Synthesis of the inner cell wall layer of the chlamydomonad flagellate,Gloeomonas kupfferi

Summary An antibody to the inner wall layer ofGloeomonas kupfferi was isolated and used in a developmental analysis of cell wall processing, secretion and extracellular assembly. The focus of the processing of this matrix layer is the endomembrane system, in particular the Golgi apparatus (GA) and contractile vacuole (CV). During interphase, inner wall materials are processed in the GA, packaged in trans face vesicles and transported to the CV, the final internal depository of wall precursors until release to the cell surface. During cell division, significant changes occur in the inner wall layer processing. Early on in cytokinesis, the GA does not label with our antibody, suggesting that other wall layers are being processed. In later stages of cytokinesis, the GA changes in morphology and begins to produce inner wall layer materials. These wall precursors are shuttled to the CV where they are released around the daughter cell protoplasts. The first wall layer that is formed around daughter cells is the crystalline median wall layer. Once assembled, the inner wall layer condenses upon the crystalline layer and grows in size.     

Roles for MreC and MreD proteins in helical growth of the cylindrical cell wall in Bacillus subtilis

Actin homologues of the MreB family have an important role in specifying the morphology of many non-spherical eubacteria. The mreC and mreD genes have been implicated in control of cell morphology but their precise functions are unknown. In Bacillus subtilis the MreB homologue Mbl directs helical insertion of new cell wall material in the cylindrical part of the rod-shaped cell. Depletion of either MreC or MreD abolishes the control of cell shape. In the presence of high concentrations of magnesium cells depleted of MreC or MreD can be propagated indefinitely, although they have a spheroidal shape. We show that growth of the spheroidal mutants is based on insertion of new wall material at cell division sites and that this localized growth is dependent on cell division. Under some conditions the MreC and MreD proteins localize in a helical configuration. This localization pattern resembles that of the helical cables of Mbl protein. These results suggest that MreC and MreD act in a morphogenic pathway that couples the helical cytosolic Mbl cables to the extracellular cell wall synthetic machinery, which is critical for cylindrical elongation of the rod-shaped cells.     

Balancing of the mitotic exit network and cell wall integrity signaling governs the development and pathogenicity in Magnaporthe oryzae

The fungal cell wall plays an essential role in maintaining cell morphology, transmitting external signals, controlling cell growth, and even virulence. Relaxation and irreversible stretching of the cell wall are the prerequisites of cell division and development, but they also inevitably cause cell wall stress. Both Mitotic Exit Network (MEN) and Cell Wall Integrity (CWI) are signaling pathways that govern cell division and cell stress response, respectively, how these pathways cross talk to govern and coordinate cellular growth, development, and pathogenicity remains not fully understood. We have identified MoSep1, MoDbf2, and MoMob1 as the conserved components of MEN from the rice blast fungus Magnaporthe oryzae. We have found that blocking cell division results in abnormal CWI signaling. In addition, we discovered that MoSep1 targets MoMkk1, a conserved key MAP kinase of the CWI pathway, through protein phosphorylation that promotes CWI signaling. Moreover, we provided evidence demonstrating that MoSep1-dependent MoMkk1 phosphorylation is essential for balancing cell division with CWI that maintains the dynamic stability required for virulence of the blast fungus.     

Dispersed mode of Staphylococcus aureus cell wall synthesis in the absence of the division machinery

We have developed several new fluorescent staining procedures that enabled us to study the synthesis of cell wall material in the spherical Gram-positive bacterium Staphylococcus aureus. The results obtained support previous proposals that these cells synthesize new wall material specifically at cell division sites, in the form of a flat circular plate that is subsequently cleaved and remodelled to produce the new hemispherical poles of the daughter cells. We have shown that formation of the septal peptidoglycan is dependent on the key cell division protein FtsZ, which recruits penicillin-binding protein (PBP) 2. Unexpectedly, in FtsZ-depleted cells, the cell wall synthetic machinery becomes dispersed and new wall material is made in dispersed patches over the entire surface of the cells, which increase in volume by up to eightfold before lysing. The results have implications for understanding the nature of S. aureus morphogenesis and for inhibitors of cell division proteins as drug targets.     

