Evaluation of L-929 postmitotic daughter cell spreading during their migration on a substrate

Spreading of postmitotic daughter cells was examined using time-lapse microscopy. The work was performed on unsynchronized cells of a permanent L-929 cell line. The study aimed at formalizing the comparison of the moving cell area and estimating whether the area of migrating cells was changed randomly or nonrandomly. Two new parameters are proposed for comparison of cell morphology: the identity indicator and the synchronism indicator. To calculate these parameters, time-dependent changes of the area in cell pairs were measured. The first indicator shows the degree of coincidence between the absolute area values in the cell pairs, whereas the second indicator shows synchronism in the changes of cell areas and does not depend on their absolute values. The low indicators were a high similarity in the time-dependent changes of the cell area. The indicators were shown to be approximately 1.5-fold lower for the pairs of the postmitotic daughter cells than those for any other pair of the cells. The results point to a nonrandom character of the changes of cultured cell morphology.     

The outcome of phagocytic cell division with infectious cargo depends on single phagosome formation

Given that macrophages can proliferate and that certain microbes survive inside phagocytic cells, the question arises as to the post-mitotic distribution of microbial cargo. Using macrophage-like cells we evaluated the post-mitotic distribution of intracellular Cryptococcus yeasts and polystyrene beads by comparing experimental data to a stochastic model. For beads, the post-mitotic distribution was that expected from chance alone. However, for yeast cells the post-mitotic distribution was unequal, implying preferential sorting to one daughter cell. This mechanism for unequal distribution was phagosomal fusion, which effectively reduced the intracellular particle number. Hence, post-mitotic intracellular particle distribution is stochastic, unless microbial and/or host factors promote unequal distribution into daughter cells. In our system unequal cargo distribution appeared to benefit the microbe by promoting host cell exocytosis. Post-mitotic infectious cargo distribution is a new parameter to consider in the study of intracellular pathogens since it could potentially define the outcome of phagocytic-microbial interactions.     

Patterned cell development in the secondary phloem of dicotyledonous trees: a review and a hypothesis

The secondary phloem of dicotyledonous trees and shrubs is constructed of sieve tube cells (S) and their companion cells, as well as parenchyma (P) and fibre (F) cells. Different species have characteristic sequences of these S, P and F cells within the radial files of their phloem. The sequences are recurrent, and are evidence of rhythmic cell determination and differentiation. A model was devised to account for the sequences found in various dicot tree species. It is based on the pattern of radial displacement of cells through a gradient of morphogen which supports secondary phloem development. According to this model, each tree species shows a particular pattern of post-mitotic cellular displacement along each radial file as a result of a corresponding sequence of periclinal division in the cambial initial and its descendents. The divisions and displacements ensure that at each timestep (equivalent to an interdivisional interval) each cell resides in a specific location within the morphogenic gradient. Cells then emerge from the post-mitotic zone of cell determination, having acquired different final positional values. These values lie above a series of thresholds that permit the respective determination and subsequent differentiation of one or other of the three cell types S, P and F. The recurrent nature of the sequences of the three cell types within each radial cell file, as well as their tangential banding, are a consequence of a shared rhythmic spatio-temporal pattern of periclinal cambial divisions. With a single set of morphogen parameters required for cell determination, and using three positions for cambial cell divisions, all the cellular sequences of secondary phloem illustrated in the literature can be accounted for.This is an invited article.     

