Micrasterias cells as a model system for research on morphogenesis.

Micrasterias species have been the subject of numerous experimental studies on cell shape formation in the last 40 years. Chemical and physical treatment during different developmental stages, as well as investigations of ultrastructure by means of various different preparation methods, have yielded information about some principles of morphogenesis in the symmetric, highly ornamented Micrasterias cell. The basic symmetry of a Micrasterias cell is determined prior to mitosis and is established without nuclear control thereafter. Normal cell development, however, may occur only under the conditions of continuous protein synthesis throughout the cell cycle. A prepattern for the later cell shape seems to be present at the plasma membrane at the early stages of septum formation. It is realized by a local, patterned distributed incorporation of cell wall material that is delivered by Golgi-produced vesicles. The areas where fusions take place between the primary wall material containing vesicles and the plasma membrane are defined by inward ionic currents that are carried at least in part by calcium. These areas develop into lobes during the following course of cell growth. Cell shaping in Micrasterias cells is thus mediated by both an enhanced extension of the cell wall and an additional incorporation of wall material in the areas of the lobes. Numerous studies have indicated that actin plays an important role in morphogenesis, whereas microtubules do not participate in this process but are involved mainly in nuclear migration. The present review shows that although a wealth of details concerning Micrasterias morphogenesis has already been elucidated, two main questions, i.e., the method of septum formation and the splitting of the lobes, remain to be answered.     

Extensibility of isolated cell walls in the giant tip-growing cells of the xanthophycean alga Vaucheria terrestris

Apical cell wall fragments isolated from the giant-cellular xanthophycean alga Vaucheria terrestris sensu Götz were inflated with silicone oil by applying internal pressure ranging from 0.1 to 0.7 MPa, and the time-course of cell wall deformation was recorded and analyzed by videomicroscopy. Cell wall extensibility in the tip-growing region was estimated by the pressure required for cell wall extension, the amount of total extension until cell wall rupture and the rate of cell wall extension. Apical cell walls exhibited gradual extension, or creep, during inflation, which was eventually followed by rupture at the apical portion, whereas no appreciable extension was found in the cylindrical basal portion of the cell wall fragment. Besides the largest extension observed around the tip, substantial extension was also observed along the subapical region of the cell wall. The wall extensibility was dependent on the buffer pH used for infiltration before inflation. The optimum pH for the extension was about 8.0, but the cell wall was much less extensible after infiltration with an acidic buffer. Cell wall extensibility was dependent on the pH of the buffer used before inflation, regardless of that used in the previous infiltration. Moreover, pretreatment of the cell wall with a protease caused considerable loosening of cell walls, but affected the pH dependence of cell wall extensibility little. These results indicate that the extensibility of the cell walls in the giant tip-growing cells of the alga is distinct from that of plant cells that exhibit "acid growth" in its dependence on environmental pH and the role of cell wall proteins.     

Ontogeny of external glands in the bladderwortUtricularia monanthos

Summary The development of external glands on traps and stolons ofU. monanthos has been studied using transmission electron microscopy. During early differentiation of the epidermis some cells remain narrow and develop a protuberance which subsequently divides into a terminal and a pedestal cell, with the remainder of the original cell forming the basal epidermal cell of the gland. The lateral wall of the pedestal cell soon becomes densely impregnated throughout its thickness, and this is followed by the formation of discontinuous cuticular deposits within the primary wall of the terminal cell. The outer wall of the terminal cell then usually undergoes extensive secondary wall thickening beginning with the formation of ingrowths which for a period characterize the cell as a transfer cell. Later, at the stage when traps begin capturing prey, these ingrowths are overlain by further layers of secondary wall material. Concomitantly, in the pedestal cell, wall ingrowths become fully differentiated on the outer transverse wall and persist throughout the remaining life of the gland.The function of external glands during early ontogeny is discussed. At the stage when the terminal cell is differentiated as a transfer cell it is suggested that the gland is mainly responsible for absorbing solutes from the external medium. Once traps commence capturing prey the gland may become modified for a rôle in water secretion, facilitated by the differentiation of the pedestal cell as a transfer cell, and by the formation of a thick outer wall in the terminal cell.     

