Control of morphogenesis in Arthrobacter crystallopoiets: effect of cyclic adenosine 3'',5''-monophosphate.

The intracellular levels of cyclic adenosine 3',5'-monophosphate (cyclic AMP) were measured at various intervals during growth and morphogenesis in Arthrobacter crystallopoietes. Cyclic AMP levels remained relatively constant throughout growth in spherical cells grown in glucose-based media. Immediately after inoculation of spheres from glucose- to succinate-containing media, a 30-fold increase in intracellular cyclic AMP was detected. This dramatic rise in cyclic AMP preceded the observed change in cellular morphology from spheres to rods. The cyclic AMP level in rod-shaped cells rapidly dropped to a relatively stable concentration during the exponential growth phase. At the onset of stationary phase and rod-to-sphere morphological transition, a second peak of cyclic AMP was observed. Neither of these two peaks was detectable in a morphogenetic mutant that grew only as spheres. The intracellular levels of cyclic AMP in this mutant remained constant throughout exponential growth and decreased slightly during stationary phase. Effects of exogenously added cyclic nucleotides and their derivatives to both parent and mutant cultures were investigated. The data presented indicate that dramatic changes in intracellular cyclic AMP levels occur just before the morphological transitions characteristic of the morphogenetic cycle in A. crystallopoietes. It is suggested that cyclic AMP is a contributing factor in the regulatory phenomenon associated with morphogenesis in this bacterium.     

Intracellular Substrates for Endogenous Metabolism During Long-Term Starvation of Rod and Spherical Cells of Arthrobacter crystallopoietes

Cells of Arthrobacter crystallopoietes, harvested during growth as spheres and as rods, were starved by shaking at 30 C in phosphate buffer for 30 days, during which time they maintained 100% viability. Changes in cellular components and the activity of specific enzyme pathways were monitored. A glycogen-like polysaccharide comprised 40% of the dry weight of growing spherical cells and 10% of the dry weight of rod cells. This material was utilized at approximately the same rate, on a percentage basis, during starvation of both cell forms. The rods degraded intracellular protein at approximately twice the rate of the spheres. At the end of 30 days, the rods had degraded 40% and the spheres 20% of their initial content of protein. Ribonucleic acid (RNA) was degraded significantly more rapidly in the rods. After 30 days starvation, 85 and 32% of the initial RNA of rods and spheres, respectively, had been depleted. Magnesium ion followed this same general pattern; the rods lost 65% and the spheres 45% of their initial content during 28 days of starvation. Deoxyribonucleic acid increased by 20% during the first few hours of starvation of both cell forms and then remained constant. The ability of glucose-, succinate-, and 2-hydroxypyridine (2-HP)-grown cells to oxidize glucose remained constant during 14 days of starvation. The ability of succinate-grown cells to oxidize succinate decreased rapidly during the first few hours of starvation to a rate which remained constant for 14 days. Cells adapted to growth on 2-HP completely lost their ability to oxidize this substrate after 3 days starvation.     

Macromolecular synthesis and cell division during morphogenesis of Arthrobacter crystallopoietes

The sphere-rod-sphere morphology cycle of Arthrobacter crystallopoietes was accompanied by changes in the rate of growth and the rates of DNA, RNA and protein synthesis. The patterns of macromolecule synthesis resembled those found in other bacteria during a step-up followed by a step-down in growth rate. During the step-up in growth spherical cells grew into rods and macromolecules were synthesized in the absence of cell division. During stepdown, successive rounds of septation produced progressively smaller cells which did not separate and remained in chains. The morphology of the cells was dependent on the growth rate and could be altered by changing the dilution rate in a malate-limited chemostat. Gradual transitions in morphology and gradual increases in macromolecule content of the cells occurred as the growth rate was increased in the chemostat. Sphere to rod morphogenesis occurred when DNA synthesis was inhibited by treatment with mitomycin C or by thymine starvation. The DNA-deficient rods did not divide and eventually lysed. DNA, RNA and protein synthesis were continuously required for the reductive division of rods to spheres.Abbreviations MS mineral salts - GS mineral salts plus glucose - CA casamino acids - GSCA mineral salts plus glucose plus casamino acids - cAMP cyclic adenosine-3,5-monophosphate - RNA ribonucleic acid - DNA deoxyribonucleic acid     

