Cell Division Symmetry Research Articles

A Novel Validation Algorithm Allows for Automated Cell Tracking and the Extraction of Biologically Meaningful Parameters

Automated microscopy is currently the only method to non-invasively and label-free observe complex multi-cellular processes, such as cell migration, cell cycle, and cell differentiation. Extracting biological information from a time-series of micrographs requires each cell to be recognized and followed through sequential microscopic snapshots. Although recent attempts to automatize this process resulted in ever improving cell detection rates, manual identification of identical cells is still the most reliable technique. However, its tedious and subjective nature prevented tracking from becoming a standardized tool for the investigation of cell cultures. Here, we present a novel method to accomplish automated cell tracking with a reliability comparable to manual tracking. Previously, automated cell tracking could not rival the reliability of manual tracking because, in contrast to the human way of solving this task, none of the algorithms had an independent quality control mechanism; they missed validation. Thus, instead of trying to improve the cell detection or tracking rates, we proceeded from the idea to automatically inspect the tracking results and accept only those of high trustworthiness, while rejecting all other results. This validation algorithm works independently of the quality of cell detection and tracking through a systematic search for tracking errors. It is based only on very general assumptions about the spatiotemporal contiguity of cell paths. While traditional tracking often aims to yield genealogic information about single cells, the natural outcome of a validated cell tracking algorithm turns out to be a set of complete, but often unconnected cell paths, i.e. records of cells from mitosis to mitosis. This is a consequence of the fact that the validation algorithm takes complete paths as the unit of rejection/acceptance. The resulting set of complete paths can be used to automatically extract important biological parameters with high reliability and statistical significance. These include the distribution of life/cycle times and cell areas, as well as of the symmetry of cell divisions and motion analyses. The new algorithm thus allows for the quantification and parameterization of cell culture with unprecedented accuracy. To evaluate our validation algorithm, two large reference data sets were manually created. These data sets comprise more than 320,000 unstained adult pancreatic stem cells from rat, including 2592 mitotic events. The reference data sets specify every cell position and shape, and assign each cell to the correct branch of its genealogic tree. We provide these reference data sets for free use by others as a benchmark for the future improvement of automated tracking methods.

Microtubules and the tax payer

Plant microtubules have evolved into a versatile tool to link environmental signals into flexible morphogenesis. Cortical microtubules define the axiality of cell expansion by control of cellulose orientation. Plant-specific microtubule structures such as preprophase band and phragmoplast determine symmetry and axiality of cell divisions. In addition, microtubules act as sensors and integrators for stimuli such as mechanic load, gravity, but also osmotic stress, cold and pathogen attack. Many of these functions are specific for plants and involve specific proteins or the recruitment of proteins to new functions. The review aims to ventilate the potential of microtubule-based strategies for biotechnological application by highlighting representative case studies. These include reorientation of cortical microtubules to increase lodging resistance, control of microtubule dynamics to alter the gravity-dependent orientation of leaves, the use of microtubules as sensitive thermometers to improve adaptive cold tolerance of chilling and freezing sensitive plants, the reduction of microtubule treadmilling to inhibit cell-to-cell transport of plant viruses, or the modulation of plant defence genes by pharmacological manipulation of microtubules. The specificity of these responses is controlled by a great variety of specific associated proteins opening a wide field for biotechnological manipulation of plant architecture and stress tolerance.