Anther Wall of Cymbidium Goeringii with Special Reference to the Tapetum

The structure of the anther wall in Cymhidum goeringii (Rchb. f.)Rchb.f, was followed through two developmental stages using light and electron microscope: The peculiar anatomical pattern of the anther wall and the cytological characteristics of its tissues are described. Tapetum is the most notable feature in the anther wall. The specialized characteristics of the tapetal cells may be summerized as followings: (1) The tapetum may be distinguished into outer and inner tapetum, but they constitute the continuous layers surrounding the pollinium. The inner tapetum is derived from the connective tissue. (2) The outer and inner tapetum are composed of 2--3 cell layers. They have the same ultrastructural features and functions. (3) There are many lipid-containg plastids in the tapetal cells during their developmental stage. This is the main source of lipid bodies in the tapetal cells during their final developmental stage. The lipid-containing plasmids are derived from the extension of the nuclear membrane. (4) After the formation of tetrads, the secretion and the autolysates from of the tapetal cells not only deposition the surface of the pollinium to form the film, but also flow into the inner region of the polinia between the retrads contributing to their adhesion. The results of this study provide some new data of the tapetal structure and function in the angiosperms.     

Native cell wall organization shown by cryo-electron microscopy confirms the existence of a periplasmic space in Staphylococcus aureus

The current perception of the ultrastructure of gram-positive cell envelopes relies mainly on electron microscopy of thin sections and on sample preparation. Freezing of cells into a matrix of amorphous ice (i.e., vitrification) results in optimal specimen preservation and allows the observation of cell envelope boundary layers in their (frozen) hydrated state. In this report, cryo-transmission electron microscopy of frozen-hydrated sections of Staphylococcus aureus D2C was used to examine cell envelope organization. A bipartite wall was positioned above the plasma membrane and consisted of a 16-nm low-density inner wall zone (IWZ), followed by a 19-nm high-density outer wall zone (OWZ). Observation of plasmolyzed cells, which were used to artificially separate the membrane from the wall, showed membrane vesicles within the space associated with the IWZ in native cells and a large gap between the membrane and OWZ, suggesting that the IWZ was devoid of a cross-linked polymeric cell wall network. Isolated wall fragments possessed only one zone of high density, with a constant level of density throughout their thickness, as was previously seen with the OWZs of intact cells. These results strongly indicate that the IWZ represents a periplasmic space, composed mostly of soluble low-density constituents confined between the plasma membrane and OWZ, and that the OWZ represents the peptidoglycan-teichoic acid cell wall network with its associated proteins. Cell wall differentiation was also seen at the septum of dividing cells. Here, two high-density zones were sandwiched between three low-density zones. It appeared that the septum consisted of an extension of the IWZ and OWZ from the outside peripheral wall, plus a low-density middle zone that separated adjacent septal cross walls, which could contribute to cell separation during division.     

Reinitiation of Cell Wall Growth After Threonine Starvation of Streptococcus faecalis

Cultures of Streptococcus faecalis ATCC 9790 were starved of threonine for 10 hr and then allowed to reinitiate growth in a fresh complete medium. On regrowth, culture turbidity began to increase within 10 min, but the ability of cells to autolyze did not begin to increase until after 30 min. Ultrastructural studies of regrowth of the initially thick-walled cells showed, at about 30 min, centripetal linear extension of new thin cross wall. This was followed, at about 40 min, by a notching, splitting, and peeling apart of the base of the cross wall. After this, extension of new thin peripheral wall from the nascent cross wall appeared to push old thick wall toward the poles. After the first cell division, asymmetric cells with one initial generation thick-walled pole and one second generation thin-walled pole were seen. After two divisions, thick-walled hemispheres were still seen, suggesting conservation of old wall during this time. A small fraction of the initial cell population exhibited aberrations and difficulties in reinitiating linear wall extension and were useful in the establishment of a model for the reinitiation of linear wall extension.     