A new slant to the Z ring and bacterial cell branch formation

Rod-shaped bacteria such as Escherichia coli accurately maintain their shape from generation to generation. The cytoskeletal proteins MreB and FtsZ, which respectively guide parallel growth of the sidewall and perpendicular growth of the division septum, are important to maintain a straight sidewall and uniformly rounded cell poles. FtsZ normally assembles into a ring at the cell midpoint, called the Z ring, which is oriented perpendicular to the cell's axis and is thus in perfect position to guide growth of a perpendicular septum. In this issue of Molecular Microbiology, Potluri et al. show that low molecular weight penicillin binding proteins, particularly PBP5, have a role in maintaining the perpendicular geometry of the Z ring and subsequent septum in E. coli. When these factors are absent or perturbed, division septa are readily deformed, which results in abnormal cell poles that often bifurcate over time to generate branches. The data suggest that cellular branching in E. coli is specifically induced by aberrant septation events caused by mis-oriented Z rings and not by deformation of a growing cell pole or emergence of new tips from the sidewall, which are likely mechanisms of branching in other bacterial families.     

Isolation and characterization of morphogenetic mutants of Arthrobacter crystallopoietes

Mutants of Arthrobacter crystallopoietes that exhibited altered ability to undergo the normal sphere-to-rod-to-sphere morphogenetic cycle were isolated. The procedure used to isolate these mutants involved velocity sedimentation in a sterile sucrose gradient to separate morphogenesis-deficient spherical cells from rod-shaped cells capable of normal morphogenesis. Three classes of mutants were obtained: (i) those that cannot form rods, (ii) those that cannot form long rods, and (iii) those that form long rods but exhibit more extensive rudimentary branching than the wild type. The isolation and characterization of these mutants are described, and the use of these mutants in the study of the morphogenetic cycle of arthrobacters is discussed.     

Regulation of cyclic AMP levels in Arthrobacter crystallopoietes and a morphogenetic mutant.

The extracellular levels of cyclic AMP (cAMP), cAMP phosphodiesterase activity, and adenylate cyclase activity were measured at various intervals during growth and morphogenesis of Arthrobacter crystallopoietes. There was a significant rise in the extracellular cAMP level at the onset of stationary phase, and this rise coincided with a decrease in intracellular cAMP. The phosphodiesterase activity measured in vitro increased in the early exponential phase of growth as intracellular cAMP decreased, and, conversely, prior to the onset of stationary phase the phosphodiesterase activity decreased as the intracellular cAMP levels increased. Adenylate cyclase activity was greater in cell extracts prepared from cells grown in a medium where morphogenesis was observed. Pyruvate stimulated adenylate cyclase activity in vitro. A morphogenetic mutant, able to grow only as spheres in all media tested, was shown to have altered adenylated cyclase activity, whereas no significant difference compared to the parent strain was detectable in either the phosphodiesterase activity or the levels of extracellular cAMP. The roles of the two enzymes, adenylate cyclase and phosphodiesterase, and excretion of cAMP are discussed with regard to regulation of intracellular cAMP levels and morphogenesis.     

