Shape and orientation of nuclei of embryonal fibroblast-like cells cultured on substrates with ordered relief]

Embryo fibroblasts of hamster, rat, mouse, and were cultured 24 hrs on polyvinylchloride plates with regular relief of grooves with cylindric surfaces in between. Most cells were accumulated on the middle part of spaces between the grooves, their nuclei were elongated and oriented in the direction of grooves. Nuclear elongation was assessed quantitatively by the ratio of long to short nuclear axes. Contact orientation was measured as deviation of the long nuclear axes from the grooves direction. Both contact orientation and elongation considered respectively as "orientation reaction" is maximal and the "form reaction", increased with increasing cylinder curvature (i. e. when the radii diminished from 333 to 61 mcm). Both reactions are well expressed in cultures of the human and (less distinctly) the rat cells. In murine cells, the "orientation reaction" is maximal and the "form reaction" is weak while for the hamster cells the opposite is true. Therefore, both types of reaction seem to be independent characteristics of cells.     

Meiotic cytokinetic apparatus in the formation of the linear spore tetrads of Conocephalum japonicum (Bryophyta)

Most bryophytes produce tetrahedral spore tetrads. However, linear spore tetrads have been reported to occur in Conocephalum japonicum (Thunb.) Grolle. In this study, the distribution of microtubules (MTs) during meiosis in C. japonicum was examined to determine the division pattern resulting in a linear tetrad. Spore mother cells in the pre-meiotic stage were cylindrical with randomly distributed cytoplasmic MTs. In the prophase-metaphase transition, spindle MTs replaced cytoplasmic MTs and a barrel-shaped spindle with two flattened poles developed. Cortical MT arrays were not detectable throughout meiosis. Although a phragmoplast appeared between sister nuclei in telophase-I, it disappeared without expanding to the parental cell wall. Metaphase-II spindles oriented parallel to the long axis of the cell and in tandem to each other resulted in a linear arrangement of telophase nuclei. Radial arrays of MTs developed from the nuclear surfaces and three phragmoplasts appeared among the four nuclei to produce four spores. Two phragmoplasts separating the paired sister nuclei appeared prior to the appearance of a phragmoplast between non-sister nuclei. The MT cycle is basically the same as that reported in meiosis of C. conicum, which produces non-linear tetrads. A morphometric study indicated that the difference in the division pattern between C. conicum and C. japonicum is due to a difference in the shape of spore mother cells. The cylindrical shape of sporocytes of C. japonicum restricts the orientation of spindles and phragmoplasts so that the four resultant spores are arranged linearly. Received: 22 April 1998 / Accepted: 15 May 1998     

Flow microfluorometric analysis of sperm DNA content: effect of cell shape on the fluorescence distribution.

The DNA content of individual sperm from populations of acriflavine-stained cells was investigated by analysis of fluorescence frequency distributions obtained with high-resolution flow-systems instruments. Sperm with spherical or cylindrical heads from three mollusk species produce narrow, symmetric fluorescence distributions. Flat sperm heads from six eutherian species produce asymmetric distributions consisting of a peak with a lateral extension to higher fluorescence values. The unexpected shape of these distributions was shown to be due to the flat geometry and high refractive index of the sperm heads in conjunction with the orthogonal axes of flow, excitation, and detection in the flow-systems instruments. The theoretical and experimeytal results indicate that the lateral extension can be eliminated either by controlling the sperm orientation with planar flow conditions or by accounting for sperm orientation by means of orientation sensing.     