Wnt Signaling Regulates Symmetry of Division of Neural Stem Cells in the Adult Brain and in Response to Injury

Neural stem cells comprise a small population of subependymal cells in the adult brain that divide asymmetrically under baseline conditions to maintain the stem cell pool and divide symmetrically in response to injury to increase their numbers. Using in vivo and in vitro models, we demonstrate that Wnt signaling plays a role in regulating the symmetric divisions of adult neural stem cells with no change in the proliferation kinetics of the progenitor population. Using BAT-gal transgenic reporter mice to identify cells with active Wnt signaling, we demonstrate that Wnt signaling is absent in stem cells in conditions where they are dividing asymmetrically and that it is upregulated when stem cells are dividing symmetrically, such as (a) during subependymal regeneration in vivo, (b) in response to stroke, and (c) during colony formation in vitro. Moreover, we demonstrate that blocking Wnt signaling in conditions where neural stem cells are dividing symmetrically inhibits neural stem cell expansion both in vivo and in vitro. Together, these findings reveal that the mechanism by which Wnt signaling modulates the size of the stem cell pool is by regulating the symmetry of stem cell division.

Phenotypic plasticity and effects of selection on cell division symmetry in Escherichia coli.

Aging has been demonstrated in unicellular organisms and is presumably due to asymmetric distribution of damaged proteins and other components during cell division. Whether the asymmetry-induced aging is inevitable or an adaptive and adaptable response is debated. Although asymmetric division leads to aging and death of some cells, it increases the effective growth rate of the population as shown by theoretical and empirical studies. Mathematical models predict on the other hand, that if the cells divide symmetrically, cellular aging may be delayed or absent, growth rate will be reduced but growth yield will increase at optimum repair rates. Therefore in nutritionally dilute (oligotrophic) environments, where growth yield may be more critical for survival, symmetric division may get selected. These predictions have not been empirically tested so far. We report here that Escherichia coli grown in oligotrophic environments had greater morphological and functional symmetry in cell division. Both phenotypic plasticity and genetic selection appeared to shape cell division time asymmetry but plasticity was lost on prolonged selection. Lineages selected on high nutrient concentration showed greater frequency of presumably old or dead cells. Further, there was a negative correlation between cell division time asymmetry and growth yield but there was no significant correlation between asymmetry and growth rate. The results suggest that cellular aging driven by asymmetric division may not be hardwired but shows substantial plasticity as well as evolvability in response to the nutritional environment.

Mafb Restricts M-CSF Instructed Lineage Choice in HSC

AbstractAbstract SCI-15HSC need to enter the cycle to continuously regenerate mature blood cells in a correctly balanced ratio or to replenish the stem cell pool under stress conditions. Cell division of HSC may thus result in self-renewal divisions or the production of more differentiated progeny. Although such downstream progenitors still retain a high degree of multi-potency, recent advances in their characterization also suggest that early diversification into cells with distinct lineage bias can occur at the most primitive stem and precursor cell level. Whereas multiple cellular regulators have been identified that affect self-renewal of HSC, regulators that selectively control lineage specific commitment divisions of HSC are only beginning to be elucidated. Both transcription factor and cytokine signaling may play important roles in lineage engagement but it has been a long-standing debate, whether cytokine signaling has instructive or permissive effects on lineage commitment. In this context we reported that deficiency for the monocytic transcription factor MafB specifically sensitized HSC populations to M-CSF induced cell division and myeloid lineage commitment. We observed that MafB deficiency resulted in a specific up-regulation of the early myeloid transcription factor PU.1 and a dramatically enhanced myeloid specific repopulation activity that did not affect self-renewal or differentiation into other lineages. Our results point to a role for MafB in the maintenance of a balanced lineage potential of HSC by selectively restricting myeloid commitment divisions that give rise to PU.1+ progenitors in response to M-CSF signaling. Together this suggests that the potential of stem cells to produce differentiated progeny of a specific lineage bias can be subject to control by integrated cytokine/transcription factor circuits, where variation in cell intrinsic sensitivity limits like those set by MafB can render external cues such as M-CSF instructive.Stem cell self renewal or differentiation is associated with two different types of cell divisions, namely symmetric divisions that generate two identical daughter cells, or asymmetric divisions that produce a daughter cell with maintained stem cell properties and a daughter cell committed to a specific differentiation pathway. Our observation that reduced MafB levels specifically increased M-CSF stimulated asymmetric divisions giving rise to PU.1−/PU.1+ daughter pairs provides a conclusive explanation why increased myeloid commitment of MafB−/− HSC does not come at the expense of self-renewal or other lineages. The micro-environment can play a critical role in specifying the symmetry of cell divisions, but so far, investigations have focused on niche micro-environments that support HSC self-renewal. It is unknown whether environments also exist that support HSC commitment to specific lineages by enabling asymmetric divisions at the niche border. Using video-imaging approaches and genetic manipulation of the stem cell and stromal cell compartments we explore how cues from the microenvironment can affect asymmetric HSC division and lineage commitment. Disclosures:No relevant conflicts of interest to declare.

