Biomedical subjects
Publications and source records attributed to N Paweletz.
Cells treated with 2,4-dinitrophenol, a metabolic inhibitor, show a strong retardation of anaphase movement. At the ultrastructural level these cells reveal nuclear envelope reformation without concurrent decondensation of the chromosomes which indicates that these are possibly two dissociable late mitotic events.
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.
The interaction between centrosomes and kinetochores was studied in multinucleate cells induced by Colcemid treatment or by random cell fusion. Except for prematurely condensed chromosomes (PCC) of the G2-phase, PCCs do not develop their own spindle area. Perhaps the maturation promoting factor (MPF) fails to activate these centrosomes. In such PCCs, the kinetochore-centrosome interaction was found to be non-specific: sometimes only a few chromosomes of a group could establish connections with centrosomes, sometimes chromosomes from the same PCC group developed microtubule (MT) attachment with different centrosomes (not the pair), and sometimes kinetochores of PCC groups failed to interact with MTs. These findings explain the abnormal mitotic behaviour of PCCs as seen in the light microscope. These PCCs develop micronuclei or normal nuclei by nuclear re-formation in telophase. All the different PCC groups revealed kinetochores with kinetochore plates. It was shown that transformation of presumptive kinetochores to a trilaminar kinetochore does not depend on nuclear envelope breakdown or on the degree of chromosome condensation. This may be induced by the MPF which may initiate different events like chromosome condensation, nuclear envelope breakdown and kinetochore transformation by secondary factors. Other observations like establishment of connections by different chromosome groups to a common centrosome, kinetochore attachment of PCCs to different centrosomes, interaction of one kinetochore with two centrosomes, kinetochores being stretched and bent to receive microtubules and finally the failure of some kinetochores to develop MT attachment, all strongly suggest that the kinetochores serve as the point of termination rather than the nucleation sites of kinetochore MTs.
In large multinucleate cells the nuclei enter mitosis and reach metaphase almost synchronously by interaction of the different parts of the cell, but some degrees of postmetaphase asynchrony still persist. Apart from chromosome movements, the important postmetaphase events are re-formation of the nuclear envelope, chromosome decondensation, and back-formation of the spindle. From ultrastructural studies of multinucleate cells showing asynchronous mitotic progression beyond metaphase, we observed that nuclear envelope re-formation takes place nearly synchronously in all chromosome groups as soon as one group has reached telophase and while others are still in earlier mitotic stages. This indicates that nuclear envelope re-formation is an inducible event independent of the degree of condensation or decondensation of the chromatin and may depend on a factor(s) opposite in behavior to the maturation-promoting factor.
We describe a technique by which macromolecules can be microinjected into mammalian cells in monolayer cultures. This technique employs erythrocyte ghosts as the vehicle for microinjection, calcium as attachment agent and polyethylene glycol as fusogen. The use of calcium allows a reduction of the time of exposure to polyethylene glycol, and results in a high injection efficiency and a high cell viability when followed by incubation in a buffer free of divalent cations. Injecting over 90% of the cells, a reduction of cell viability is not observed and the mitotic index is never lower than 2.3%. Light and electron microscopy suggest that erythrocyte ghost-cell fusion is only a short event.
The kinetochore plate which develops after nuclear envelope breakdown in normal cells can be seen to be formed on condensed chromosomes still enclosed in the nuclear envelope in fused multinucleate cells where some nuclei show delayed envelope breakdown caused by nuclear interaction. This suggests that neither nuclear envelope breakdown nor assembly of microtubules is directly related to the formation of the kinetochore plate. Furthermore, it can be clearly observed in these cells that the kinetochores do not have any special association with the nuclear envelope.
We have located the active ribosomal cistrons together with their primary transcripts and the associated proteins and enzymes forming the typical 'christmas tree' configuration in the dense nucleolar components. Our results can thus correlate the structural and functional organization of the nucleolus.
A mouse monoclonal antibody generated against Drosophila intermediate filament proteins (designated Ah6/5/9 and referred to herein as Ah6) is found to cross-react specifically with centrosomes in sea urchin eggs and with a 68-kDa antigen in eggs and isolated mitotic apparatus. When preparations stained with Ah6 are counterstained with a human autoimmune serum whose anti-centrosome activity has been established, the immunofluorescence images superimpose exactly. A more severe test of the specificity of the antibody demands that it display all of the stages of the centrosome cycle in the cell cycle: the flattening and spreading of the compact centrosomes followed by their division and the establishment of two compact poles. The test was made by an experimental design that uses a period of exposure of the eggs to 2-mercaptoethanol. This treatment allows observation of the stages of the centrosome cycle--separation, division, and bipolarization--while the chromosomes are arrested in metaphase. Mitosis is arrested in the presence of 0.1 M 2-mercaptoethanol. Chromosomes remain in a metaphase configuration while the centrosomes divide, producing four poles perpendicular to the original spindle axis. Microtubules are still present in the mitotic apparatus, as indicated by immunofluorescence and transmission electron microscopy. When 2-mercaptoethanol is removed, the chromosomes reorient to the poles of a tetrapolar (sometimes tripolar) mitotic apparatus. During the following cycle, the blastomeres form a monopolar mitotic apparatus. The observations of the centrosome cycle with the Ah6 antibody display very clearly all the stages that have been seen or deduced from work with other probes. The 68-kDa antigen that reacts with the Ah6 monoclonal antibody to Drosophila intermediate filament proteins must be a constant component of sea urchin centrosomes because it is present at all stages of the centrosome cycle.