Nuclear distributions of NUP62 and NUP214 suggest architectural diversity and spatial patterning among nuclear pore complexes

The shape of nuclei in many adherent cultured cells approximates an oblate ellipsoid, with contralateral flattened surfaces facing the culture plate or the medium. Observations of cultured cell nuclei from orthogonal perspectives revealed that nucleoporin p62 (NUP62) and nucleoporin 214 (NUP214) are differentially distributed between nuclear pore complexes on the flattened surfaces and peripheral rim of the nucleus. High resolution stimulated emission depletion (STED) immunofluorescence microscopy resolved individual NPCs, and suggested both heterogeneity and microheterogeneity in NUP62 and NUP214 immunolabeling among in NPC populations. Similar to nuclear domains and interphase chromosome territories, architectural diversity and spatial patterning of NPCs may be an intrinsic property of the nucleus that is linked to the functions and organization of underlying chromatin.     

Electric field-induced orientation of l- and dl-phosphatidylcholine bilayers

Membrane orientation induced by an alternating electric field has been examined for the l-enantiomer and racemic dipalmitoylphosphatidylcholine (DPPC) bilayers. The orientation effect was measured by bending curvature of hairpin-like deformation of the multilamellar cylindrical tubes with varying field-strength, frequency and tube size. It has been observed that both l- and dl-DPPC tubes are similar in the profiles of field-strength dependence and frequency dependence on the curvature deformation, but different in the deformed curvatures. dl-DPPC tubes deform largely as compared with l-DPPC tubes. The square of the deformed curvature of dl-DPPC tubes is larger than that of l-DPPC by about 37% on average. The result indicates that the racemic membrane is responsive to the electric field as compared with the l-enantiomer membrane. This suggests that a hybrid arrangement of head groups of the racemic lipid leads an effective response of the membrane due to the head group orientation.     

Stereologic correlates of steroid receptor concentration in invasive ductal breast cancer

The hypothesis was tested that morphometric parameters of tumor cell nuclei correlate with the steroid receptor concentration in mammary carcinoma. In 50 consecutive mastectomy specimens with a diagnosis of invasive ductal cancer in which estrogen receptor (ER) and progesterone receptor (PR) concentrations had been assayed quantitatively, morphometric measurements were performed on four visual fields of two sections per case. The fields were sampled from the most cellular regions of the tumor. The number of tumor cell nuclear profiles per tissue area, the nuclear profile area and the long and short nuclear profile axes and their ratios were measured with a semiautomatic image analysis system. Estimates of the number of tumor cell nuclei per tissue volume (Nv) and of the mean tumor cell nuclear volume (V) were obtained by standard stereologic techniques. Association between the morphometric and biochemical parameters was tested by Spearman's rank correlation coefficient. Nv correlated positively with the steroid receptor concentration whereas V correlated negatively with both ER and PR concentrations. A correlation of the receptor concentrations to the standard deviation of the nuclear area or the mean ratio of the nuclear axes could not be demonstrated. These results suggest that receptor-rich tumors have a large number of small tumor cell nuclei whereas receptor-poor tumors have a small number of large tumor cell nuclei per tissue volume in the actively proliferating, highly cellular regions. These differences are not accompanied by significant changes in nuclear size variability or nuclear shape.     

Effects of nuclear isolation on psoralen affinity for chromatin

We have tested the effects of nuclear isolation on intercalation of TMP (a psoralen) at specific sequences and in total DNA of cultured human cells. DNA in nuclei photobound about 20% more TMP than in cells and about 10% as much as purified DNA. In contrast, a transcribed ras gene and a randomly selected polymorphic sequence each bound about 20% more TMP than total DNA in cells. However, in nuclei, as in purified DNA, both sequences were just as sensitive as total DNA. Apparently, chromatin in cells exists within diverse TMP-binding environments and some of this diversity was lost upon nuclear isolation.     