不同品种苹果采后后熟软化过程中细胞壁多糖的降解

以2种苹果为试材,提取了不同贮藏时期果实的细胞壁物质和8种细胞壁多糖组分,并采用气相色谱法分析了细胞壁多糖组分的单糖组成。结果表明,在贮藏过程中,‘金星’苹果果肉的硬度下降明显,在贮藏第10天前后出现明显的乙烯释放量高峰,而耐贮藏性‘富士’苹果在贮藏期间只释放极少量的乙烯。‘金星’苹果的Na2CO3-1溶性果胶多糖组分的减少尤为显著。这些结果表明,苹果果实Na2CO3-1溶性果胶多糖组分侧链成分的酶降解,是引起苹果细胞壁多糖网络结构的变化,进而导致果实软化的重要原因之一。     

Silicification of auxospores in the araphid diatom Tabularia fasciculata (Bacillariophyta)

We report on auxospore wall structure and development in the araphid pennate diatom Tabularia fasciculata. Similar to most other pennates, these auxospores showed a typical bidirectional elongation, but unexpectedly bore no transverse perizonium, and with no detectable silicon during much of their expansion. Energy dispersive X-ray spectroscopy (EDS) analyses segregated auxospores into two types: (1) those containing no detectable silicon and (2) those with measureable amounts. Both types were of similar size. Silica precipitation began throughout the auxospore at or near maximal length, but initially was detectable in isolated regions throughout the structure. Following this initial condition, silicon was consistently detectable throughout auxospores of comparable size and corresponded to deposition of longitudinal perizonium (visible through the thin organic outer layer of the wall in some auxospores), followed by the deposition of the initial valves. Our results raise the question as to how the tubular shape of bidirectionally expanding auxospores up to ∼90 μm long is maintained in the absence of transverse siliceous elements restricting isodiametric expansion of the cell, which are present in all other known pennate auxospores and all but one other diatom. Our study is the first to systematically examine mineral elements of the auxospore wall analytically.     

STUDIES ON THE OXIDATIVE METABOLISM OF SACCHAROMYCES CEREVISIAE : I. Observations on the Fine Structure of the Yeast Cell

Vegetative cells of Saccharomyces cerevisiae were fixed with potassium permanganate followed by uranyl nitrate, embedded in methacrylate, and studied in electron micrographs of thin sections. Details of the structure of the cell wall, cytoplasmic membrane, nucleus, vacuole, and mitochondria are described. Cell membranes, about 70 to 80 A thick, have been resolved into two dense layers, 20 to 25 A thick, separated by a light layer of the same dimensions, which correspond in thickness and appearance to the components of the "unit membrane" as described by Robertson (15). The cell wall is made up of zones of different electron opacity. Underlying the cell wall is the cytoplasmic membrane, a sinuous structure with numerous invaginations. The nucleoplasm, often of uneven electron opacity, is enclosed in a pair of unit membranes in which nuclear pores are apparent. The vacuole, limited by a single unit membrane, is usually irregular in outline and contains some dense material. Rod-shaped mitochondria, 0.4 to 0.6 µ in length and 0.2 to 0.3 µ in diameter, are smaller in size, but similar in structure to some of those described in plant and animal cells. Attempts to use osmium tetroxide as fixative were unsuccessful, a result similar to that obtained by other workers. It is suggested that yeast cells are impermeable to osmium tetroxide, except when grown under specific conditions.     

Relationship of a Wall-Associated Enzyme with Specific Layers of the Cell Wall of a Gram-Negative Bacterium

In untreated cells of the marine pseudomonad studied here, alkaline phosphatase was found to be located in the periplasmic space, at the cell surface, and in the medium into which it had been shed during growth. Washing in 0.5 M NaCl, which removed the loosely bound outer layer, caused a shift of periplasmic enzyme to the outer aspect of the double-track layer and released some of the cell surface-associated enzyme. When the double-track layer of the cell wall was partially deranged, large amounts of this cell wall-associated enzyme were released, and, when the double-track was removed from the cells to produce mureinoplasts, alkaline phosphatase was released into the menstruum. There was no significant association of the enzyme with the peptidoglycan layer of the cell wall, which is the outermost structure of the mureinoplast, and no association of the enzyme with the cytoplasmic membrane of these modified cells. This study has shown that alkaline phosphatase is specifically associated with the outer layers of the cell walls of cells of this organism and is retained within the cell wall by virtue of this association.     

