Alterations in the cell wall of Spirillum serpens VHL early in its association with Bdellovibrio bacteriovorus 109D

In both freeze-etched and critical-point dried preparations examined by transmission and scanning electron microscopy, respectively, the outer surfaces of the cells of Spirillum serpens VHL assume a wrinkled appearance 10–15 min after challenge by Bdellovibrion bacteriovorus 109D. This wrinkling effect is believed (on circumstantial evidence) to be caused by the bdellovibrio's disruption of the cell wall lipoprotein of the Spirillum. With the exception of those topological changes caused by wrinkling, the outer membrane of the Spirillum cell wall retains a normal appearance as viewed in freeze-etched preparations, even after the Spirillum cell has been converted into a bdelloplast. Although the peptidoglycan layer of the Spirillum cell presumably is weakened somewhat by the invading Bdellovibrio, evidence obtained from freeze-fractured preparations of Spirillum bdelloplasts suggests that the peptidoglycan remains as a discrete cell wall layer, even though the Spirillum cell wall apparently has lost much of its rigidity. That the peptidoglycan backbone remains essentially intact, even after the Spirillum cell has been entered by the Bdellovibrio, is supported by the observation that the soluble amino sugar content of the culture medium, as determined by chemical analysis, does not rise even 5.0 h after the association of the Bdellovibrio with the Spirillum has begun.     

Cell division in a minimal bacterium in the absence of ftsZ

Mycoplasma genomes exhibit an impressively low amount of genes involved in cell division and some species even lack the ftsZ gene, which is found widespread in the microbial world and is considered essential for cell division by binary fission. We constructed a Mycoplasma genitalium ftsZ null mutant by gene replacement to investigate the role of this gene and the presence of alternative cell division mechanisms in this minimal bacterium. Our results demonstrate that ftsZ is non‐essential for cell growth and reveal that, in the absence of the FtsZ protein, M. genitalium can manage feasible cell divisions and cytokinesis using the force generated by its motile machinery. This is an alternative mechanism, completely independent of the FtsZ protein, to perform cell division by binary fission in a microorganism. We also propose that the mycoplasma cytoskeleton, a complex network of proteins involved in many aspects of the biology of these microorganisms, may have taken over the function of many genes involved in cell division, allowing their loss in the regressive evolution of the streamlined mycoplasma genomes.     

The Problem of Nucleolar Number in Cell Nuclei and in Sections of Cell Nuclei

In this paper the mean number N of nucleoli of cell nuclei and the relative frequency PN of cell nuclei with N nucleoli are investigated. To solve the first problem, the stereological model of convex shaped nuclei and nucleoli and the assumption of randomly-isotropically distributed objects in space are used. The model is developed for non-vanishing thickness T of tissue sections. The relationship between the unknown number N of nucleoli in cell nuclei and the mean number n of nucleoli in nuclear sections determined by counting is N = n (D+T)/(d + T). Here D and d are the mean values of caliper diameter for nuclei and for nucleoli, resp. The second problem is illustrated by means of some biomedical examples: The relative frequency PN of cell nuclei with N nucleoli can be approximated by a generalized Poisson distribution in all investigated cases. Therefore the mean nucleolar number N is the essential parameter to describe the frequencies of cell nuclei with different numbers of nucleoli.     

Cell size and chloroplast size in relation to chloroplast replication in light-grown wheat leaves

As part of an investigation into the control of chloroplast replication the number and size of chloroplasts in mesophyll cells was examined in relation to the size of the cells. In first leaves of Triticum aestivum L. and T. monococcum L. the number of chloroplasts in fully expanded mesophyll cells is positively correlated with the plan area of the cells. The linear relationship between chloroplast number per cell and cell plan area is also consistent over a fivefold range of cell size in isogenic diploid and tetraploid T. monococcum. In T. aestivum the chloroplast number per unit cell plan area varies among cells in relation to the size of the chloroplasts. Those cells containing chloroplasts with a relatively small face area have a correspondingly higher density of chloroplasts, and consequently, the total chloroplast area per unit cell plan area is very similar in all the cells. The results indicate that the proportion of the cell surface area covered by chloroplasts is precisely regulated, and that this is achieved during cell development by growth and replication of the chloroplasts.     