Sublethal High Hydrostatic Pressure Treatment Reveals the Importance of Genes Coding Cytoskeletal Protein in Escherichia Coli Morphogenesis

We studied morphologic changes after sublethal high hydrostatic pressure treatment (HPT) of Escherichia coli K-12 strains in which genes related to the cytoskeleton, cell wall, and cell division had been deleted. Some long filamentous and swelling cells were observed in wild-type bacteria, while some spherical, branched, or collapsed cells were observed in deletion mutants. In particular, ΔzapA and ΔrodZ showed distinguished morphologies. ZapA supports FtsZ, a cytoskeletal protein, forming ring with ZapB. RodZ, a cytoskeletal protein, interacts with MreB, also a cytoskeletal protein, and both factors are necessary for maintaining the rod shape of the cell. These results showed that insufficient formation of FtsZ rings induced cell elongation and that insufficient formation of MreB induced a branched and collapsed cell shape. Therefore, the correct formation of the bacteria cytoskeleton by FtsZ rings and MreB is important for keeping normal cell shape during growth after HPT, and the polymerization of cytoskeletal proteins was a critical target of sublethal HPT. These results indicate that sublethal HPT induces bacterial cell morphologic change and provide important information on the role of genes involved in morphogenesis. Therefore, sublethal HPT may be a good tool for studying the morphogenesis of bacterial cells.     

Sphere-rod morphogenesis in Arthrobacter crystallopoietes. II. Peptides of the cell wall peptidoglycan

Cell walls of Arthrobacter crystallopoietes grown as spheres and as rods were solubilized by treatment with the B enzyme from Chalaropsis, an N-acetylmuramidase. The neutral glycopeptides were then isolated by chromatography on ECTEOLA cellulose. The glycopeptides, consisting of disaccharide-peptide units interlinked by peptide cross-bridges, were fractionated by gel filtration on Sephadex columns into oligomers of various sizes. The size distribution ranged from monomers with no cross-bridges to polymers with a high degree of polymerization, but did not differ significantly between cell walls from cells grown as spheres or rods. Some small differences in the distribution of C- and N-terminal amino acids were found. Analyses revealed that all the peptide bridges in the glycopeptide fractions from rod cell walls were formed by one l-alanine residue. In sphere cell walls, l-alanine was also found, but, in addition, higher oligomers of the glycopeptide contained glycine in their cross-bridges. These results were confirmed by determinations of C- and N-terminal amino acids released after lysostaphin and AL-1 enzyme digestions and by Edman degradations. Models representing the structures of the sphere and rod cell walls are presented. These structures indicate that the sphere cell wall is probably a more loosely knit macromolecule than is the rod cell wall.     

Adenosine triphosphate pool levels and endogenous metabolism in Arthrobacter crystallopoietes during growth and starvation

The adenosine triphosphate (ATP) content of Arthrobactery crystallopoietes was measured during growth, starvation and recovery from starvation. During exponential growth of the cells as spheres in a glucose salts medium, the level of ATP per cell remained constant at 8.0×10^-10 g/cell. Morphogenesis to rodshaped cells and an increased growth rate following addition of casein hydrolysate was accompanied by an almost two-fold increase in the ATP level. As division of the rod-shaped cells proceeded, the level of ATP declined. After growing as rods for 12–14 h the cells underwent fragmentation to spheres during which time the ATP level again increased to the original value of 8.0×10^-10 g/cell. As the spherical cells resumed growth on the residual glucose, their ATP content declined for a short period and then remained relatively constant. During starvation of sphere or rod-shaped cells for one week, the ATP level declined by approximately 70% during the first 40–50 h and then remained constant. The endogenous metabolism rate of spherical cells declined during the first 10–20 h of starvation and then remained constant at approximately 0.02% of the cell carbon being utilized per h. Addition of glucose to spherical cells which had been starved for one week increased both the ATP content per cell and their rate of endogenous metabolism. The ATP content fluctuated and then remained at a level higher than maintained during starvation while endogenous metabolism quickly declined.Non-Standard Abbreviations ATP adenosine triphosphate - GS glucose mineral salts - HC casein hydrolysate - PVP polyvinylpyrrolidone - DMSO dimethylsulfoxide - MOPS morpholinopropane sulfonic acid - EDTA ethylene diaminetetraacetic acid     

Zebrafish neural tube morphogenesis requires Scribble-dependent oriented cell divisions