Role of the polycomb group gene BMI1 in normal and leukemic hematopoietic stem and progenitor cells

The polycomb group gene BMI1 fulfills essential roles in both normal and leukemic stem cells. The underlying molecular mechanisms are beginning to become unraveled and an overview of the current knowledge on BMI1 signaling in normal and leukemic stem cells will be presented here. In addition to a role in bypassing senescence and the orchestration of the symmetry of hematopoietic stem cell divisions, it has recently become clear that BMI1 also functions in the protection against oxidative stress. In the absence of BMI1, reactive oxygen species accumulate, associating with activation of DNA damage response pathways and increased apoptosis. BMI1-mediated control over reactive oxygen species levels might occur independently of the INK4a/ARF pathway, but rather involves impaired mitochondrial functions. In human hematopoietic malignancies, BMI1 is frequently overexpressed, which associates with poor prognosis. Down modulation of BMI1 impairs self-renewal and long-term expansion of leukemic stem cells. Understanding molecular mechanisms by which BMI1 affects stem cell fate will increase our insights into the biology of hematopoietic stem cells and will also aid in understanding the process of leukemic transformation and ultimately in the identification of drugable targets that might facilitate the eradication of leukemic stem cells.

Caloric restriction causes symmetric cell division and delays aging in Escherichia coli

AbstractAging is one of the most intriguing processes of biology and despite decades of research, many aspects of aging are poorly understood. Aging is known to occur in bacteria and yeast that divide with morphological asymmetry. Morphologically symmetrically dividing bacteria such as Escherichia coli were assumed not to age until they were shown to divide with functional asymmetry leading to aging and death of some of the cells even in exponentially growing cultures. In asymmetrically dividing E. coli the newly synthesized components are presumed to occupy one pole so that after division one of the daughter cells receives newly synthesized components whereas the other retains the older components. Mathematical models predicted that at the population level, asymmetric growth should result in higher growth rate and symmetric growth in higher growth yield. Therefore, arguably symmetric cell division should be selected in low nutrient environments and asymmetric division in nutrient rich environments. A further prediction was that lower substrate concentrations should strengthen repair mechanisms and suppress aging whereas higher substrate concentrations suppress repair and enhance aging. We show here that E. coli divides more symmetrically under caloric restriction, that both genetic selection and phenotypic plasticity are important determinants of cell division symmetry and also that the proportion of cells that stop dividing and therefore are presumably dead is significantly lower in symmetrically dividing cultures. However, contrary to the prediction, symmetry was not always accompanied by reduced growth rate. These results demonstrate that asymmetry of division in E. coli is not hardwired but responsive to the nutritional environment. This provides a new perspective on why caloric restriction increases lifespan in organisms ranging from microbes to mammals. Symmetry of division may be a mechanism spanning across the width of life forms but regulating aging in different ways in different forms.

Distinct behaviors of neural stem and progenitor cells underlie cortical neurogenesis.