PHOTOTROPISM OF SELAGINELLA: THE DIFFERENTIAL RESPONSE TO LIGHT

Selaginella kraussiana (Kunze) A. Braun has a plagiotropic dorsiventral stem with two rows of small leaves on the dorsal surface and two rows of large leaves inserted laterally. Stem tips exhibit a differential phototropic response. When stem tips are placed in their normal horizontal orientation and the dorsal surfaces are illumianted, the tips bend only 20” below the horizon and away from light. Stem curvature is limited to a zone 450 μm long located 1.5 mm behind the shoot apex. The dorsal cortical cells within this zone of curvature are about 1.44 times longer than the ventral cortical cells. Illumination of the ventral surface of the stem tips elicits a strong phototropic response. The stems bend from 123–158° below the horizon and toward light, and the zone of curvature increases in length to 10 mm of the explant. The curvature is large enough so that the previously shaded dorsal leaves of the stem tips become redirected toward the light. This phototropic response is promoted by white and blue light, whereas red or far-red light has no effect. When stem tips are cultured in total darkness, the length of the zone of curvature is 8.0 mm but the stems bend only 50–67°. Treatment of the small dorsal leaves with phenylacetic acid inhibits phototropic curvature, and the phototropic response is unaffected by gravity.     

Hit rates and radiation doses to nuclei of bone lining cells from alpha-particle-emitting radionuclides.

Factors relating the local concentration of a bone-seeking alpha-particle emitter to the mean hit rate have been determined for nuclei of bone lining cells using a Monte Carlo procedure. Cell nuclei were approximated by oblate spheroids with dimensions and location taken from a previous histomorphometric study. The Monte Carlo simulation is applicable for planar and diffuse labels at plane or cylindrical bone surfaces. Additionally, the mean nuclear dose per hit, the dose mean per hit, the mean track segment length and its second moment, the percentage of stoppers, and the frequency distribution of the dose have been determined. Some basic features of the hit statistics for bone lining cells have been outlined, and the consequences of existing standards of radiation protection with regard to the hit frequency to cell nuclei are discussed.     

Synthesis of adenosine triphosphate in isolated nuclei and intact cells

1. It has previously been demonstrated that nuclei isolated from normal and neoplastic lymphoid cells are capable of oxygen-dependent ATP synthesis. In this paper it is shown that also the corresponding intact cells can synthesize ATP under those conditions in which nuclei can synthesize ATP. 2. In nuclei isolated from liver, kidney, rhabdomyosarcoma and osteosarcoma, oxygen-dependent ATP synthesis could not be demonstrated. The cells isolated from these tissues or tumours could not synthesize ATP either. The alternatives that such nuclei lost their ability for oxidative phosphorylation during the isolation procedure or that the process does not occur in these nuclei were explored. 3. Janus Green B, a vital stain for mitochondria, was used as a differential inhibitor of mitochondrial and nuclear ATP synthesis in intact cells. 4. Oxidative phosphorylation in mitochondria isolated from cells that had been incubated with various concentrations of Janus Green B (1–10μm) was seriously uncoupled, whereas at these concentrations oxygen-dependent ATP synthesis in isolated nuclei and in isolated cells were only inhibited to a small extent. 5. The results suggest that oxygen-dependent ATP synthesis in isolated cells measured under `nuclear' conditions and in the presence of Janus Green B and Ca²⁺ is mainly due to nuclear oxygen-dependent ATP synthesis. The stimulation of cellular ATP synthesis by glucose was completely inhibited by Janus Green B. 6. It is tentatively concluded that the stimulation of ATP synthesis in isolated cells by glucose, which is not found in isolated nuclei, represents mitochondrial ATP synthesis, and nuclear and mitochondrial ATP synthesis can then be studied differentially in the intact cell. The possibility is considered that oxygen-dependent nuclear ATP synthesis is not a general property of cell nuclei.     