Analysis of the myogenic lineage in chick embryos: I. Studies on the terminal cell division

An antibody prepared against the MM isozyme of creatine phosphokinase (M-CK) stained multinucleated myotubes and post-mitotic mononucleated myoblasts in mass cultures of myogenic cells taken from the breast muscles of 11-day chick embryos. No cycling cells bound the antibody. Single cells isolated either directly from the embryo or from mass cultures were seeded at clonal density and allowed to undergo one division. The resulting pairs of cells were stained with the antibody and were scored as (a) both members of the pair M-CK⁺; (b) both M-CK^?; or (c) mixed (one M-CK⁺ and one M-CK^?). No mixed pairs were observed. Conditioned medium did not induce all myogenic pairs to differentiate and growth medium did not keep myogenic pairs in the cell cycle. About 10% of clonal pairs established from 10 h cultures were M-CK⁺, while about 27% of pairs established from 30 h mass cultures were M-CK⁺. These results indicate that (1) the myogenic lineage ends in a symmetrical division whose products are two post-mitotic M-CK⁺ cells; (2) the expression of the muscle phenotype is not determined exclusively by the environment; (3) the terminal cells are the product of an intrinsic program or cell lineage in which only the last cells can synthesize muscle-specific proteins.     

DISTRIBUTION OF LABELED CHROMATIN : I. M1 and M2 Anaphases of Diploid and Tetraploid Cultured Mammalian Cells

The question of whether distribution of chromatids to daughter cells in mitosis is a random or nonrandom process was investigated by study of the distribution of labeled chromatin in anaphase pairs at M₁ and M₂ after a pulse of tritiated thymidine. Diploid and tetraploid rat and diploid human fibroblast-like cells in serial monlayer culture were synchronized by two different methods to "purify" M₁ and M₂ anaphases: metaphase shake, and FUdR block to DNA synthesis followed by exogenous thymidine. Exposed grains of NTB-2 emulsion were counted over M₁ and M₂ anaphase pairs. An analysis (by pair) of diploid M₂ anaphase grain counts showed two discrete populations of daughters with less and with more radioactivity. A similar analysis of diploid M₁ and tetrapolid M₂ anaphases showed a single grain-count distribution. These findings may support a nonrandom model of chromatid segregation for diploid mammalian cells but do not rule out random segregation until sound mathematical models are formulated for expected random grain distributions in M₂ anaphases of cells with differing numbers of chromosomes.     

Transformation of the flagella and associated flagellar components during cell division in the coccolithophoridPleurochrysis carterae

Summary Immunofluorescence microscopy, conventional and high voltage transmission electron microscopy were used to describe changes in the flagellar apparatus during cell division in the motile, coccolithbearing cells ofPleurochrysis carterae (Braarud and Fagerlund) Christensen. New basal bodies appear alongside the parental basal bodies before mitosis and at prophase the large microtubular (crystalline) roots disassemble as their component microtubules migrate to the future spindle poles. By prometaphase the crystalline roots have disappeared; the flagellar axonemes shorten and the two pairs of basal bodies (each consisting of one parental and one daughter basal body) separate so that each pair is distal to a spindle pole. By late prometaphase the pairs of basal bodies bear diminutive flagellar roots for the future daughter cells. The long flagellum of each daughter cell is derived from the parental basal bodies; thus, the basal body that produces a short flagellum in the parent produces a long flagellum in the daughter cell. We conclude that each basal body in these cells is inherently identical but that a first generation basal body generates a short flagellum and in succeeding generations it produces a long flagellum. At metaphase a fibrous band connecting the basal bodies appears and the roots and basal bodies reorient to their interphase configuration. By telophase the crystalline roots have begun to reform and the rootlet microtubules have assumed their interphase appearance by early cytokinesis.Abbreviations CR1, CR2 crystalline roots 1 and 2 - CT cytoplasmic tongue microtubules - DIC differential interference contrast light microscopy - H haptonema - HVEM high voltage transmission electron microscopy - IMF immunofluorescence microscopy - L left flagellum/basal body - M metaphase plate - MT microtubule - N nucleus - R right flagellum/basal body - R1, R2, R3 roots 1, 2, and 3 - TEM transmission electron microscopy     

A Highlights from MBoC Selection: Spatial distribution of cell–cell and cell–ECM adhesions regulates force balance while main-taining E-cadherin molecular tension in cell pairs

Mechanical linkage between cell–cell and cell–extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell–cell and cell–ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell–cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell–cell and cell–ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell–cell pairs resulted in shorter junction lengths and constant cell–cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell–cell forces and was evenly distributed along cell–cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.     

