on the condensed chromosomes show no attachments to microtubules. Later, a few microtubules may be attached to the kinetochores. The chromosomes and microtubules are contained in a "clear zone", a large compact accumulation of membranes which displaces yolk particles and mitochondria, but not ribosomes, from that region of the cell. No bipolar spindle is formed.

Scanning electron microscopic observations on cells grown in vitro. V. Human diploid cells can attach also with their upper surface.

When small glass particles are seeded on top of human diploid cells in a monolayer the cells attach firmly to the glass with their upper surface. In combination with this attachment fingerlike protrusions and small leading lamellae are formed de novo from the normally completely protrusion-free upper surface. These results are in contradition to findings from Vasiliev and Gelfand [10]. Our results indicate that attachment and adhesiveness must not unequivocally be in relation to the presence and formation of cellular protrusions.

Scanning electron microscopic observations on cells grown in vitro. VIII. Cell-cell interactions between HeLa cells and WI38 fibroblasts.

Adherent HeLa 1 and non-adherent HeLa S cells were seeded onto a preexisting monolayer of WI38 fibroblasts. Both cell types were able to attach to and to spread on top of the fibroblasts. Furthermore, both cell types were able to migrate actively from these fibroblasts if they managed to contact the underlying glass support. Both cell types were capable of penetrating the monolayer, under-lapping the fibroblast cells and finally destroying the monolayer. The cells' behavior was different when the monolayer consisted of alcohol-fixed or irradiated WI38 cells. In this case spreading on top of the treated fibroblasts resembled the concentric spreading seen on glass or plastic. This was in contrast to the spreading on untreated cells where the tumor cells became more or less spindle shaped. Moreover, the HeLa cells appeared to be less mobile and destruction of the monolayer was never observed. From all this we concluded: i: HeLa 1 and HeLa S cells have more than one attachment mechanism. ii: the spreading behavior is strongly influenced by the underlying support. iii: the upper cell surface of fibroblasts supports the spreading and movement of other cell types. iv: movement of HeLa cells and consequently the destruction of the monolayer is promoted by the intact fibroblasts.

Scanning electron microscopic observation on cells grown in vitro. VI. aggregate formation in confrontation cultures of human diploid and tumor cells.

Two human cell lines, HeLa carcinoma cells and Wi38 lung fibroblasts have been used in confrontation experiments to study different adhesion processes including cellular motility, sorting out and invasion by SEM and statistics. Aggregates of one cell type were co-cultured with resuspended cells of the other type for up to 96 hours and vice versa. Homotypic aggregation kinetics as a control showed a saturation of the time-dependent increase in aggregate diameter and a mirror-like decrease in single cell number for both cell lines. In the heterotypic aggregation process, dissociated HeLa cells covered the surface of Wi38 aggregates without showing any particular distribution pattern. They partially penetrated the outer fibroblast layers and invasive actions were detected. Suspended Wi38 lung cells also adhered to the tumor cell aggregates. After a period of non-coordinated, random settlement (8 to 18 h or co-cultivation), the fibroblasts sorted out to form a network around the tumor aggregates in the form of sheets of cells arranged in parallel. The underlying HeLa cells then broke through the Wi38 layers, thus performing an inside-out invasion. Finally, all the aggregates were again covered by HeLa cells.

Fine structural studies of early mitotic stages in untreated and nocodazole-treated HeLa cells.

When cells in mitosis are treated with nocodazole, a microtubule-disrupting drug, it can be shown in comparison to untreated cells that microtubules are responsible for the polarized formation of indentations, folds, tubes, and crypts of the nuclear envelope in prophase nuclei. No translocation of chromosomes within the nucleus takes place. Microtubules are not necessary for chromosome condensation, nuclear envelope breakdown, the formation of trilaminar kinetochores, and the orientation of sister-kinetochores within one chromosome in relation to each other. The orientation of kinetochores in relation to the mitotic poles, however, is mediated by microtubules. The data shown here support the working hypothesis about chromosome translocation in prophase nuclei which was presented in an earlier paper.

Scanning electron microscopic observations on cells grown in vitro. VII. HeLa cells can penetrate Wi38 fibroblasts.