cAMP-mediated increase in the critical cell size required for the G1 to S transition in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, cyclic AMP is required for cellular growth. In this study we show that cAMP also specifically inhibits the G1-S transition of the S. cerevisiae cell cycle by increasing the critical cell size required at start, the major yeast cell cycle control step. In fact: (a) addition of cAMP delays the time of entering into the S budded phase of small G1 cells, while it is ineffective on large fast-growing cells. (b) If cell growth is strongly depressed, cAMP permanently inhibits cell cycle commitment of cells arrested at the α-factor-sensitive step. The cell fraction inhibited by cAMP is inversely correlated with the average cell size of treated populations. (c) The critical protein content (P_s) and the critical cell volume (V_B) required for budding in unperturbed exponentially growing yeast populations are largely increased by cAMP. On these bases, we propose a new cAMP role at start.     

Coordination of cell proliferation and cell expansion in the control of leaf size in Arabidopsis thaliana

Size is an important parameter in the characterization of organ morphology and function. To understand the mechanisms that control leaf size, we previously isolated a number of Arabidopsis thaliana mutants with altered leaf size. Because leaf morphogenesis depends on determinate cell proliferation, the size of a mature leaf is controlled by variation in cell size and number. Therefore, leaf-size mutants should be classified according to the effects of the mutations on the cell number and/or size. A group of mutants represented by angustifolia3/grf-interacting factor1 and aintegumenta exhibits an intriguing cellular phenotype termed compensation: when the leaf cell number is decreased due to the mutation, the leaf cell size increases, leading to compensation in leaf area. Several lines of genetic evidence suggest that compensation is probably not a result of the uncoupling of cell division from cell growth. Rather, the evidence suggests an organ-wide mechanism that coordinates cell proliferation with cell expansion during leaf development. Our results provide a key, novel concept that explains how leaf size is controlled at the organ level.     

<Emphasis Type="Italic">FORMOSA</Emphasis> controls cell division and expansion during floral development in <Emphasis Type="Italic">Antirrhinum</Emphasis><Emphasis Type="Italic">majus</Emphasis>

Control of organ size is the product of coordinated cell division and expansion. In plants where one of these pathways is perturbed, organ size is often unaffected as compensation mechanisms are brought into play. The number of founder cells in organ primordia, dividing cells, and the period of cell proliferation determine cell number in lateral organs. We have identified the Antirrhinum FORMOSA (FO) gene as a specific regulator of floral size. Analysis of cell size and number in the fo mutant, which has increased flower size, indicates that FO is an organ-specific inhibitor of cell division and activator of cell expansion. Increased cell number in fo floral organs correlated with upregulation of genes involved in the cell cycle. In Arabidopsis the AINTEGUMENTA (ANT) gene promotes cell division. In the fo mutant increased cell number also correlates with upregulation of an Antirrhinum ANT-like gene (Am-ANT) in inflorescences that is very closely related to ANT and shares a similar expression pattern, suggesting that they may be functional equivalents. Increased cell proliferation is thought to be compensated for by reduced cell expansion to maintain organ size. In Arabidopsis petal cell expansion is inhibited by the BIGPETAL (BPE) gene, and in the fo mutant reduced cell size corresponded to upregulation of an Antirrhinum BPE-like gene (Am-BPE). Our data suggest that FO inhibits cell proliferation by negatively regulating Am-ANT, and acts upstream of Am-BPE to coordinate floral organ size. This demonstrates that organ size is modulated by the organ-specific control of both general and local gene networks. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fibroblast-myocyte interactions in in vitro cardiomyogenesis