Taking shape: control of bacterial cell wall biosynthesis

The characteristic shape of a bacterial cell is a function of the three dimensional architectures of the cell envelope and is determined by the balance between lateral wall extension and synthesis of peptidoglycan at the division septum. The three dimensional patterns of cell wall synthesis in the bacterium Bacillus subtilis is influenced by actin-like proteins that form helical coils in the cell and by the MreCD membrane proteins that link the cytoskeletal elements with the penicillin-binding proteins that carry out peptidoglycan synthesis. Recent genetic studies have provided important clues as to how these proteins are arranged in the cell and how they function to regulate cell shape.     

Structural changes in the cell wall of Schizosaccharomyces pombe during cell division

Streiblová, Eva (Czechoslovak Academy of Sciences, Prague, Czechoslovakia), I. Málek, and K. Beran. Structural changes in the cell wall of Schizosaccharomyces pombe during cell division. J. Bacteriol. 91:428-435. 1966.-Individual stages of growing and dividing cells of Schizosaccharomyces pombe were studied by means of fluorescence and electron microscopy with the use of metal-shadowed isolated walls, replicas, and ultrathin sections. Vegetative cells were found to contain division scars (six at the most); their formation and structure are described. More data on the growth of arthrospores were obtained. New structural observations were made on the architecture of the cell wall (original wall ring, polar cell wall, plug wall band, additional wall ring). Structural changes of cell surfaces and lateral walls during fission are represented schematically to the fourth generation. The question of origin of the septum is discussed, and on this basis the entire structure of the cell wall is interpreted.     

Regenerated cell wall of carrot protoplasts isolated from suspension-cultured cells

Protoplasts of Daucus carota L. cultured in a synthetic liquid medium resumed cell division after about 4 days of cultivation. During this lag period, nucleic acid and protein showed only slight increases but the protoplasts commenced cell-wall regeneration soon after the removal of lytic enzymes. The originally spherical protoplasts became ellipsoidal before they underwent division. Radioactive glucose and myo-inositol were readily utilized by the protoplasts. Most of the radioactivity, however, appeared in extracellular polysaccharides and only a small portion was deposited in the regenerated wall. The sugar composition of new cell wall, as studies by chemical analysis and incorporation of labelled precursors, was shown to be considerably different from that of normal cell wall.     

LP28, a lily pollen-specific LEA-like protein, is located in the callosic cell wall during male gametogenesis

. LP28, a pollen-specific LEA-like protein identified in Lilium longiflorum purportedly related to the desiccation tolerance of pollen, was localized during male gametogenesis using immuno-electron microscopy. At premeiotic interphase, LP28 label is absent from the microsporocyte. LP28 label was first detected in the cell wall of the microsporocyte at meiotic prophase I. LP28 gradually increased as the cell wall thickened. In the dyad, after the first meiotic division, LP28 label also appeared in the septum. In the tetrad, after the second meiotic division, LP28 was detected throughout the cell wall, including the septa. Immunolabeling of callose during meiosis indicated that the appearance and localization of LP28 was very similar to that of callose. After the microspores were released from the tetrad by digesting the callosic cell wall, LP28 was not found in the microspores. In bicellular pollen, just after microspore mitosis, LP28 appeared in the generative cell wall, which also consisted of callose. After pollen germination, LP28 also accumulated in the callosic layer of the elongated pollen tube wall and the callose plug. Thus, LP28 colocalized with the callosic cell wall during male gametogenesis. The possible role of LP28 with respect to wall formation during meiosis and pollen development is discussed.     

More than just lysins: peptidoglycan hydrolases tailor the cell wall

Enzymes that degrade the peptidoglycan (PG) cell wall layer called PG hydrolases or autolysins are often thought of as destructive forces. Phages employ them to lyse their host for the release of virion particles and some bacteria secrete them to eliminate (lyse) their competition. However, bacteria also harness the activity of PG hydrolases for important aspects of growth, division, and development. Of course, using PG hydrolases in this capacity requires that they be tightly regulated. While this has been appreciated for some time, we are only just beginning to understand the mechanisms governing the activities of these 'tailoring' enzymes. This review will focus on recent advances in this area with an emphasis on the regulation of PG hydrolases involved in cell division.     