When HeLa cells are seeded on a preexisting monolayer of Wi38 fibroblasts, the HeLa cells "try" to get into direct contact with the glass substrate. Once on top of a flat fibroblast the HeLa cell can either migrate from the fibroblast to settle between two fibroblasts on glass or somehow stimulate the underlying cells to retract. In a few cases the HeLa cell directly penetrates the fibroblast, "melting" its way through the underlying cell. The mechanism of this phenomenon which has never been described before in this combination is discussed.

Nuclear asynchrony in multinucleate rat kangaroo cells.

Multinucleate (MN) cells were induced in PtK1 cells by colcemid treatment. A large percentage of cells developed nuclear asynchrony both in relation to DNA synthesis and mitosis within one cell cycle. Asynchrony could be traced even in metaphase and anaphase cells in which interphase nuclei, PCC of S-phase nuclei and less condensed prophase-like chromosomes could be observed along with normally condensed chromosomes. The occurrence of such abnormalities in these large MN cells may be explained on the basis of an uneven distribution of inducer molecules of DNA synthesis and mitosis due to cytoplasmic compartmentation. The less condensed form of all the chromosomes except chromosome 4 could be traced in asynchronous metaphase. The failure of the less condensed chromosomes to undergo complete condensation does not always appear to result from late entry of nuclei containing these chromosomes into G2 phase. It is likely that chromosome 4 carries gene(s) for chromosome condensation, as this chromosome itself never appears in a less condensed form. The inducers for chromosome condensation may not always be available at equal concentrations to all chromosomes located in separate nuclei, thus they may sometimes fail to undergo complete condensation before other nuclei reach the end of prophase, when the nuclear envelopes of all nuclei present in the cell break down simultaneously.

Localization of proteasomal antigens during different phases of the cell cycle in HeLa cells.

We localized two different proteasome-associated epitopes with the use of two specific monoclonal antibodies, MCP21 and anti-p25, in interphase and mitotic HeLa cells cultured in vitro. We provide evidence for an association of those antigens with the spindle poles in metaphase and with the midbody region in anaphase and telophase. Triton-nonextractable structures are also labeled in interphase. Moreover, the labeling pattern of interphase cells obtained with the monoclonal antibodies MCP21 and anti-p25 differs considerably, and in the case of MCP21 displays clear cell cycle-specific changes: nuclear labeling is absent or very weak in G1 and S phases of the cell cycle, whereas it is strong in G2 phase. This might reflect the existence of different subpopulations of proteasomes in the cell.

Mitosis arrested by deuterium oxide. Light microscopic, immunofluorescence and ultrastructural characterization.

Dynamics and correctness of mitotic division of PtK1 cells grown in vitro in the presence of deuterium oxide were assessed by morphological criteria. Incubation of PtK1 monolayers for 2 h in media supplemented with 25% to 75% D2O retarded in a dose-dependent manner the interphase/prophase transition, thus substantially decreasing the fraction of prophase cells. The progression of mitosis was also arrested at the metaphase/anaphase transition resulting in an accumulation of prometaphase and metaphase cells. After a prolonged incubation (12-24 h) in 75% D2O, prophase cells reappeared, often displaying a peculiar, "overmatured" pattern of chromosome condensation. In the presence of D2O an "overmatured" prophase cell is able to transform into a prometaphase cell and then directly into a multinucleate/micronucleate cell. Arrested prometaphase and metaphase cells developed restitution nuclei when incubated up to 24 h in the media containing 25% to 75% of heavy water. Immunofluorescence and electron microscopic studies revealed that the treatment with deuterium oxide profoundly disturbed development of the mitotic spindle. On the basis of these observations, a possible mechanism of the antimitotic action of deuterium oxide is discussed. We postulate that centrosomally controlled transformation of the microtubular cytoskeleton into the mitotic spindle is impaired by D2O. This delays the progression of prophase and disturbs congression of chromosomes into a regular metaphase plate.

Disorganization of mitosis in HeLa cells by deuterium oxide.