Oscillations in continuous cultures of budding yeast: a segregated parameter analysis

Sustained oscillations have been observed in continuous cultures of Saccharomyces cerevisiae. These oscillations appear spontaneously under aerobic conditions and may constitute a severe limitation for process control. We have found that oscillations arise only in a well defined range of dilution rates and dissolved oxygen values. The period of the oscillations is related, but not equal, to the mass doubling time, and shows a relation ship with both the parent cells and daughter cells generation times. At high dilution rates two oscillatory regimens, with different periods, are observed. The analysis of the budding index shows a marked degree of synchronization of the culture, however significant differences, both in phase and in amplitude, are ob served if the budding index of parent cells and of daughter cells are considered separately. The complex changes of the cell population are clearly demonstrated by the continuous and periodic modification of both cell volume distributions and protein distributions. Ethanol is always accumulated before the drop of dissolved oxygen concentration and one of the peaks of budding index. We propose a model that explains the insurgence of these oscillation as a consequence of changes in cell cycle parameters due to alternate growth in glucose and in ethanol.     

On the influence of substrate morphology and surface area on phytofauna

The independent effects and interactions between substrate morphology and substrate surface area on invertebrate density or biomass colonizing artificial plant beds were assessed in a clear-water and a turbid playa lake in Castro County, Texas, USA. Total invertebrate density and biomass were consistently greater on filiform substrates than on laminar substrates with equivalent substrate surface areas. The relationship among treatments (substrates with different morphologies and surface areas) and response (invertebrate density or biomass) was assessed with equally spaced surface areas. Few statistically significant interactions between substrate morphology and surface area were detected, indicating that these factors were mostly independent from each other in their effect on colonizing invertebrates. Although infrequently, when substrate morphology and surface area were not independent, the effects of equally spaced changes in substrate surface area on the rate of change of phytofauna density or biomass per unit of substrate surface area were dependent upon substrate morphology. The absence of three-way interactions indicated that effects of substrate morphology and substrate area on phytofauna density or biomass were independent of environmental conditions outside and inside exclosures. Handling editor: D. Harper     

Visualization of detyrosination along single microtubules reveals novel mechanisms of assembly during cytoskeletal duplication in trypanosomes

We have been able to use immunogold labeling with monoclonal antibodies specific for tyrosinated alpha-tubulin to define new microtubule assembly within the T. brucei pellicular cytoskeleton. Using this approach, we have been able to visualize and define the detyrosination gradient along single microtubules in vivo. New microtubules are seen to invade the cytoskeletal array early in the cell cycle between old microtubules. In post-mitotic cells, a unique form of microtubule assembly occurs, with very short microtubules being intercalated in the array. We propose that these are nucleated by lateral interaction with the MAPs on existing adjacent microtubules. This construction pattern suggests a templated morphogenesis of microtubule arrays with semi-conservative distribution to the daughter cells.     

Microvilli and blebs as sources of reserve surface membrane during cell spreading

The increase in surface area that occurs as cells spread from the rounded to the flattened state has been examined in synchronized BHK 21 cells in the scanning electron microscope. Rounded cells, whether in mitosis or dissociated and freshly seeded in culture, are covered with a mixture of folds, blebs, and microvilli. As cells spread, these protuberances disappear, first in the flattening marginal region and progressively submarginally until the entire cell surface is virtually smooth. The estimated surface area of rounded post-mitotic daughter cells, taking microvilli into account, is close to that of fully spread cells 4 h after mitosis. Likewise, rounded early mitotic mother cells, which are also covered with microvilli, have approximately the same surface as fully spread cells just prior to mitosis. These findings suggest that cells possess a membrane reserve in their microvilli and other protuberances which can be utilized for spreading and initiating cell locomotion.     