Characterization of a cytokinesis defective (cyd1) mutant of Arabidopsis.

Although several mutations and genes affecting plant cytokinesis have been identified, mutant screens are not yet saturated and knowledge about gene function is still limited. A novel Arabidopsis mutation, cytokinesis defective1 (cyd1), was identified by partial or missing cell walls in stomata. Stomata with incomplete or no cytokinesis still differentiate and some contain swellings of the outer wall not found in the wild type. The incomplete walls are correctly placed opposite stomatal wall thickenings suggesting that the mutation interferes with the execution of cytokinesis rather than with the placement of the division site. Cytokinesis defects are also detectable in other cell types throughout the plant, defects which include cell wall protrusions, two or more nuclei in one cell, and reduced cell number. The extent of cytokinetic partitioning correlates with nuclear number in abnormal stomata. Many cyd1 epidermal cells, stomata and pollen are larger, and trichomes have more branches. cyd1 is partially lethal with poor seed set and some defective ovules, but many plants are fertile despite abnormalities in vegetative and reproductive development such as missing, reduced, fused or misshapen leaves and floral organs. cyd1 appears to be the only cytokinesis mutant described where defects are known to occur in both mature vegetative and reproductive organs. Thus, the CYD1 gene product appears to be necessary for the execution of cytokinesis throughout the shoot. The examination of stomata by microscopy may be a useful screen for the directed isolation of additional cytokinesis mutations that are not embryo or seedling lethal     

Effect of culture in a rotating wall bioreactor on the physiology of differentiated neuron-like PC12 and SH-SY5Y cells.

A variety of evidence suggests that nervous system function is altered during microgravity, however, assessing changes in neuronal physiology during space flight is a non-trivial task. We have used a rotating wall bioreactor with a high aspect ratio vessel (HARV), which simulates the microgravity environment, to investigate the how the viability, neurite extension, and signaling of differentiated neuron-like cells changes in different culture environments. We show that culture of differentiated PC12 and SH-SY5Y cells in the simulated microgravity HARV bioreactor resulted in high cell viability, moderate neurite extension, and cell aggregation accompanied by NO production. Neurite extension was less than that seen in static cultures, suggesting that less than optimal differentiation occurs in simulated microgravity relative to normal gravity. Cells grown in a mixed vessel under normal gravity (a spinner flask) had low viability, low neurite extension, and high glutamate release. This work demonstrates the feasibility of using a rotating wall bioreactor to explore the effects of simulated microgravity on differentiation and physiology of neuron-like cells.     

Ultrastructural changes in the cell walls of ripening apple and pear fruit

Ultrastructural changes in the cell walls of “Calville de San Sauveur” apples (Malus sylvestris Mill) and “Spadona” pear (Pyrus communis L.) fruit were followed during ripening. In apple, structural alterations in cell walls became apparent at advanced stages of softening and showed predominantly dissolution of the middle lamella. In pears softening was also associated with the dissolution of the middle lamella, and in addition a gradual disintegration of fibrillar material throughout the cell wall. In fully ripe fruit almost all of the fibrillar arrangement in the cell wall was lost. Application of enzyme solutions containing polygalacturonase and cellulase to tissue discs from firm pear fruit led to ultrastructural changes observed in naturally ripening pears. In apple polygalacturonase alone was sufficient to dissolve the middle lamella region of the cell walls, as was also found to occur in naturally ripening fruit. In both apple and pear the cell wall areas containing plasmodesmata maintained their structural integrity throughout the ripening process. At advanced stages of ripening vesicles appeared in the vicinity of plasmodesmata.     