How control of subcellular events in single cells determines morphogenesis on the scale of the tissue is largely unresolved. The stereotyped cross-midline mitoses of progenitors in the zebrafish neural keel provide a unique experimental paradigm for defining the role and control of single-cell orientation for tissue-level morphogenesis in vivo. We show here that the coordinated orientation of individual progenitor cell division in the neural keel is the cellular determinant required for morphogenesis into a neural tube epithelium with a single straight lumen. We find that Scribble is required for oriented cell division and that its function in this process is independent of canonical apicobasal and planar polarity pathways. We identify a role for Scribble in controlling clustering of α-catenin foci in dividing progenitors. Loss of either Scrib or N-cadherin results in abnormally oriented mitoses, reduced cross-midline cell divisions, and similar neural tube defects. We propose that Scribble-dependent nascent cell-cell adhesion clusters between neuroepithelial progenitors contribute to define orientation of their cell division. Finally, our data demonstrate that while oriented mitoses of individual cells determine neural tube architecture, the tissue can in turn feed back on its constituent cells to define their polarization and cell division orientation to ensure robust tissue morphogenesis.     

Regulation of mammalian cell growth by autocrine growth factors: analysis of consequences for inoculum cell density effects

Effects of inoculum cell density on mammalian cell growth in culture have been observed in a variety of experimental systems. Although these effects have been attributed generally to medium conditioning by the cells, there has previously been no quantitative theory proposed for this phenomenon based on developments in molecular and cell biology. In this article, we offer such a theory founded on the regulatory action of autocrine growth factors. A particularly relevant example of these is platelet- derived growth factor (PDGF), which is produced by fibroblastic cells in response to stimulation by transforming growth factor beta (TGFbeta), a common serum constituent, and provides a mitogenic signal for the same cells. A simple mathematical model for the production, diffusive transport, and binding of autocrine growth factors to cell surface receptors, coupled to a model for the dependence of cell proliferation on growth factor receptor binding allows prediction of initial cell population growth rate as a function of inoculum cell density. We focus on situations involving anchorage-dependent cell growth, in which the cells are attached to a surface. A number of clear results are obtained, most notably the following: 1) for cells cultured on spherical microcarrier bead surfaces, the inoculum cell density needed to produce a given growth rate is linearly proportional to the bead radius; and 2) all other factors being equal, the inoculum cell density on a unit surface area basis needed to produce a given growth rate is greater for spherical microcarrier surfaces than for flat culture dish surfaces. These two results are consistent with the experimental observations of Hu and coworkers(1,2) for fibroblast growth in minimal medium plus serum. The model also allows elucidation of the influence of other system parameters, both biological and physical, on initial cell proliferation rate and the inoculum cell density dependence.     

Effect of growth substrates on morphology of Nocardia corallina.

Cells of Nocardia corallina ATCC 4273 form multiply branched coenocytic mycelia and subsequent fragment to spherical cells when grown on solidified complex media. In liquid shake cultures using complex media the organisms grow into pleomorphic but seldomly branched rods, divide as rods and then the rods fragment to spheres as the stationary phase is reached. In a defined liquid medium with glucose as carbon source, the organisms divide entively as spheres at a doubling time of 44 hrs. The addition of L-tyrosine, some fatty acids and tricarboxylic acid cycle intermediates or fructose to the glucose medium caused the cells to grow at considerably faster growth rates (2.8-8.5 hrs doubling times) and to undergo the shphre-rod-shpere growth cycle. Other amino acids, fatty acids or surgars added singly to the glucose medium did not produce the sphere to rod morphology change. Some amino acids when added to the medium in pairs effected sphere to rod morphopoiesis. None of these amino acids alone were effectors. Some of the culture grew as rods and the remainder as spheres when isoleucine and valine were added to the glucose medium. No other amino acid combination tested gave this result. The reason for the mixed growth response was traced to inhomogeneity of the parent culture. The life cycle of N. corallina is illustrated in a series of photomicrographs of two slide cultures.     