When in dissociated cell culture, heart muscle cells and fibroblasts taken from 8-day chick embryos do not remain unaffected by one another. Instead they interact from early incubation on. The interaction of dissociated heart cells appears to form the basis of cardiomyogenesis in vitro as obtained by various laboratories. Our cinematographs of monolayer cultures show six different phenomena: 1. When a fibroblast, wandering in a heart cell monolayer culture, enters the sphere of influence of a myocyte, contact between the two cell types occurs. To this effect the fibroblast changes its direction or its leading lamella ramifies. 2. The area of the muscle cell contacted by the fibroblast tends to dart out. The resulting myocyte process advances along the fibroblast. 3. Similar processes can be pulled out by the retracting fibroblast, and be guided passively to neighboring myocytes. 4. Thin threads produced in like manner and often repeatedly contacted by the handling lamella of the fibroblast subsequently broaden and develop into intercellular bridges. 5. A conspicuous reaction of myocytes after contact with fibroblasts is the accelerated initiation of the spreading phenomenon in which the muscle cell polarizes and starts to beat. 6. After contact, a myocyte can be transported to a neighboring muscle cell by a fibroblast. These six interactions result in the development of synchronously pulsating muscle cell units.     

Early Morphogenesis of Glugea-lnduced Xenomas in Laboratory-Reared Flounder

Glugea stephani-infecled submucosal cells of laboratory-reared winter flounder, Pseudopleuronectes americanus, change to unique giant cell xenomas. These cells hypertrophy while the intracellular Glugea propagate to high numbers in the cytoplasm and eventually overrun the host cell. The xenomas slowly reach 20-25 muml;m (at 17°C) by day 10 after parasite invasion. These xenomas (eight often examined by electron microscopy) are coated with a mucus-like envelope onto which is attached a layer of endothelial ceils. The 10-day xenomas display (a) host cell endonuclear polyploidization and amitosis, (b) limited parasite growth and reproduction, and (c) a host cell cytoplasm structure similar to that seen in undifferentiated phagocytes. Glugea parasites do not induce obvious cell degenerative effects in 10-day xenomas; the 20-day xenomas are hypertrophied to 70-100 m?m. These cells are characterized by (a) a host cell component denuded of endoplasmic reticulum and phagosome membrane, (b) a reduction in host cell ribosomes and the disappearance of host cell cytoskeletal assemblages, including microtubules, and (c) a significant increase in vegetative Glugea within xenomas. Evidence indicates cytoplasmic calcium of the host cell xenoma is perturbed by the intracellular Glugea; the alterations in the host cell calcium domains may be a big factor in the induction of the block of mitosis in the host cell and in the disruption in its cytoskeletal controls.     

On the average cellular volume in synchronized cell populations

As was done by Sinclair and Ross (1969(, we consider a cellular population that consists initially (at time zero) ofN ₀ newborn cells, all with the same volumev _o. It is assumed that the occurrence of cell division is determined only by a cell’s age, and not by its volume. The frequency function of interdivision times, τ, is denoted byf(τ). If cell death is negligible, the expected number of cells,N(t), will increase according to the laws of a simple age-dependent branching process. The expression forN(t) is obtained as a sum over all generations; thevth term of this sum, in turn, is a multiple convolution integral, reflecting the life history ofvth generation cells (i.e., the lengths of thev successive interdivision periods plus the age of the cell at timet). Assuming that cell volume is a given function of cell age, e.g., linear or exponential, and that cellular volume is exactly halved at each division, it is possible to calculate the volume of a cell with a given life history, and thus the average cellular volume of the whole population as a function of time. If at time zero the volumes differ from cell to cell, the final equation must be modified by averaging over initial volumes. In the case of linear volume increase with age, a very simple asymptotic expression is found for the average cellular volume ast→∞. The case of exponential volume increase with age also leads to a simple asymptotic formula, but the resulting volume distribution is unstable. The mean cellular volume at birth and the second moment of the volume distribution can be calculated in a similar manner. Work supported by the U.S. Atomic Energy Commission.     