Neocortical precursor cells undergo symmetric and asymmetric divisions while producing large numbers of diverse cortical cell types. In Drosophila, cleavage plane orientation dictates the inheritance of fate-determinants and the symmetry of newborn daughter cells during neuroblast cell divisions. One model for predicting daughter cell fate in the mammalian neocortex is also based on cleavage plane orientation. Precursor cell divisions with a cleavage plane orientation that is perpendicular with respect to the ventricular surface (vertical) are predicted to be symmetric, while divisions with a cleavage plane orientation that is parallel to the surface (horizontal) are predicted to be asymmetric neurogenic divisions. However, analysis of cleavage plane orientation at the ventricle suggests that the number of predicted neurogenic divisions might be insufficient to produce large amounts of cortical neurons. To understand factors that correlate with the symmetry of cell divisions, we examined rat neocortical precursor cells in situ through real-time imaging, marker analysis, and electrophysiological recordings. We find that cleavage plane orientation is more closely associated with precursor cell type than with daughter cell fate, as commonly thought. Radial glia cells in the VZ primarily divide with a vertical orientation throughout cortical development and undergo symmetric or asymmetric self-renewing divisions depending on the stage of development. In contrast, most intermediate progenitor cells divide in the subventricular zone with a horizontal orientation and produce symmetric daughter cells. We propose a model for predicting daughter cell fate that considers precursor cell type, stage of development, and the planar segregation of fate determinants.

Morphometry and growth of three Synechococcus-like picoplanktic cyanobacteria at different culture conditions

Three phycocyanin-rich strains of Synechococcus-like picoplanktic cyanobacteria, isolated from the plankton of Czech oligotrophic to eutrophic freshwater reservoirs, were investigated in crossed gradients of light and temperature and in combination with two different culture media (BG-11 and WC). The strains exhibited similar growth and reproduction patterns and displayed overlapping ranges of cell size (1.5 × 0.8 μm) under standardized laboratory conditions (18 μmol m−2 s−1; 20°C). However, strains behavior differed in the crossed gradients. All strains preferred BG-11 medium, where also remarkable size changes could be observed. Length, width, cell abundance and growth rate of two strains were positively correlated with temperature and nutrients, whereas the impact of light intensity was insignificant. Maximum cell elongation (involution cells up to 19 μm) occurred in two strains only in BG-11 medium at highest temperature (28°C) and highest irradiance (53 μmol m−2 s−1). Cell dimensions in WC medium were constant under most conditions given. The third strain was influenced by all three factors, from which light and nutrients played pivotal role. The length of the lag-phase for all strains appeared to be temperature dependent (negative correlation). Despite the fact that the cell volume in all strains increased more than five times under the lowest light and low temperature (6 μmol m−2 s−1, <15°C) in both media, the length/width ratio remained unchanged. The strains differed in the degree of cell enlargement and cell division symmetry as well as in optimum temperature and light dependence. Based on this experimental work two strains could be identified as Synechococcus sp. and one as Cyanobium sp., which can be used as a support for the following genetical analyses.

82: Regulation of initial self-renewing divisions of hematopoietic stem cells by human mesenchymal stromal cells

To identify the signals controlling cell division symmetry and self-renewal of hematopoietic stem cells (HSC) we have studied the cell-cell contact between human HSC and human mesenchymal stem cells (MSC) derived from the marrow, the latter serving as a surrogate “niche” in vitro. By applying novel time lapse microscopy to monitor cell divisions of HSC and correlating cell division symmetry with LTC-IC or myeloid-lymphoid initiating cell (ML-IC) assays at a single cell level, we have previously demonstrated that only vital human MSC were able to drive asymmetric divisions and maintain self-renewal.