Radial microtubule organization by histone H1 on nuclei of cultured tobacco BY-2 cells

In acentriolar higher plant cells, the surface of the nucleus acts as a microtubule-organizing center, substituting for the centrosome. However, the protein factors responsible for this microtubule organization are unknown. The nuclear surfaces of cultured tobacco BY-2 cells possess particles that generate microtubules. We attempted to isolate the proteins in these particles to determine their role in microtubule organization. When incubated with plant or mammalian tubulin, some, but not all, of the isolated nuclei generated abundant microtubules radially from their surfaces. The substance to induce the formation of radial microtubules was confirmed by SDS-PAGE to be a protein with apparent molecular mass of 38 kDa. Partial analysis of the amino acid sequences of the peptide fragments suggested it was a histone H1-related protein. Cloning and cDNA sequence analysis confirmed this and revealed that when the recombinant protein was incubated with tubulin, it could organize microtubules as well as the 38-kDa protein. Histone H1 and tubulin formed complexes immediately, even on ice, and then clusters of these structures were formed. These clusters generated radial microtubules. This microtubule-organizing property was confined to histone H1; all other core histones failed to act as organizers. On immunoblot analysis, rabbit antibodies raised against the 38-kDa protein cross-reacted with histone H1 proteins from tobacco BY-2 cells. These antibodies virtually abolished the ability of the nucleus to organize radial microtubules. Indirect immunofluorescence showed that the antigen was distributed at the nuclear plasm and particularly at nuclear periphery independently from DNA.     

Quantitative evaluation of cell orientation in culture.

A quantitative method to assess mutual orientation of cells in cultures on a substrate includes the following operations: (1) the cellular groups to be evaluated are chosen; (2) position of the long axis for each nucleus of the group is determined; (3) the axis OX is arbitrary chosen for every group and the angles alphai between the long axis of every nucleus i and the axis OX are measured. Every nucleus i corresponds to a vector of unit length ei with the angles 2alpha. D, the mean of the vectors ei for every cell group is calculated. This value of D is compared with a set of values of D computed according to a model of mutual orientation studies in a simulation experiment. In this model the group of n vectors consists of a fraction of Kn parallel vectors (o less than or equal to K less than or equal to I) and of (I minus K)n randomly oriented vectors. K corresponding to the computed D which is equal to the experimental value of D is considered as an index of orientation for the group. Contact orientation with respect to the relief of the substrate may be evaluated as a root mean square deviation sigma0 to the angles between the long axes of cell nuclei and the direction of relief. Examples of the measurements of K and sigma0 in cell cultures are given.     

The degree of coupling of nuclear rotation in binucleate 3T3 cells

In order to test the existence of mechanical coupling between the rotational movements of two adjacent nuclei, we prepared binucleate 3T3 cells and observed their nuclear movements by near infrared microscopy and recorded them with time-lapse video techniques. We found that 49 out of 110 (44%) of the selected binucleate cells expressed nuclear rotation. Rotation could occur in just one of the nuclei while the second nucleus remained stationary (31/110) or in both nuclei simultaneously (18/110). In almost all cases where both nuclei rotated simultaneously (15/110) they did so at different speeds and in opposite directions. The nuclei were observed to rotate in the same direction in only three of the examples. The results are consistent with a weak mechanical interaction between a rotating nucleus and its neighbor. Consistent with our previous observations in mononucleate cells, we did not find a characteristic position of the centrosphere or a special distribution of the microtubules or the intermediate filaments in binucleate cells with rotating nuclei. There was an absence of long, well-formed microfilament bundles beneath the nuclei during rotation, even in the local region beneath the rotating nucleus in those cells with one rotating and one stationary nucleus. Also consistent with observations of mononucleate cells, nuclear rotation was inhibited by treatment with colcemid, although the ability of the nuclei to rotate was eventually restored when the colcemid-containing medium was replaced with normal medium.     