Orientation of microfibrils and microtubules in cortical parenchyma cells of poplar during elongation growth

Three types of microfibrillar orientation, namely parallel, perpendicular and oblique to the main cell axis were found not only in the innermost surface of but also throughout the developing wall. Furthermore, three types of microtubule orientation, namely parallel, perpendicular and oblique to the main cell axis, were found, coinciding with those of microfibrils. As a whole, the wall was shown to be a crossed polylamellate structure. These observations suggest that the orientation of microfibrils is determined at the time of wall formation, and not influenced by the extension of the wall.     

THE ULTRASTRUCTURE AND HISTOCHEMISTRY OF A NEMATODE-INDUCED GIANT CELL

The development of giant cells induced by the nematode Meloidogyne in tomato roots has been followed under controlled growth conditions and the ultrastructure and histochemistry of these structures have been examined. Entry of the nematode larvae into the roots took place within 24 hours; giant cell formation started on the 4th day and involved breakdown of the cell walls accompanied by thickening of a surrounding giant cell wall and an increase in density and area of the cytoplasm. The nuclei increased in number by simultaneous mitosis throughout a single giant cell. The peak of cytoplasmic density was reached after moulting and during egg production. The rate of protein synthesis in the giant cell is correlated with the rate of growth of the nematode. The giant cell wall is a thick, irregularly surfaced structure which contains all the normal polysaccharide components of a cell wall. The cytoplasm is rich in protein and RNA and contains mitochondria, proplastids, Golgi bodies, and a dense endoplasmic reticulum. The nuclei are large and irregular in shape and contain large nucleoli and a number of Feulgen-positive bodies scattered irregularly along the nuclear envelope. The nucleolus contains RNA and fat as well as Feulgen-positive granules which are revealed after treatment with ribonuclease. It consists of a dense outer cortex surrounding a much lighter central core and is connected at times with the Feulgen-positive bodies in the nucleus. Speculation is provided on the role of these bodies in cytoplasmic protein synthesis.     

THE FINE STRUCTURE OF DIFFERENTIATING XYLEM ELEMENTS

Differentiating xylem elements of Avena coleoptiles have been examined by light and electron microscopy. Fixation in 2 per cent phosphate-buffered osmium tetroxide and in 6 per cent glutaraldehyde, followed by 2 per cent osmium tetroxide, revealed details of the cell wall and cytoplasmic fine structure. The localized secondary wall thickening identified the xylem elements and indicated their state of differentiation. These differentiating xylem elements have dense cytoplasmic contents in which the dictyosomes and elements of rough endoplasmic reticulum are especially numerous. Vesicles are associated with the dictyosomes and are found throughout the cytoplasm. In many cases, these vesicles have electron-opaque contents. "Microtubules" are abundant in the peripheral cytoplasm and are always associated with the secondary wall thickenings. These microtubules are oriented in a direction parallel to the microfibrillar direction of the thickenings. Other tubules are frequently found between the cell wall and the plasma membrane. Our results support the view that the morphological association of the "microtubules" with developing cell wall thickenings may have a functional significance, especially with respect to the orientation of the microfibrils. Dictyosomes and endoplasmic reticulum may have a function in some way connected with the synthetic mechanism of cell wall deposition.     

Down-regulation of POLYGALACTURONASE 1 alters firmness, tensile strength and water loss in apple (Malus x domestica) fruit

ABSTRACT: BACKGROUND: While there is now a significant body of research correlating apple (Malus x domestica) fruit softening with the cell wall hydrolase ENDO-POLYGALACTURONASE1 (PG1), there is currently no direct evidence of its function. This study examined the effect of down regulation of PG1 expression in 'Royal Gala' apples, a cultivar that typically has high levels of PG1, and softens during fruit ripening. RESULTS: PG1-suppressed 'Royal Gala' apples harvested from multiple seasons were firmer than controls after ripening, and intercellular adhesion was higher. Cell wall analyses indicated changes in yield and composition of pectin, and a higher molecular weight distribution of CDTA-soluble pectin. Structural analyses revealed more ruptured cells and free juice in pulled apart sections, suggesting improved integrity of intercellular connections and consequent cell rupture due to failure of the primary cell walls under stress. PG1-suppression also had reduced expansion of cells in the hypodermis of ripe apples, resulting in more densely packed cells in this layer. This change in morphology appears to be linked with reduced transpirational water loss in the fruit. CONCLUSIONS: These findings confirm PG1's role in apple fruit softening and suggests that this is achieved in part by reducing cellular adhesion. This is consistent with previous studies in shown in strawberry but not in tomato. In apple PG1 also appears influence other fruit texture characters such as juiciness and water loss.     