Human Mesenchymal Stromal Cells Regulate Initial Self-Renewing Divisions of Hematopoietic Progenitor Cells by a β1-Integrin-Dependent Mechanism

In previous reports, we have demonstrated that only direct cell-cell contact with stromal cells, such as the murine stromal cell line AFT024, was able to alter the cell division kinetics and self-renewing capacity of hematopoietic progenitor cells (HPC). Because beta(1)-integrins were shown to be crucial for the interaction of HPC with the bone marrow microenvironment, we have studied the role of beta(1)-integrins in the regulation of self-renewing cell divisions. For this purpose, we used primary human mesenchymal stromal (MS) cells as in vitro surrogate niche and monitored the division history and subsequent functional fate of individually plated CD34(+)133(+) cells in the absence or presence of an anti-beta(1)-integrin blocking antibody by time-lapse microscopy and subsequent long-term culture-initiating cell (LTC-IC) assays. beta(1)-Integrin-mediated contact with MS cells significantly increased the proportion of asymmetrically dividing cells and led to a substantial increase of LTC-IC. Provided that beta(1)-integrin-mediated contact was available within the first 72 hours, human MS cells were able to recruit HPC into cell cycle and accelerate their division kinetics without loss of stem cell function. Activation of beta(1)-integrins by ligands alone (e.g., fibronectin and vascular cell adhesion molecule-1) was not sufficient to alter the cell division symmetry and promote self-renewal of HPC, thus indicating an indirect effect. These results have provided evidence that primary human MS cells are able to induce self-renewing divisions of HPC by a beta(1)-integrin-dependent mechanism.

Fission yeast cytoskeletons and cell polarity factors: connecting at the cortex

Cell polarity is a fundamental property of cells from unicellular to multicellular organisms. Most of the time, it is essential so that the cells can achieve their function. The fission yeast Schizosaccharomyces pombe is a powerful genetic model organism for studying the molecular mechanisms of the cell polarity process. Indeed, S. pombe cells are rod-shaped and cell growth is restricted at the poles. The accurate localization of the cell growth machinery at the cell cortex, which involves the actin cytoskeleton, depends on cell polarity pathways that are temporally and spatially regulated. The importance of interphase microtubules and cell polarity factors acting at the cortex of cell ends in this process has been shown. Here, we review recent advances in knowledge of molecular pathways leading to the establishment of a cellular axis in fission yeast. We also describe the role of cortical proteins and mitotic cytoskeletal rearrangements that control the symmetry of cell division.

Mesenchymal Stem Cells Regulate the Cell Division Symmetry of Hematopoietic Stem Cells by an Integrin Dependent Mechanism.

Asymmetric cell division is the basal assumption for the generation of a hematpoietic system. On the basis of the murine stromal cell line AFT024 we could demonstrate that cell division symmetry is governed by intercellular contacts between hematopoietic stem cells (HSC) and niche cells. In the adult bone marrow the niche is primarily represented by osteoblasts, whereas during fetal hematopoiesis and pathological extramedullary hematopoiesis in the adulthood mesenchymal stem cells (MSC) are thought to hold this function. Nevertheless data demonstrating an alteration of cell division symmetry by MSC as well as an determination of the type of cellular interaction regulating this process are lacking so far. Therefore the cell division symmetry of individual human PKH-26 labelled CD34+/133+ HSC in contact and non-contact cultures with MSC was monitored for 7d by means of time lapse fluorescence microscopy. For comparison, cultures without feeder layer were monitored. The experiments were performed both, in the absence and presence of early acting cytokines (LIF, IL-6, MIP-1α, G-CSF, TPO, SCF, Flt-3L) promoting the maintenance of primitive progenitors. We found that the vitality and number of dividing cells was exclusively dependent on soluble factors with the highest vitality and number of dividing cells in cytokine augmented MSC cultures (89.9 ± 6.4% vital cells and 98.8 ± 2.7% dividing cells) and the lowest in cultures with neither stromal nor cytokine support (67.9 ± 0.7% vital cells and 41.8 ± 2.5% dividing cells). Nevertheless the percentage of asymmetrically dividing cells was twice as much in MSC contact cultures (22.7 ± 0.7%) than in all other culture conditions (9.1 ± 3.4 to 12.2 ± 2.3%). This demonstrates for the first time that MSC can alter cell division symmetry by a contact dependent mechanism. To identify the type of cellular interactions between HSC and MSC regulating the cell division symmetry, we monitored the latter in prescence of a function blocking β1-integrin antibody that reduced the percentage of asymmetrically dividing cells in MSC contact cultures from 22.7 ± 0.7% to 8.9 ± 4.2%. This was accompanied by a reduction of adherent HSC from 31.6 ± 6.1% to 9.7 ± 4.1% and a loss of functionally primitive cells as determined by a LTC-IC assay. Taken together these data demonstrate for the first time that MSC can alter the cell division symmetry of HSC by a mechanism that is partially dependent on β1-integrin mediated cell contacts.