Piwi expression in archeocytes and choanocytes in demosponges: insights into the stem cell system in demosponges

SUMMARY Little is known about the stem cells of organisms early in metazoan evolution. To characterize the stem cell system in demosponges, we identified Piwi homologs of a freshwater sponge, Ephydatia fluviatilis, as candidate stem cell (archeocyte) markers. EfPiwiA mRNA was expressed in cells with archeocyte cell morphological features. We demonstrated that these EfPiwiA‐expressing cells were indeed stem cells by showing their ability to proliferate, as indicated by BrdU‐incorporation, and to differentiate, as indicated by the coexpression of EfPiwiA with cell‐lineage‐specific genes in presumptive committed archeocytes. EfPiwiA mRNA expression was maintained in mature choanocytes forming chambers, in contrast to the transition of gene expression from EfPiwiA to cell‐lineage‐specific markers during archeocyte differentiation into other cell types. Choanocytes are food‐entrapping cells with morphological features similar to those of choanoflagellates (microvillus collar and a flagellum). Their known abilities to transform into archeocytes under specific circumstances and to give rise to gametes (mostly sperm) indicate that even when they are fully differentiated, choanocytes maintain pluripotent stem cell‐like potential. Based on the specific expression of EfPiwiA in archeocytes and choanocytes, combined with previous studies, we propose that both archeocytes and choanocytes are components of the demosponge stem cell system. We discuss the possibility that choanocytes might represent the ancestral stem cells, whereas archeocytes might represent stem cells that further evolved in ancestral multicellular organisms.     

d-canavanine affects peptidoglycan structure,morphogenesis and fitness in Rhizobiales

The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for maintenance of cell shape and survival. Many bacteria alter their PG chemistry as a strategy to adapt their cell wall to external challenges. Therefore, identifying these environmental cues is important to better understand the interplay between microbes and their habitat. Here, we used the soil bacterium Pseudomonas putida to uncover cell wall modulators from plant extracts and found canavanine (CAN), a non-proteinogenic amino acid. We demonstrated that cell wall chemical editing by CAN is licensed by P. putida BSAR, a broad-spectrum racemase which catalyses production of dl -CAN from l -CAN, which is produced by many legumes. Importantly, d -CAN diffuses to the extracellular milieu thereby having a potential impact on other organisms inhabiting the same niche. Our results show that d -CAN alters dramatically the PG structure of Rhizobiales (e.g., Agrobacterium tumefaciens, Sinorhizobium meliloti), impairing PG crosslinkage and cell division. Using A. tumefaciens, we demonstrated that the detrimental effect of d -CAN is suppressed by a single amino acid substitution in the cell division PG transpeptidase penicillin binding protein 3a. Collectively, this work highlights the role of amino acid racemization in cell wall chemical editing and fitness.     

Ultrastructure of the nephridio-circulatory connections in Tubulanus annulatus (Nemertini,Anopla)

Summary The protonephridial terminal organs in the nemertean Tubulanus annulatus form an integral part of the blood vessel wall. Both endothelial and muscle-cell layers of the vessel's wall are discontinued at the site of each terminal organ. The terminal organs are usually composed of from one to three terminal cells enclosing a central lumen provided with many microvilli and separated from the blood vessel's lumen by a membranous filtration area. The latter is perforated by numerous winding clefts formed by interdigitation of minute cytoplasmic pedicels arising from processes issued by each of the involved terminal cells. Ultrafiltration of blood plasma takes place across a filtration membrane which spans the cleft system and the basal lamina of the terminal cells. Fluid is propelled into the lumen of the terminal organs through the activity of ciliary bundles, one for each terminal cell involved, perhaps supplemented by vascular turgor. All efferent conduits of the protonephridium have profuse infoldings of the luminal cell surfaces and/or numerous pinocytotic pits suggestive of reabsorption of substances from the primary urine.Abbreviations BL basal lamina - C cilium - CP coated pit - CT collecting tubule - CV inzcoated vesicle - D dictyosome - E endothelial cell - F fenestration of endothelial cell - FA filtration area - FM filtration membrane - G glycogen granule - LV lateral vessel - M mitochondrion - MC muscle cell - MV microvillus - N nucleus of terminal cell - NE nucleus of endothelial cell - NP nephridiopore - PC protonephridial capillary cell - PT protonephridial tubule - R rootlet - TC terminal cell     

Micronuclei and apoptosis in glioma and neuroblastoma cell lines and role of other lesions in the reconstruction of cellular radiosensitivity