A membrane model of plant cell extension

A theory is presented for the mechanics of plant cell wall extension and is based on the analogy of the cell wall with a membrane structure made of material capable of large non-linear deformations. These wall deformations may be elastic, elastic-plastic or visco-elastic. Mathematical analyses of such membrane structures show that there is, generally, a critical internal pressure at which dimensional instability occurs. This instability is characterized by a sudden drop in internal pressure accompanied by a large increase in the physical proportions of the membrane structure. The theory proposes that cell wall extension occurs when the cell turgor pressure reaches this critical instability value. The cell wall thus stretched is fixed by biochemical synthesis of wall material. Osmotic regulation re-establishes the turgor pressure and the instability cycle repeats itself as long as the critical instability pressure of the cell is below the osmotic pressure of the cell contents. Equalization of these pressures stops cell extension. The rate of cell extension depends on the frequency of the instability cycle and is thus dependent on the various rate processes associated with the instability cycle. The theory appears to be able to explain most of the known facts regarding cell extension such as the influence of temperature and the action of some growth substances.     

Electron microscope study of the rod-to-coccus shape change in a temperature-sensitive rod- mutant of Bacillus subtilis.

The changes in cell morphology of Bacillus subtilis rodB during a temperature shift from 20 to 42 degrees C, in the absence of added anions, are described. At 20 degrees C the organisms grow as rods but gradually become spherical in shape when placed at 42 degrees C. The shape change is initiated by an increase in diameter at the cell equator, resulting in a bulged morphology, which is further modified to the morphology of a coccus. This change may involve a modification of the pattern of normal cylindrical extension such that incorporation of newly synthesized wall leads only to increase in diameter, perhaps from a growth zone of limited extent. The pattern of surface growth was followed by reconstructing the sequence of cross wall formation and pole construction in rods grown at 20 degrees C and in organisms incubated at 42 degrees C for 75 and 150 min. In thin section, wall forming the septum and nascent poles can be distinguished from the surface distal to the division site by the presence of raised tears, perhaps analogous to the wall bands of streptococci. By using an analog rotation technique involving the three-dimensional reconstruction of cells by mathematical rotation of axial thin sections about their longitudinal axis, it is shown that the proportion of septal wall increases during the shape change. In the coccal forms, all surface growth may arise from septal growth sites.     

Golgi inheritance under a block of anterograde and retrograde traffic

In mitosis, the Golgi complex is inherited following its dispersion, equal partitioning and reformation in each daughter cell. The state of Golgi membranes during mitosis is controversial, and the role of Golgi-intersecting traffic in Golgi inheritance is unclear. We have used brefeldin A (BFA) to perturb Golgi-intersecting membrane traffic at different stages of the cell cycle and followed by live cell imaging the fate of Golgi membranes in those conditions. We observed that addition of the drug on cells in prometaphase prevents mitotic Golgi dispersion. Under continuous treatment, Golgi fragments persist throughout mitosis and accumulate in a Golgi-like structure at the end of mitosis. This structure localizes at microtubule minus ends and contains all classes of Golgi markers, but is not accessible to cargo from the endoplasmic reticulum or the plasma membrane because of the continuous BFA traffic block. However, it contains preaccumulated cargo, and intermixes with the reforming Golgi upon BFA washout. This structure also forms when BFA is added during metaphase, when the Golgi is not discernible by light microscopy. Together the data indicate that independent Golgi fragments that contain all classes of Golgi markers (and that can be isolated from other organelles by blocking anterograde and retrograde Golgi-intersecting traffic) persist throughout mitosis.