Egg quality determinants in cod (Gadus morhua L.): egg performance and lipids in eggs from farmed and wild broodstock

Lipids and essential fatty acids, particularly the highly unsaturated fatty acids, 20:5n-3 (eicosapentaenoic acid; EPA), 22:6n-3 (docosahexaenoic acid; DHA) and 20:4n-6 (arachidonic acid, AA) have been shown to be crucial determinants of marine fish reproduction directly affecting fecundity, egg quality, hatching success, larval malformation and pigmentation. In Atlantic cod (Gadus morhua L.) culture, eggs from farmed broodstock can have much lower fertilization and hatching rates than eggs from wild broodstock. The present study aimed to test the hypothesis that potential quality and performance differences between eggs from different cod broodstock would be reflected in differences in lipid and fatty acid composition. Thus eggs were obtained from three broodstock, farmed, wild/fed and wild/unfed, and lipid content, lipid class composition, fatty acid composition and pigment content were determined and related to performance parameters including fertilization rate, symmetry of cell division and survival to hatching. Eggs from farmed broodstock showed significantly lower fertilization rates, cell symmetry and survival to hatching rates than eggs from wild broodstock. There were no differences in total lipid content or the proportions of the major lipid classes between eggs from the different broodstock. However, eggs from farmed broodstock were characterized by having significantly lower levels of some quantitatively minor phospholipid classes, particularly phosphatidylinositol. There were no differences between eggs from farmed and wild broodstock in the proportions of saturated, monounsaturated and total polyunsaturated fatty acids. The DHA content was also similar. However, eggs from farmed broodstock had significantly lower levels of AA, and consequently significantly higher EPA/AA ratios than eggs from wild broodstock. Total pigment and astaxanthin levels were significantly higher in eggs from wild broodstock. Therefore, the levels of AA and phosphatidylinositol, the predominant AA-containing lipid class, and egg pigment content were positively related to egg quality or performance parameters such as fertilization and hatching success rates, and cell symmetry.

The fate of isolated blastomeres with respect to germ cell formation in the amphipod crustacean Parhyale hawaiensis

Germ cells may be specified through the localization of germ line determinants to specific cells in early embryogenesis, or by inductive signals from neighboring cells to germ cell precursors in later embryogenesis. Such determinants can be produced and localized during or after oogenesis, either autonomously by oocytes or by associated nutritive cells. In Drosophila, each oocyte is connected to nurse cells by cytoplasmic bridges, and determinants synthesized in nurse cells are transported through these bridges to the oocyte. However, the Drosophila model may not be applicable to all arthropods, since in many species of all four extant arthropod classes, gametogenesis functions without nurse cells. In this paper, I use immunodetection of Vasa protein to study germ cell development in the amphipod crustacean Parhyale hawaiensis, a species whose ovaries lack nurse cells and whose eggs lack obvious polarity. Previous cell lineage analyses have shown that all three germ layers and the germ line are exclusively specified by third cleavage. In the present study, I use a molecular marker to follow germ cell development during P. hawaiensis embryogenesis. I determine the capacity of individual blastomeres to form germ cells by isolating blastomeres at early cleavage stages and provide experimental evidence for localized germ cell determinants at the two-cell stage in P. hawaiensis. These experiments indicate that many aspects of early amphipod development, including timing and symmetry of cell division, the transition from holoblastic to superficial cleavage, and possibly some gastrulation movements, are cell autonomous following first cleavage.