Clinical implications and utility of field cancerization

Cancer begins with multiple cumulative epigenetic and genetic alterations that sequencially transform a cell, or a group of cells in a particular organ. The early genetic events might lead to clonal expansion of pre-neoplastic daughter cells in a particular tumor field. Subsequent genomic changes in some of these cells drive them towards the malignant phenotype. These transformed cells are diagnosed histopathologically as cancers owing to changes in cell morphology. Conceivably, a population of daughter cells with early genetic changes (without histopathology) remain in the organ, demonstrating the concept of field cancerization. With present technological advancement, including laser capture microdisection and high-throughput genomic technologies, carefully designed studies using appropriate control tissue will enable identification of important molecular signatures in these genetically transformed but histologically normal cells. Such tumor-specific biomarkers should have excellent clinical utility. This review examines the concept of field cancerization in several cancers and its possible utility in four areas of oncology; risk assessment, early cancer detection, monitoring of tumor progression and definition of tumor margins.     

Oxygen, pH value, and carbon source induced changes of the mode of oscillation in synchronous continuous culture of Saccharomyces cerevisiae

Oscillations of measured process parameters occur in continuous cultures of Saccharomyces cerevisiae owing to a partial synchronization of budding. Intentional changes of the oxygen concentration, pH value, and carbon source cause effects on the period length similar to those known from variations of the dilution rate. The generation times of parent and daughter cells frequently differ in synchronous culture. To analyze the oscillation the term mode IJ of oscillation is used, which is defined as the ratio IJ of the generation times of parent and daughter cells. When the dissolved oxygen concentration was reduced to zero, the mode of oscillation changed within two periods from mode 12 to mode 11, caused by a decrease of the generation time of daughter cells and an increase of that of the parent cells. When the pH value was slowly reduced from 5.0 to 3.9, a change from mode 112 to mode 13 was observed. Mode 13, representing one parent and three daughter cell populations (the start of budding of each of the three being delayed by one period), denotes an elongated generation time of the daughter cells compared to mode 112, marked by one parent and two different daughter cell classes. When the carbon source galactose was replaced by glucose a mode change from mode 12 to mode 11 was observed. This alteration of the mode was found to be dependent on the status of the cell cycle at the time when the carbon source is changed. The population distribution in batch cultures with glucose or galactose as a substrate was analysed by dyeing the DNA and counting the bud scars. Galactose provoked higher growth rates for the older cells. According to the model for stationary synchronous growth parameters like DO, pH value or the type of carbon source can be varied within a certain range without effecting the period length. If the variation imposes a certain stress, the culture switches to a new mode. These kinds of parameters therefore provide selective measures to influence the period lengths and the modes of oscillation.     

Transmission of radiation-induced acentric chromosomal fragments to micronuclei in normal human fibroblasts

A simplifying assumption made when calculating the probability of a chromosomal aberration resulting in a micronucleus is that virtually all radiation-induced micronuclei result from acentric fragments. In the present study we used antibodies to chromosomal centromeres (kinetochores) to determine the frequency of centric versus acentric micronuclei in normal human fibroblasts exposed to 6 Gy of 60Co gamma rays while they were in density-inhibited growth. Up to 14% of the micronuclei induced by this exposure contained one or more kinetochores; i.e., they were not composed of acentric chromatin. By deleting kinetochore-positive micronuclei from the analysis, and by reconstructing micronucleus frequencies based on the fraction of cells that had divided following radiation exposure, a direct comparison between micronuclei and acentric chromosome fragments was made. On that basis, the probability of an acentric fragment becoming a visible micronucleus in either daughter cell of a dividing pair was estimated to be about 0.6. The distribution of acentric fragments among mitotic cells conformed to Poisson expectation, while the distribution of micronuclei among daughter cells was significantly overdispersed. The phenomenon of overdispersion is discussed in connection with proposed cellular processes that effect a nonrandom segregation of acentric fragments.     