Changing of the cell division axes drives vulva evolution in nematodes

Vulval epithelial tubes invaginate through concerted cell migration, ring formation, stacking of rings and intra-ring cell fusion in the nematodes Caenorhabditis elegans, Oscheius tipulae and Pristionchus pacificus. The number of rings forming the invaginations is invariantly seven, six, and eight, respectively. We hypothesize that each ring is formed from pairs of symmetrically positioned primordial vulval cells following three premises: If the final cell division is left-right, the daughters will fuse, migrate and form only one ring. If these cells do not divide, one ring will form. If the final division is anterior-posterior, two rings will form. We test the ring hypothesis and found coincidence between the patterns of vulva cell divisions and the number of rings for 12 species. We find heterochronic variations in the timing of division, migration and fusion of the vulval cells between species. We report a unique ring-independent pathway of vulva formation in Panagrellus redivivus. C. elegans lin-11(n389) mutation results in cell fate transformations including changes in the orientation of vulval cell division. lin-11 animals have an additional ring, as predicted by the ring hypothesis. We propose that the genetic pathway determining how vulval cells invaginate evolves through ring-dependent and ring-independent mechanisms.     

Cell division orientation and planar cell polarity pathways

The orientation of cell division has a crucial role in early embryo body plan specification, axis determination and cell fate diversity generation, as well as in the morphogenesis of tissues and organs. In many instances, cell division orientation is regulated by the planar cell polarity (PCP) pathways: the Wnt/Frizzled non-canonical pathway or the Fat/Dachsous/Four-jointed pathway. Firstly, using asymmetric cell division in both Drosophila and C. elegans, we describe the central role of the Wnt/Frizzled pathway in the regulation of asymmetric cell division orientation, focusing on its cooperation with either the Src kinase pathway or the heterotrimeric G protein pathway. Secondly, we describe our present understanding of the mechanisms by which the planar cell polarity pathways drive tissue morphogenesis by regulating the orientation of symmetric cell division within a field of cells. Finally, we will discuss the important avenues that need to be explored in the future to better understand how planar cell polarity pathways control embryo body plan determination, cell fate specification or tissue morphogenesis by mitotic spindle orientation.     

Oriented cell division in vertebrate embryogenesis

Tissue morphogenesis depends on the spatial arrangement of cells during development. A number of mechanisms have been described to contribute to the final shape of a tissue or organ, ranging from cell intercalation to the response of cells to chemotactic cues. One such mechanism is oriented cell division. Oriented cell division is determined by the position of the mitotic spindle. Indeed, there is increasing evidence implicating spindle misorientation in tissue and organ misshaping, which underlies disease conditions such as tumorigenesis or polycystic kidneys. Here we review recent studies addressing how the direction of tissue growth is determined by the orientation of cell division and how both extrinsic and intrinsic cues control the position of the mitotic spindle.     

The co-ordination of cell division, differentiation and morphogenesis in the shoot apical meristem: a perspective

Whether morphogenesis is cell division-driven or organismal-based has been a long-running debate in plant biology. This article is a summary of a series of experiments aimed at distinguishing these alternate views by local manipulation of parameters of cell division frequency, orientation, and growth within the shoot apical meristem. These data, put in the context of other investigations in this area, support an organismal view of plant morphogenesis and support the idea that the cell wall plays a key role in the mechanism by which this is achieved. At the same time, the data indicate that the intimate but variable relationship between cell growth and division within the organism means that cell proliferation can indirectly influence this process, leading to a context-dependent influence on morphogenesis. Finally, cell growth and proliferation are intimately related with the process of differentiation as cells exit the meristem. In the final part of the article the molecular mechanism by which these basic cellular parameters are intertwined is discussed.     