Symmetry of Initial Cell Divisions Among Primitive Hematopoietic Progenitors Is Independent of Ontogenic Age and Regulatory Molecules

We have developed a time-lapse camera system to follow the replication history and the fate of hematopoietic stem cells (HSC) at a single-cell level. Combined with single-cell culture, we correlated the early replication behavior with colony development after 14 days. The membrane dye PKH26 was used to monitor cell division. In addition to multiple, synchronous, and symmetric divisions, single-sorted CD34(+)/CD38(-) cells derived from fetal liver (FLV) also gave rise to a daughter cell that remained quiescent for up to 8 days, whereas the other daughter cell proliferated exponentially. Upon separation and replating as single cells onto medium containing a cytokine cocktail, 60.6% +/- 9.8% of the initially quiescent cells (PKH26 bright) gave rise again to colonies and 15.8% +/- 7.8% to blast colonies that could be replated. We have then determined the effects of various regulatory molecules on symmetry of initial cell divisions. After single-cell sorting, the CD34(+)/CD38(-) cells derived from FLV were exposed to flt3-ligand, thrombopoietin, stem cell factor (SCF), or medium containing a cytokine cocktail (with SCF, interleukin-3, interleukin-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin). Whereas mitotic rate, colony efficiency, and asymmetric divisions could be altered using various regulatory molecules, the asymmetric division index, defined as the number of asymmetric divisions versus the number of dividing cells, was not altered significantly. This observation suggests that, although lineage commitment and cell proliferation can be skewed by extrinsic signaling, symmetry of early divisions is probably under the control of intrinsic factors.

Unequal cleavage in leech embryos: zygotic transcription is required for correct spindle orientation in subset of early blastomeres.

Leech embryos undergo invariant sequences of equal and unequal cell divisions to give rise to identifiable progeny cells. While many of the early cleavages are under maternal control, the divisions of a subset of early blastomeres (the large cells of the D' lineage) are perturbed after the inhibition of zygotic transcription. Analysis of the different types of cells produced in embryos injected with the transcriptional inhibitor, alpha-amanitin, revealed that the symmetry of cell division is perturbed in these large D'-derived cells during this early period of development. These cells, which would normally undergo a series of equal and unequal cleavages, always undergo equal cleavages after the inhibition of zygotic transcription. It appears that zygotically transcribed gene product(s) are required in the large cells of the D' lineage to orient the mitotic spindles properly for these unequal cell cleavages.

A Monte Carlo Simulation of the Escherichia coliCell Cycle

A Monte-Carlo simulation of the division cycle of an individual bacterium has been developed to test theories concerning the control of cell cycle events and for the analysis of cell cycle data. The model is based on the work of Cooper and Helmstetter and other theories concerning the control of DNA replication initiation, chromosome segregation and cell division. Variability in the molecular events that initiate a particular cell cycle event is incorporated using a Monte Carlo approach. The model results are compared with experimental data from a number of different sources and collected using a number of different analytical techniques. This model is able to accurately represent the cell size distributions of an exponentially growing population as determined by a Coulter Counter and the DNA distributions as determined by flow cytometry. The model is also able to accurately simulate the cell age distribution and chromosome replication and segregation patterns as determined by the membrane-elution technique. Results from parameter sensitivity analysis indicate that variability in the symmetry of cell division and in the cell size at initiation of chromosome replication have the most significant impact on the cell size, age and DNA distributions. Results from the membrane-elution simulation support the hypothesis that total cell protein is synthesized exponentially during the division cycle and that cell size control is a plausible explanation for control of DNA replication. The simulation results for chromosome segregation agree with experimental data and support chromosome strand segregation models. In the future, this simulation should be useful in testing complex theories about molecular control of cell cycle events against experimental data.