It is now well established that micronuclei frequency does not always rank cell lines according to radiosensitivity. There is, however, a growing interest in reconstructing cellular radiosensitivity (measured by colony assay) from concurrent micronucleus and apoptosis data. Using a variety of radiosensitive and radioresistant cell lines, we have derived a missing parameter –P _oe , the probability of cell death by other events such as small deletions, chromosome aberrations, late apoptosis and necrosis which are undetectable by micronucleus and apoptosis assays performed at a single time point. In the radioresistant glioma cell lines G120, G60, G28, G44 and G62 (SF2 ≥0.59), a characteristic threshold dose exists above which cell loss due to undetectable lesions occurs. In the radiosensitive SK-N-SH and KELLY cell lines (SF2 ≤0.43), the P _oe parameter is positive at very low doses, reaches a maximum and declines at higher doses. In the radiation resistant G28 cells, P _oe was found to be below zero for doses up to 6 Gy. In the G62, G44 and G120 cell lines, the threshold doses to induce P _oe events were 0.87, 3.04 and 3.85 Gy, respectively. Cell death by undetectable lesions is a cell-specific and time-dependent variable. Micronucleus and apoptosis assays performed concurrently and at a specific time point miss cell death due to other events and this may be the reason why reconstruction of cellular radiosensitivity from micronucleus and apoptosis data fails. Received: 8 March 2001 / Accepted: 1 September 2001     

Analysis of Events Associated with Serum Deprivation-Induced Apoptosis in C3H/Sol8 Muscle Satellite Cells

Satellite cells are the source of new muscle fibers in postnatal skeletal muscle growth and regeneration. Regulation of satellite cell survival is of fundamental importance in maintaining normal muscle function. Here we describe and characterize a tissue culture model of satellite cell apoptosis. Kinetic studies indicate that serum deprivation triggers a set of sequential events: early cell death, transient cell cycle traverse, and delayed cell death. The satellite cell death occurs by apoptosis based on the internucleosomal DNA laddering,in situDNA end-labeling, and the requirements forde novoprotein synthesis and extracellular calcium influx. The transient period of cell cycle progression (7–11 h after serum withdrawal) is accompanied by temporal induction of members of the immediate early gene family, such as c-myc,c-fos,and SRF, and appears to precede the delayed phase of cell death. Satellite cell apoptosis can be suppressed by several growth factors and by blocking the activity of calpain, a calcium-regulated protease. The late phase of apoptosis is marked by selective activation of ubiquitin-mediated protein conjugation and degradation. This study defines several control points where satellite cell apoptosis may be genetically or pharmacologically intervened.     

A new cell division operon in Escherichia coli

Summary At 76 min on theE. coli genetic map there is a cluster of genes affecting essential cellular functions, including the heat shock response and cell division. A combination ofin-vivo andin-vitro genetic analysis of cell division mutants suggests that the cell division genefts E is the second gene in a 3 gene operon. A cold-sensitive mutant, defective in the third gene, is also unable to divide at the restrictive temperature, and we designate this new cell division genefts X. Another cell division gene,fts S, is very close to, but distinct from, the 3 genes of the operon. Thefts E product is a 24.5 Kd polypeptide which shows strong homology with a small group of proteins involved in transport. Both thefts E product and the protein coded by the first gene (fts Y) in the operon have a sequence motif found in a wide range of heterogeneous proteins, including the Ras proteins of yeast. This common domain is indicative of a nucleotide-binding site.     

De novo morphogenesis in L‐forms via geometric control of cell growth

In virtually all bacteria, the cell wall is crucial for mechanical integrity and for determining cell shape. Escherichia coli's rod‐like shape is maintained via the spatiotemporal patterning of cell‐wall synthesis by the actin homologue MreB. Here, we transiently inhibited cell‐wall synthesis in E. coli to generate cell‐wall‐deficient, spherical L‐forms, and found that they robustly reverted to a rod‐like shape within several generations after inhibition cessation. The chemical composition of the cell wall remained essentially unchanged during this process, as indicated by liquid chromatography. Throughout reversion, MreB localized to inwardly curved regions of the cell, and fluorescent cell wall labelling revealed that MreB targets synthesis to those regions. When exposed to the MreB inhibitor A22, reverting cells regrew a cell wall but failed to recover a rod‐like shape. Our results suggest that MreB provides the geometric measure that allows E. coli to actively establish and regulate its morphology.