Indicators of hemeroby for the monitoring of landscapes in Germany

The article discusses the concepts of “closeness to nature” and “hemeroby”, and outlines a method to establish two indicators of hemeroby. Until now Germany's national land use monitoring systems have lacked an indicator to capture the naturalness respectively hemeroby of the landscape. Based on digital spatial data on land use (DLM-DE) and the mapping of potential natural vegetation, these indicators have now been estimated for the whole of Germany and illustrated cartographically. The indicators have been integrated into a land use monitoring system (IOER-Monitor). A hemeroby index that considers all hemeroby classes of a reference area (e.g. administrative unit and regular grid cell) is presented as well as an indicator named “Proportion of certain natural areas”. The results on hemeroby of several time-cuts can be used to estimate the cumulative impact of land use changes on the environmental status.     

Configural changes in the plastidome ofPolytoma papillatum after completion of cytokinesis and during fusion of the gametes

Summary Changes in plastidome morphology in several definite stages of both the vegetative (asexual) and generative (sexual) life cycle ofPolytoma papillatum were examined by means of serial sectioning technique. Just after completion of cytokinesis daughter cells contain a simple through-shaped, bulky and scarcely perforated leucoplast whose opening faces the original plane of cell cleavage (=lateral part of the new daughter cell). In the course of progressive reorganization the trough-shaped leucoplast is first transformed into a hollow oviform meshwork whose reticulated frame is irregular in shape, size and distribution and whose interior is traversed at the base by two sheets, thus forming separate chambers. This then becomes the typical cup-shaped leucoplast of interphase cells, whose elongated cylindrical lateral part is thin and highly perforated and whose cup-opening faces the apical part of the cell. The changes in leucoplast morphology, probably due to reshuffling and reorganization processes, are correlated to leucoplast areas characterized both by starch and membrane breakdown and by frequent appearance at times of upheaval during leucoplast differentiation. Fusion of two leucoplasts to again form a large, highly perforated cup was also investigated; union of both gametic leucoplasts in such a spherical quadriflagellate zygote is demonstrated by a reconstructed model. Finally the relevancy of these morphological data to extranuclear inheritance is discussed.     

Multi-species outcomes in a common model of sympatric speciation

While models of sympatric speciation are motivated in part by multi-species adaptive radiations such as the Cameroon crater lake cichlids, existing models have focused on bifurcation into a single pair of daughter species. This paper shows that a familiar model of sympatric speciation, driven by intraspecific competition and assortative mating based on ecological characters values, can yield multiple daughter species if individual niche widths are sufficiently restricted. Surprisingly, the multi-species outcome is not produced by successive bifurcation events, but by simultaneous divergence resulting in a hard polytomy. This result is sensitive to a number of assumptions, whose violation may prevent speciation. In some cases when speciation fails, the population instead ends in a state that closely resembles incipient species pairs, with an ecological polymorphism and partial reproductive isolation. However, this polymorphism is stable and does not lead to complete reproductive isolation, suggesting that empirical cases of incipient species pairs may not always end in speciation.     

A kinetic method to determine the cell cycle times of chick skin fibroblast subpopulations

We have inferred, from computer simulations of clonal growth data, mean cell cycle time (Tc) for putative subpopulations of fibroblastic cells having unique replicative potentials. The growth kinetics of chick embryo fibroblast clones can be accounted for if it is assumed that: (1) there is a transient, and rather substantial, decline in mean Tc (from 34 to 12 hr) immediately following the commitment of a 'stem' cell daughter to a limited replicative lifespan; (2) the mean Tc increases progressively (from 12 to 48 hr) as 'committed' cells exhaust their remaining replicative potential; and (3) the daughters of committed cells may occasionally become abruptly post-mitotic.