The Independence of Cell Shape and Overall Form in Multicellular Algae and Land Plants: Cells Do Not Act as Building Blocks for Constructing Plant Organs

We evaluate here whether the mechanisms regulating cellular morphogenesis in planar structures are also responsible for dictating their overall shapes. A comparative survey of 15 multicellular algae and land plants was performed in order to identify the common features associated with cellular morphogenesis in their immature planar structures. The superficial layers of all such structures are partitioned into polygonal cellular arrays that are characterized by specific mean numbers of walls per cell, narrow ranges of cell sizes, and regular cell shapes. The ability to generate these polygonal arrays is independent of overall shape, evolutionary lineage, cell layer number, cell division pattern, and cell wall composition. Instead, geometric principles dictate that the superficial layers of plant structures exhibit mean numbers of ca. 6.0 walls per cell, which result from the tendency of cell plates in most dividing plant cells to avoid existing three-way vertices. In contrast, the superficial layers of algal structures exhibit mean numbers between 5.5 and 5.6 walls per cell, which depend on the ability of cleavage furrows in dividing algal cells to arise at existing three-way vertices. Sequential observations of developing prothalli of the fern Onoclea sensibilis were used to characterize the regulation of cell size and cell shape in this representative planar structure. Cell biological mechanisms appear to dictate cell size via the control over the symmetry, timing, and orientation of cell division. Biomechanical mechanisms regulate cell shape in a remarkable manner. Cell walls in the region of isodiametrical expansion pivot around their vertices so that the observed angles approach the ideal angles calculated for equiangular polygons. Additional divisions alter the wall numbers of the three cells around each vertex, but the observed angles continue to adjust so that they parallel the predicted ideal angles. Angle rotation is accompanied by differential amounts of relative wall expansion, which are inversely dependent on wall length. It appears as if the vertices of dividing cells undergoing isodiametrical expansion are attempting to reach the condition of local mechanical equilibrium. But the vertices in older cells undergoing preferential elongation along one axis tend to revert to the original rightangle configuration in the attempt to accommodate to global growth stresses. Thus, biomechanical mechanisms regulating cell shape allow the prothallus to approach mechanical stability. Last, the polygonal cellular array in planar structures is viewed as the morphogenetic equivalent of a ground state; i.e., it is generated without any evident input of specific genetic instructions. We conclude that the local regulation of cellular morphogenesis is completely independent of the global regulation of the overall form of planar structures.

The effect of DL-7-azatryptophan on heterocyst development in the cyanobacterium Anabaena cylindrica

SUMMARY: Addition of the amino acid analogue dl-7-azatryptophan (AT) to dinitrogen-fixing cultures of the cyanobacterium Anabaena cylindrica, and to cultures in which heterocyst development was induced by the removal of fixed nitrogen from the medium, resulted in the development of many adjacent (double) heterocysts. Cell division in A. cylindrica was asymmetrical, with a maximum of 10% of all cell divisions producing two daughter cells of equal size. During incubation with AT the frequency of symmetrical cell divisions remained unchanged, indicating that the preponderance of double heterocysts induced by the analogue did not result from any change in the symmetry of cell division. Incubation of cultures with AT resulted in a decrease in the number of vegetative cells between heterocysts (the interheterocyst interval). The extent of the decrease was proportional to the length of the incubation period in the presence of AT. Double heterocysts, which constitute a zero interval, developed at a time when the minimum interval was three cells in dinitrogen-fixing cultures, or nine cells in cultures induced to differentiate by the removal of fixed nitrogen from the medium. These observations have been used to formulate a model to explain the influence of AT on the control of heterocyst development and spacing. In this model the inactive form of an inhibitor of heterocyst development is produced constitutively by vegetative cells and is activated either by a co-inhibitor derived from developing or mature heterocysts, or by high concentrations of fixed nitrogen.