Real-time lineage analysis reveals oriented cell divisions associated with morphogenesis at the shoot apex of Arabidopsis thaliana

Precise knowledge of spatial and temporal patterns of cell division, including number and orientation of divisions, and knowledge of cell expansion, is central to understanding morphogenesis. Our current knowledge of cell division patterns during plant and animal morphogenesis is largely deduced from analysis of clonal shapes and sizes. But such an analysis can reveal only the number, not the orientation or exact rate, of cell divisions. In this study, we have analyzed growth in real time by monitoring individual cell divisions in the shoot apical meristems (SAMs) of Arabidopsis thaliana. The live imaging technique has led to the development of a spatial and temporal map of cell division patterns. We have integrated cell behavior over time to visualize growth. Our analysis reveals temporal variation in mitotic activity and the cell division is coordinated across clonally distinct layers of cells. Temporal variation in mitotic activity is not correlated to the estimated plastochron length and diurnal rhythms. Cell division rates vary across the SAM surface. Cells in the peripheral zone (PZ) divide at a faster rate than in the central zone (CZ). Cell division rates in the CZ are relatively heterogeneous when compared with PZ cells. We have analyzed the cell behavior associated with flower primordium development starting from a stage at which the future flower comprises four cells in the L1 epidermal layer. Primordium development is a sequential process linked to distinct cellular behavior. Oriented cell divisions, in primordial progenitors and in cells located proximal to them, are associated with initial primordial outgrowth. The oriented cell divisions are followed by a rapid burst of cell expansion and cell division, which transforms a flower primordium into a three-dimensional flower bud. Distinct lack of cell expansion is seen in a narrow band of cells, which forms the boundary region between developing flower bud and the SAM. We discuss these results in the context of SAM morphogenesis.     

FtsZ ring formation without subsequent cell division after replication runout in Escherichia coli

In this report, we have investigated cell division after inhibition of initiation of chromosome replication in Escherichia coli. In a culture grown to the stationary phase, cells containing more than one chromosome were able to divide some time after restart of growth, under conditions not allowing initiation of chromosome replication. This shows that there is no requirement for cell division to take place within a certain time after initiation of chromosome replication. Continued growth without initiation of replication resulted in filamented cells that generally did not have any constrictions. Interestingly, FtsZ rings were formed in a majority of these cells as they reached a certain cell length. These rings appeared and were maintained for some time at the cell quarter positions on both sides of the centrally localized nucleoid. These results confirm previous findings that cell division sites are formed independently of chromosome replication and indicate that FtsZ ring assembly is dependent on cell size rather than on the capacity of the cell to divide. Disruption of the mukB gene caused a significant increase in the region occupied by DNA after the replication runout, consistent with a role of MukB in chromosome condensation. The aberrant nucleoid structure was accompanied by a shift in FtsZ ring positioning, indicating an effect of the nucleoid on the positioning of the FtsZ ring. A narrow cell length interval was found, under and over which primarily central and non-central FtsZ rings, respectively, were observed. This finding correlates well with the previously observed oscillatory movement of MinC and MinD in short and long cells.     

Cycling in a crowd: Coordination of plant cell division,growth, and cell fate

The reiterative organogenesis that drives plant growth relies on the constant production of new cells, which remain encased by interconnected cell walls. For these reasons, plant morphogenesis strictly depends on the rate and orientation of both cell division and cell growth. Important progress has been made in recent years in understanding how cell cycle progression and the orientation of cell divisions are coordinated with cell and organ growth and with the acquisition of specialized cell fates. We review basic concepts and players in plant cell cycle and division, and then focus on their links to growth-related cues, such as metabolic state, cell size, cell geometry, and cell mechanics, and on how cell cycle progression and cell division are linked to specific cell fates. The retinoblastoma pathway has emerged as a major player in the coordination of the cell cycle with both growth and cell identity, while microtubule dynamics are central in the coordination of oriented cell divisions. Future challenges include clarifying feedbacks between growth and cell cycle progression, revealing the molecular basis of cell division orientation in response to mechanical and chemical signals, and probing the links between cell fate changes and chromatin dynamics during the cell cycle.

Plant cell cycle and division are linked to specific cell fates and respond to growth-related cues, such as metabolic state, cell size, cell shape, and mechanical stress.     

Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.