Nuclear proteins of quiescent Xenopus laevis cells inhibit DNA replication in intact and permeabilized nuclei

Quiescent cells from adult vertebrate liver and contact-inhibited or serum-deprived tissue cultures are active metabolically but do not carry out nuclear DNA replication and cell division. Replication of intact nuclei isolated from either quiescent Xenopus liver or cultured Xenopus A6 cells in quiescence was barely detectable in interphase extracts of Xenopus laevis eggs, although Xenopus sperm chromatin was replicated with approximately 100% efficiency in the same extracts. Permeabilization of nuclei from quiescent Xenopus liver or cultured Xenopus epithelial A6 cells did not facilitate efficient replication in egg extracts. Moreover, replication of Xenopus sperm chromatin in egg extracts was strongly inhibited by a soluble extract of isolated Xenopus liver nuclei; in contrast, complementary-strand synthesis on single-stranded DNA templates in egg extracts was not affected. Inhibition was specific to endogenous molecules localized preferentially in quiescent as opposed to proliferating cell nuclei, and was not due to suppression of cdk2 kinase activity. Extracts of Xenopus liver nuclei also inhibited growth of sperm nuclei formed in egg extracts. However, the rate and extent of decondensation of sperm chromatin in egg extracts were not affected. The formation of prereplication centers detected by anti-RP-A antibody was not affected by extracts of liver nuclei, but formation of active replication foci was blocked by the same extracts. Inhibition of DNA replication was alleviated when liver nuclear extracts were added to metaphase egg extracts before or immediately after Ca++ ion-induced transition to interphase. A plausible interpretation of our data is that endogenous inhibitors of DNA replication play an important role in establishing and maintaining a quiescent state in Xenopus cells, both in vivo and in cultured cells, perhaps by negatively regulating positive modulators of the replication machinery.     

Improved Visualization of Cell Nuclei in Unstained Cultured Cells

Nuclear morphology is useful in tissue culture studies in determining the presence and grade of transformed cells as well as in determining the heterogeneity of the cell population (Grogan el al. 1981, Hustin 1976, Siracky et al. 1978, Siracky 1979). The ratio of long and short nuclear axes provides a useful numerical expression of nuclear shape (Hustin 1976). Clear visualization of nuclei is critical for making the necessary measurements.     

Electronic structure of Q-A in reaction centers from Rhodobacter sphaeroides. I. Electron paramagnetic resonance in single crystals.

The magnitude and orientation of the electronic g-tensor of the primary electron acceptor quinone radical anion, Q-A, has been determined in single crystals of zinc-substituted reaction centers of Rhodobacter sphaeroides R-26 at 275 K and at 80 K. To obtain high spectral resolution, EPR experiments were performed at 35 GHz and the native ubiquinone-10 (UQ10) in the reaction center was replaced by fully deuterated UQ10. The principal values and the direction cosines of the g-tensor axes with respect to the crystal axes a, b, c were determined. Freezing of the single crystals resulted in only minor changes in magnitude and orientation of the g-tensor. The orientation of Q-A as determined by the g-tensor axes deviates only by a few degrees (< or = 8 degrees) from the orientation of the neutral QA obtained from an average of four different x-ray structures of Rb. sphaeroides reaction centers. This deviation lies within the accuracy of the x-ray structure determinations. The g-tensor values measured in single crystals agree well with those in frozen solutions. Variations in g-values between Q-A, Q-B, and UQ10 radical ion in frozen solutions were observed and attributed to different environments.     

Degree of cell spreading on a substrate as the factor determining the nature of cell microrelief in suspension

Quantitative ratio of various types of cell surface microrelief was determined in suspensions prepared from mouse monolayer cultures of embryo fibroblasts grown on different solid substrates: with high (Falcon) or low poly(2-hydroxyethylmethacrylate) adhesiveness; with flat or cylindrical (53-mu curvature radius) surfaces (polyvinylchloride). The electron microscopy revealed that poorly spread cells (on low adhesive or cylindrical substrata) in suspensions had the microvillous surface relief much more often than the cells brought to suspension from highly adhesive or flat substrata. Thus, the lower the degree of cell spreading on the substratum, the higher the probability for the cell to acquire the microvillous relief in suspended state. The microvillous relief of transformed cells in suspensions is, probably, due to their poor spreading on substrata in the monolayer cultures.     