We assessed, by light and electron microscopy, the influence of deuterium oxide on the dynamics of mitosis and on the morphology of the mitotic apparatus in HeLa cells grown in vitro. A 2-h incubation of HeLa monolayers with low concentrations of D2O (1%-25%) in the medium increased the frequency of multipolar divisions up to 20 times the control level. Substitution of 10% and 25% D2O for H2O induced changes in the proportions of mitotic phases. These changes could be fully reversed to the control pattern after 1 h of recovery in non-deuterated medium. Fifty % D2O strongly inhibited, and 75% D2O blocked the cell cycle before prophase and at (pro-)metaphase. In cells treated with 50% D2O conspicuous morphological changes of the interphase chromatin as well as ultrastructural abnormalities of all mitotic phases were regularly observed. Overall, these results confirmed the antimitotic activity of deuterium oxide and revealed that it could also influence the cell cycle before mitosis. It is suggested that interference with diverse cellular constituents rather than a specific influence on microtubule turnover could be responsible for the disorganization of the cell cycle in HeLa cells by D2O.

The behavior of centrosomes in multinucleate cells formed after colcemid treatment.

Multinucleate PtK1 cells were generated by treating the cells with colcemid for up to 60 h. Cells with scattered chromosomes reconstructed nuclear envelopes around these chromosomes. After recovery of up to 36 h these multinucleate cells went into mitosis. In such cells mainly two types of spindles are found: a bipolar spindle with some "accessory" half-spindles and multipolar mitotic apparatus with several equally-sized half-spindles ordered in an irregular way. Ultrastructural studies revealed centrosomes within such spindles which had not developed a microtubular connection to chromosomes and obviously could not act as mitotic pole. This result is interpreted in the way that centrosomes undergo a maturation cycle. Immature centrosomes cannot form mitotic poles. The asynchrony of the cycles of the multiple centrosomes seems to be generated by an uneven distribution of special factor(s).

Lack of detectable kinetochores on some chromosomes in mouse x human hybrids.

When treated with an anti-kinetochore antibody present in the sera of scleroderma (var. CREST) patients, most chromosomes exhibit kinetochore dots at the position of the centromere. In this paper we report that some chromosomes in the mouse x human somatic cell hybrid fail to show these dots. In the early passages in a hybrid, HYG-1, the frequency of such chromosomes was higher (0.85%) than in later passages (0.45%) studied after five months of continuous culturing. In parallel, the mean number of human chromosomes in the hybrid also dropped. The somewhat hypodiploid parental cell lines, when similarly treated, showed only a rare chromosome without kinetochore dots. Immunoblots of the proteins showed that the sera used for kinetochore detection recognized all major centromere proteins (CENPs). Electron microscopy of some offlying metaphase chromosomes in another hybrid, HR61, exhibited a lack of trilamellar kinetochores. This study suggests that akinetochoric chromosomes might provide a novel mechanism responsible for chromosome loss and genesis of aneuploidy. In early passages, some cells in the hybrid showed detached kinetochores. These autonomous kinetochores could be seen in clusters and involved some mouse chromosomes also. Potential significance of these autonomous kinetochores in generating compound centromeres is discussed.

Fine structural studies of the bipolarization of the mitotic apparatus in the fertilized sea urchin egg. II. Bipolarization before the first mitosis.

After fusion of the two pronuclei the former sperm head centrosome is attached to the envelope of the zygote nucleus while the former mitochondrial centrosome is only loosely associated with it. These two centrosomes are not yet in opposite positions but are separated from each other by spreading centrosomal material. This spreading is mediated by microtubules. It is concluded that the attached centrosome remains stationary while the motile one is moved around the nuclear surface to an antipodal position, 180 degrees from the other. The first bipolarization process which occurs prior to the breakdown of the nuclear envelope is compared to the second and all other bipolarizations: Similarities and dissimilarities can be found, but similar or identical mechanisms for both processes are assumed.

Fine structural studies of the bipolarization of the mitotic apparatus in the fertilized sea urchin egg. I. The structure and behavior of centrosomes before fusion of the pronuclei.