Towards a comprehensive view of the bacterial cell wall

Direct in vivo visualization, in full atomic detail, of the microbial cell wall and its stress-bearing structural architecture remains one of the prime challenges in microbiology. In the meantime, molecular modeling can provide a framework for explaining and predicting mechanisms involved in morphogenesis, bacterial cell growth and cell division, during which the wall and its major structural component--murein--have to protect the cell from osmotic pressure and multiple tensile forces. Here, we illustrate why the scaffold concept of murein architecture provides a more comprehensive representation of bacterial cell wall physiology than previous models.     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Characterization of the ftsZ gene from Mycoplasma pulmonis, an organism lacking a cell wall.

The ftsZ gene is required for cell division in Escherichia coli and Bacillus subtilis. In these organisms, FtsZ is located in a ring at the leading edge of the septum. This ring is thought to be responsible for invagination of the septum, either causing invagination of the cytoplasmic membrane or activating septum-specific peptidoglycan biosynthesis. In this paper, we report that the cell division gene ftsZ is present in two mycoplasma species, Mycoplasma pulmonis and Acholeplasma laidlawii, which are eubacterial organisms lacking a cell wall. Sequencing of the ftsZ homolog from M. pulmonis revealed that it was highly homologous to other known FtsZ proteins. The M. pulmonis ftsZ gene was overexpressed, and the purified M. pulmonis FtsZ bound GTP. Using antisera raised against this purified protein, we could demonstrate that it was expressed in M. pulmonis. Expression of the M. pulmonis ftsZ gene in E. coli inhibited cell division, leading to filamentation, which could be suppressed by increasing expression of the E. coli ftsZ gene. The implications of these results for the role of ftsZ in cell division are discussed.     

Space and time in the plant cell wall: relationships between cell type, cell wall rheology and cell function

BACKGROUND: The biomechanical behaviour of plant cells depends upon the material properties of their cell walls and, in many cases, it is necessary that these properties are quite specific. Additionally, physiological regulation may require that target cells responding to hormonal signals or environmental factors are able to modulate these characteristics. ARGUMENT: This paper uses a rheological analysis of creep of elongating sunflower (Helianthus annuus) sunflower hypocotyls to demonstrate that the mechanical behaviour of plant cell walls is complex and involves multiple layered processes that can be distinguished from one another by the time-scale over which they lead to a change in tissue dimensions, their sensitivity to pH and temperature, and their responses to changes in spatial arrangement of the cell wall brought about by treatment with high M(r) PEG. Furthermore, it appears possible to regulate individual rheological processes, with limited effect on others, in order to modulate growth without affecting tissue structural integrity. It is proposed that control of the water content of the cell wall and therefore the space between cell wall polymers may be one mechanism by which differential regulation of cell wall biomechanical properties is achieved. This hypothesis is supported by evidence showing that enzyme extracts from growing tissues can cause swelling in cell wall fragments in suspension. IMPLICATIONS: The physiological implications of this complexity are then considered for growing tissues, stomatal guard cells and abscission cells. It is noted that, in each circumstance, a different combination of mechanical properties is required and that differential regulation of properties affecting behaviour over different time-scales is often necessary.     

Yeast myosin heavy chain mutant: maintenance of the cell type specific budding pattern and the normal deposition of chitin and cell wall components requires an intact myosin heavy chain gene

Recent studies with myosin heavy chain mutants in the slime mold Dictyostelium discoideum and the yeast Saccharomyces cerevisiae indicate that the myosin heavy chain gene is not essential for cell survival under laboratory growth conditions. However, cells lacking a normal myosin heavy chain gene demonstrate substantial alterations in growth and cell division. In this study, we report that a disruption mutant in the rod portion of the yeast myosin heavy chain gene, MYO1, produces abnormal chitin distribution and cell wall organization at the mother-bud neck in a high proportion of dividing cells. It is suggested that this phenotype is the cause of the cell division defect and the osmotic sensitivity of yeast MYO1 mutants. In the absence of a normal MYO1 polypeptide, yeast cells alter their cell type specific budding pattern. It is concluded that an intact myosin heavy chain gene is required to maintain the cell type specific budding pattern and the correct localization and deposition of chitin and cell wall components during cell growth and division.     