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.
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.
Latrunculin A, a marine toxin from a Red Sea sponge, is a potent inhibitor of the microfilament-mediated processes of fertilization and early development in sea urchins and in mice. Sperm from sea urchins, but not those from Limulus or mice, were affected by latrunculin, and fertilization in both sea urchins and in mice was arrested but at different stages. Sea urchin sperm treated with 2.6 microM latrunculin are unable to assemble acrosomal processes and their ability to fertilize eggs is impaired. The unwinding of the Limulus sperm acrosomal process occurs in the presence of latrunculin. Treated mouse sperm are able to fertilize mouse oocytes in vitro, suggesting that microfilaments may not be required in this mammalian sperm. In sea urchin eggs, sperm incorporation, microvillar elongation and cytokinesis are inhibited. Microtubule-mediated motility occurs normally. 20 nM latrunculin prevents the morphogenetic movements during gastrulation. It reduces the viscosity of actin gels from sea urchin egg homogenates. In unfertilized mouse oocytes, it prevents the colcemid-induced dispersion of the meiotic chromosomes; accumulations of cortical actin are noted adjacent to the scattered chromosomes. Sperm incorporation during mouse fertilization in vitro is unaffected suggesting that sperm entry may occur independent of microfilament activity in mammals. However, the apposition of the pronuclei at the center of the egg cytoplasm does not occur, providing evidence that cytoplasmic microfilaments may be required for the motions leading to pronuclear union during mouse fertilization. It inhibits the second polar body formation and cytokinesis. These results indicate that latrunculin is a potent inhibitor of microfilament-mediated processes in sperm, eggs and embryos, and that it may prove to be a powerful new drug for exploring the cellular behavior of microfilaments in the maintenance of cell shape and during motility.
Aggregates of normal human Wi38 cells are used as a three dimensional substrate to test in vitro the behavior of rat tumor cells which exhibit different invasive and metastatic capabilities in vivo. The invasive but non metastatic tumor cells colonize the Wi38 cell aggregates, invade and destroy them within three days. The non invasive but highly metastatic tumor cells settle in a limited number on the aggregates but show no further activities. Co-cultivation of these tumor cells with cell suspension of single Wi38 cells under aggregation conditions does not hinder the Wi38 cells in forming aggregates. The results show that the invasive process in the metastatic cascade needs more specific reaction partners and host environment than local tumor growth. The conditions of the first process cannot be mimicked by a simple model.
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The highly metastatic and nonmetastatic variants of the rat tumor BSp73 have been tested for motility in vitro by a phagokinetic assay. Surprisingly, the nonmetastatic variant is locomotional, whereas the highly metastatic variant exhibited stationary motility only. Soluble factors of normal rat serum and medium conditioned by regenerating normal rat lung, or solid constituents of the extracellular matrix material which was exudated by normal rat lung fibroblasts or bovine cornea endothelial cells as well as collagen type III could not stimulate locomotory ability in these tumor cells. However, by contact to rat lung cells they acquired passive mobility. The significance of these cellular properties for their metastatic behavior is discussed.
This investigation focuses on the identification, distribution, and transport of intracellular membrane systems during mitosis. The membranes of the Golgi apparatus can be identified cytochemically by staining for acid phosphatase (acPase) and thiamine pyrophosphatase (TPPase) activity. Using this approach we are able to study the disintegration of the Golgi apparatus during mitosis and to follow the dislocation as well as the organized reappearance of Golgi elements after the completion of mitosis. We are able to demonstrate that during mitosis the activity of both enzymes is strong enough to react with the substrate applied during the staining procedure. Furthermore, we observe a characteristic pattern of membrane distribution in mitotic cells. During interphase the TPPase reaction is characteristically limited to one or two cisternae of a dictyosomal stack. The acPase reaction stains the membranes of the total stack, of the GERL, of some vesicles and cisternae near the dictyosomes and lysosomes. After the mitotic breakdown of the dictyosomal stacks the forming vesicles still stain positively and are distributed over the entire cytoplasm. At late anaphase and early telophase the enzyme activity occurs not only in the reconstituting dictyosomes but also in the nuclear envelope and in some ER cisternae. The extended spectrum of membrane structures indicating Golgi enzyme activity becomes obvious. This phenomenon favors the idea that at least some functions of the Golgi apparatus persist during mitosis.
The rat tumor cell system BSp73AS/BSp73ASML was investigated for its structural aspects. The non-metastasizing line BSp73AS has less nuclear atypism than the metastasizing line BSp73ASML. Microvilli are scanty and variable in length and structure; in BSp73ASML they cover the cells densely, are short and homogeneous in size. In vitro adhesion and spreading tests show structurally flexible BSp73AS cells which flatten completely onto substrata. BSp73ASML cells remain spherical and develop only small attachment areas. In vitro aggregation of the BSp73AS cells leads to round, tightly packed aggregates, their outer cell layer is epitheloid BSp73ASML cells form clusters of mostly ball-shaped cells. Surprisingly, as judged from EM images, the intercellular junctions in BSp73ASML cell clusters are qualitatively the same as in BSp73AS cell aggregates. However, the extent of apposition between cells in BSp73AS aggregates is much higher than between cells in the BSp73ASML clusters. BSp73ASML cells lack the ability to vary their shape. This is the prominent difference revealed by these tests between the two cell lines. Hence, this fact could play an important role in their different metastatic behavior in vivo. We speculate that the ability to vary the cell shape is necessary for expansive growth in the host environment but not for the metastatic spread.
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.