During fertilization the sperm brings two centrosomes into the egg. One centrosome contains a centriole of normal length originally seen as the basal body of the sperm flagellum. Characteristically, the proximal half is enwrapped in osmiophilic material. This centrosome is attached to the centrosomal fossa, a bowl-shaped depression of the nuclear envelope of the male pronucleus. Microtubules radiate out from the osmiophilic half characterizing this structure as a centrosome and microtubule organizing center (MTOC). The second centrosome which also acts as an MTOC is attached to the mitochondrion of the sperm. At the beginning it appears as an unstructured accumulation of osmiophilic material out of which later on centriolar microtubules grow. Though this centrosome is marked by an immature centriole it is capable of organizing microtubules and of reproducing itself. This centrosome becomes loosely associated with the female pronucleus by means of microtubules. Then it separates from the mitochondrion which finally is lost. When the two pronuclei fuse, the centrosome derived from the basal body remains firmly attached to the centrosomal fossa, which has persisted in the envelope of the zygote nucleus after pronuclear fusion. Using the fossa as a marker of the position of this centrosome on the nuclear surface, we conclude that it is a stationary centrosome in the process of bipolarization for the first mitosis.

Proteasome activator (PA28) subunits, alpha, beta and gamma (Ki antigen) in NT2 neuronal precursor cells and HeLa S3 cells.

The catalytic activity of the 20S proteasome can be modulated by endogenous proteins. A proteasome activator protein termed PA28 or 11S regulator, composed of two homologous subunits (alpha and beta) and a separate but related protein termed Ki antigen or PA28gamma have been characterized. To explore the functional relationship of these proteins, NT2 clone D1 human neuronal precursor cells, as well as HeLa S3 cells were labeled by immunofluorescence and immunoelectron microscopy with three different antisera directed against peptides derived from their sequences. It was found that both PA28alpha and PA28beta antisera label the cytoplasm and the nucleoli. In contrast, the PA28gamma antiserum labels the nucleus but not the nucleoli while in the cytoplasm it labels two different classes of structures identified as microtubular-like extensions and inclusion bodies that are most likely autophagosomes. The latter do not contain proteasome delta subunit antigen. The microtubular-like structures colocalize with beta-tubulin, are dispersed by nocodazole and are not affected by brefeldin A treatment. PA28alpha and PA28beta are co-localized in the cell whereas PA28gamma has a different distribution. PA28gamma complexed with the proteasome may serve a function other than or in addition to activation and may also have a proteasome-independent function.

Deuterium oxide (heavy water) arrests the cell cycle of PtK2 cells during interphase.

Deuterium oxide (D2O, heavy water) exerts an antiproliferative effect on a variety of cells in vitro and on some organisms. This effect is mainly ascribed to a tubulin-mediated antimitotic action. We evaluated the morphology, the mitotic activity, and the dynamics of the cell cycle of PtK2 cells grown in vitro in the presence of 75% D2O for up to eight weeks by microspectrophotometric DNA measurements as well as flow cytometric analysis and a determination of mitotic indices. Substitution of heavy water for water in the culture medium initially increased the mitotic index by a (pro-) metaphase block but after 2 to 3 days of incubation no mitotic figures were seen. Analysis of cells grown for 6 days in medium containing 75% D2O revealed accumulation of cells in S/G2-phase. Extended treatment stabilized the high level of cells in this specific phase, when compared to normal growing cells. Cells grown for 1 to 6 weeks in the presence of D2O remained non-proliferating, nevertheless, they were able to divide again after recovery in non-deuterated medium. The time needed for resumption of the mitotic activity was proportional to the duration of deuterium oxide exposure. Cells incubated for 8 weeks in 75% D2O did not recommence mitotic activity. Light and electron microscopic examination revealed characteristic morphological changes of size and ciliation in PtK2 cells subjected to prolonged deuteration.(ABSTRACT TRUNCATED AT 250 WORDS)

An inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (20S proteasome) induces arrest in G2-phase and metaphase in HeLa cells.

HeLa cells were treated with different concentrations of an inhibitor of the proteasome chymotrypsin-like activity, the peptidyl aldehyde N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI). A detailed analysis, which included flow cytometry, cell counting and morphological assessment, was performed. PSI treatment induces a significant reduction of mitotic activity, accompanied by metaphase arrest of the mitotic cells. DNA flow cytometry shows an accumulation of the cells in G2+M phases of the cell cycle, which indicates the existence of a proteasome-mediated step in the G2-phase of the cell cycle. After removal of the inhibitor and supplementation with fresh medium, the cell cycle is resumed, but the mitotic cells show increased misalignment of chromosomes in the metaphase plate. PSI also induces HeLa cells to acquire a fibroblastoid phenotype.