Ethylene effects on cambial activity and cell wall formation in hypocotyls of Picea abies seedlings

Ethylene regulation of cell division in the vascular cambium and cell wall formation was studied in hypocotyls of Norway spruce ( Picea abies [L.] Karst.) seedlings. Cuttings from 6-week-old seedlings were placed in water culture to which compounds affecting the synthesis and action of ethylene were added. After a 3-week treatment period, growth, ethylene production, morphology and cell wall composition of the hypocotyls were determined. Addition of high concentrations of the potent ethylene releasing agent 2-chloroethylphosphonic acid (ethrel), which increased ethylene emission by more than twice compared to control plants, inhibited the expansion of xylem cells while stimulating the incorporation of cell wall material, especially cellulose. Addition of small amounts of ethrel, which slightly stimulated ethylene emission, led to increases in the size of xylem cells, the amount of phloem tissue and the number of intercellular spaces in the cortex, and thus to increased hypocotyl diameter. However, no significant change in cell wall composition was detected. When ethylene production was decreased by adding Co²⁺ to the nutrient solution, differentiation of new xylem was disturbed, but the rate of cell division was not affected. Although the incorporation of cell wall material was inhibited, the proportions of lignin and cellulose in the wall appeared to remain unchanged. Silver ions stimulated the expansion of both xylem and cortex cells, but had no significant effect on cell wall formation. We conclude that ethylene has a role in regulating the incorporation of wall carbohydrates.     

Some observations on cell wall structure and taxonomy ofPhymatodocis nordstedtiana (Conjugatophyceae,Chlorophyta)

Filaments ofPhymatodocis nordstedtiana Wolle were isolated from a sample of a Texan lake. Cultures were established and examined by light and scanning electron (SEM) as well as transmission electron microscopy (TEM). It is shown that the pores apparent on light microscopical examination are not of the cosmaroid type as expected. TEM examination disclosed that they are similar to those found in the generaClosterium Ralfs andPenium Bréb. Furthermore, it could be demonstrated by light and SEM microscopy that the primary cell wall is shed during cell division. The remaining secondary cell wall of the mature cell consists of interwoven bands of parallel microfibrils. A conspicuous overlap of the semicell walls clearly denotes the isthmus region. The significance of these deviations unusual for desmids is discussed. Suggestions are made that the taxonomic position ofPh. nordstedtiana should be re-evaluated.     

Insertion and fate of the cell wall in Bacillus subtilis

Cell wall assembly was studied in autolysin-deficient and -sufficient strains of Bacillus subtilis. Two independent probes, one for peptidoglycan and the other for surface-accessible teichoic acid, were employed to monitor cell surface changes during growth. Cell walls were specifically labeled with N-acetyl-D-[3H]glucosamine, and after growth, autoradiographs were prepared for both cell types. The locations of silver grains revealed that label was progressively lost from numerous sites on the cell cylinders, whereas label was retained on the cell poles, even after several generations. In the autolysin-deficient and chain-forming strain, it was found that the distance between densely labeled poles approximately doubled after each generation of growth. In the autolysin-sufficient strain, it was found that the numbers of labeled cell poles remained nearly constant for several generations, supporting the premise that completed septa and poles are largely conserved during growth. Fluorescein-conjugated concanavalin A was also used to determine the distribution of alpha-D-glucosylated teichoic acid on the surfaces of growing cells. Strains with temperature-sensitive phosphoglucomutase were used because in these mutants, glycosylation of cell wall teichoic acids can be controlled by temperature shifts. When the bacteria were grown at 45 degrees C, which stops the glucosylation of teichoic acid, the cells gradually lost their ability to bind concanavalin A on their cylindrical surfaces, but they retained concanavalin A-reactive sites on their poles. Discrete areas on the cylinder, defined by the binding of fluorescent concanavalin A, were absent when the synthesis of glucosylated teichoic acid was inhibited during growth for several generations at the nonpermissive temperature. When the mutant was shifted from a nonpermissive to a permissive temperature, all areas of the cylinder became able to bind the labeled concanavalin A after about one-half generation. Old cell poles were able to bind the lectin after nearly one generation at the permissive temperature, showing that new wall synthesis does occur in the cell poles, although it occurs slowly. These data, based on both qualitative and quantitative experiments, support a model for cell wall assembly in B. subtilis, in which cylinders elongate by inside-to-outside growth, with degradation of the stress-bearing old wall in wild-type organisms. Loss of wall material, by turnover, from many sites on the cylinder may be necessary for intercalation of new wall and normal length extension. Poles tend to retain their wall components during division and are turned over much more slowly.