Modeling apoptotic chromatin condensation in normal cell nuclei. Requirement for intranuclear mobility and actin involvement

Hallmarks of the terminal stages of apoptosis are genomic DNA fragmentation and chromatin condensation. Here, we have studied the mechanism of condensation both in vitro and in vivo. We found that DNA fragmentation per se of isolated nuclei from non-apoptotic cells induced chromatin condensation that closely resembles the morphology seen in apoptotic cells, independent of ATP utilization, at physiological ionic strengths. Interestingly, chromatin condensation was accompanied by release of nuclear actin, and both condensation and actin release could be blocked by reversibly pretreating nuclei with Ca2+, Cu2+, diamide, or low pH, procedures shown to stabilize internal nuclear components. Moreover, specific inhibition of nuclear F-actin depolymerization or promotion of its formation also reduced chromatin condensation. Chromatin condensation could also be inhibited by exposing nuclei to reagents that bind to the DNA minor groove, disrupting native nucleosomal DNA wrapping. In addition, in cultured cells undergoing apoptosis, drugs that inhibit depolymerization of actin or bind to the minor groove also reduced chromatin condensation, but not DNA fragmentation. Therefore, the ability of chromatin fragments with intact nucleosomes to form large clumps of condensed chromatin during apoptosis requires the apparent disassembly of internal nuclear structures that may normally constrain chromosome subdomains in non-apoptotic cells.     

IMMUNOFLUORESCENT LOCALIZATION OF MICROTUBULES THROUGHOUT THE CELL CYCLE IN THE GREEN ALGA MOUGEOTIA (ZYGNEMATACEAE)

Indirect immunofluorescence microscopy was used to survey the three-dimensional distribution of microtubules throughout the cell cycle in the green alga Mougeotia. The network of microtubules present in the cortex of the cells at interphase gradually disappeared before mitosis. A band of cortical microtubules reminiscent of the preprophase band of higher plants surrounded the nuclei of some preprophase cells undergoing cortical microtubule disassembly. Longitudinally oriented bundles of microtubules appeared at the future spindle poles on either side of the nuclei in prophase. These bundles disappeared gradually as the spindle microtubule arrays formed. New spindles had broad poles but these became quite pointed before anaphase. Interzonal microtubules appearing at anaphase persisted until the end of nuclear migration, by which time they were concentrated into narrow bundles on either side of the centripetally forming crosswalls. During decondensation of the chromosomes and early nuclear migration, the spindle poles persisted as sites of microtubule concentration. New arrays of microtubules radiated from these microtubule centers into the cytoplasm ahead of the migrating nuclei. After cytokinesis, reinstatement of cortical microtubules was best observed in regions of the cells remote from the nuclei and associated microtubules. In contrast to higher plants, the first detectable cortical microtubules were short and already oriented transverse to the long axes of the cells.     

A library of monoclonal antibodies to nuclear proteins from Drosophila melanogaster embryos. Characterization by a cultured cell assay

To study the structure and function of the cell nucleus, a library of 170 monoclonal antibodies was produced to nuclear antigens from 3-6 h old Drosophila embryos. In preparation for immunization, nuclei were separated, at neutral pH and in the presence of polyamines, into two fractions containing either urea-soluble non-histone nuclear proteins or histones plus small quantities of non-histone proteins complexed to DNA. The antibodies were characterized in a rapid, indirect immunofluorescent assay employing cultured Drosophila cells (Schneider's line 2). Low backgrounds and high specific fluorescence were achieved in this assay by purifying the rhodamine-labelled second antibody on a polystyrene resin and washing the cells with optimal concentrations of detergents. The assay categorized antigens according to their cellular locations: in nuclei, in nuclei plus cytoplasm, or primarily in cytoplasm. A subset of nuclear antigens reacted specifically with the nuclear envelope. In addition, some antibodies were characterized by their reactions with polytene chromosomes. The cultured cell assay provides a new, efficient method for expanding this antibody library. The monoclonal antibodies in the library now provide highly specific tools for investigating structural nuclear proteins and proteins that may be regulatory during embryonic development.