DEVELOPMENT OF MUCILAGINOUS SURFACES IN EUGLENOIDS. I. STALK MORPHOLOGY OF COLACIUM MUCRONATUM1

The envelope and stalk of Colacium mucronatum Bourr. & Chad, were examined in living cells with light microscopy and in fixed preparations with scanning electron microscopy using critically point dried (CPD) and freeze dried (FD) preparations. The envelope of palmelloid cells is formed over the entire cell surface by many individual strands attached at right angles to areas of articulation of the pellicular strips. Strands were observed to anastomose on the posterior tip of otherwise naked cells. Stalks of living cells in India ink preparations had an optically dark inner core with a lighter outer sheath. In FD stalks a definite inner core was not evident, whereas CPD stalks had an outer surface composed of thick strands which may be the collapsed and aggregated strands of the FD stalks. In both there was also an amorphous matrix. The stalk forms from the aggregation of many strands from the anterior cell tip back to a point encompassing the cell surface anterior to a cross section of the tip 9 μm diam. The outer surface of the stalk comes from the pellicular surface joining that area and the core from the cell tip in the area of the canal opening. Any possible participation of the inner canal surface in stalk formation could not be determined because of the great density of the mucilage at the cell-tip/stalk junction.     

Effects of glucanase treatment on the structure and mechanical properties of cell walls in the giant‐celled xanthophycean alga Vaucheria frigida

The cell wall of the tip‐growing cells of the giant‐cellular xanthophycean alga Vaucheria frigida is mainly composed of cellulose microfibrils (CMFs) arranged in random directions and the major matrix component into which the CMFs are embedded throughout the cell. The mechanical properties of a cell‐wall fragment isolated from the tip‐growing region, which was inflated by artificially applied pressure, were measured after enzymatic removal of the matrix component by using a protease; the results showed that the matrix component is involved in the maintenance of cell wall strength. Since glucose and uronic acid are present in the matrix component of Vaucheria cell walls, we measured the mechanical properties of the cell wall after treatment with endo‐1,3‐ß‐glucanase and observed the fine structures of its surfaces by atomic force microscopy. The major matrix component was partially removed from the cell wall by glucanase, and the enzyme treatment significantly weakened the cell wall strength without affecting the pH dependence of cell wall extensibility. The enzymatic removal of the major matrix component by using a protease released polysaccharide containing glucose and glucuronic acid. This suggests that the major matrix component of the algal cell walls contains both proteins (or polypeptides) and polysaccharides consisting of glucose and glucuronic acid as the main constituents.     

Unterschiedliche Zusammensetzung von Stiel- und Hutzellwand bei Acetabularia mediterranea

Summary The main constituents of the cell wall of the unicellular green alge Acetabularia mediterranea are mannose, glucose, galactose and rhamnose. There are, however, striking differences in the composition of the cell wall of the stalk and the cap. The glucose and galactose content of the cap wall is much higher than that of the stalk wall. Rhamnose is found in the cap wall only. The different composition of stalk and cap wall is also manifest in cells which were enucleated prior to cap formation. Therefore, regulation of the enzymatic processes which are responsible for the different composition of stalk and cap wall take place in enucleated cells also.     

Brittle stalk 2 encodes a putative glycosylphosphatidylinositol-anchored protein that affects mechanical strength of maize tissues by altering the composition and structure of secondary cell walls

A spontaneous maize mutant, brittle stalk-2 (bk2-ref), exhibits dramatically reduced tissue mechanical strength. Reduction in mechanical strength in the stalk tissue was highly correlated with a reduction in the amount of cellulose and an uneven deposition of secondary cell wall material in the subepidermal and perivascular sclerenchyma fibers. Cell wall accounted for two-thirds of the observed reduction in dry matter content per unit length of the mutant stalk in comparison to the wildtype stalk. Although the cell wall composition was significantly altered in the mutant in comparison to the wildtype stalks, no compensation by lignin and cell wall matrix for reduced cellulose amount was observed. We demonstrate that Bk2 encodes a Cobra-like protein that is homologous to the rice Bc1 protein. In the bk2-ref gene, a 1 kb transposon-like element is inserted in the beginning of the second exon, disrupting the open reading frame. The Bk2 gene was expressed in the stalk, husk, root, and leaf tissues, but not in the embryo, endosperm, pollen, silk, or other tissues with comparatively few or no secondary cell wall containing cells. The highest expression was in the isolated vascular bundles. In agreement with its role in secondary wall formation, the expression pattern of the Bk2 gene was very similar to that of the ZmCesA10, ZmCesA11, and ZmCesA12 genes, which are known to be involved in secondary wall formation. We have isolated an independent Mutator-tagged allele of bk2, referred to as bk2-Mu7, the phenotype of which is similar to that of the spontaneous mutant. Our results demonstrate that mutations in the Bk2 gene affect stalk strength in maize by interfering with the deposition of cellulose in the secondary cell wall in fiber cells.     

Morphological studies of the soft tissues involved in skeletal dissolution in the coralFungia fungites

Light and transmission electron microscopy were used to study mechanisms involved in the separation of the disc from the stalk in juvenileFungia fungites (Scleractinia, Fungiidae). Separation occurs because the skeleton is weakened by dissolution across a distinct plane at the junction of the stalk and disc. The tissue layer adjacent to the skeleton in the stalk was composed of typical, squamose, calicoblastic cells. In contrast, calicoblastic cells in the region of skeletal dissolution were tall and columnar. They contained many microvilli, abundant mitochondria and several different types of vesicles. It is assumed that these calicoblastic cells are actively involved in skeletal dissolution.     

Ultrastructural Features of Three Ectocommensal Protozoa Attached to the Gills of the Red Swamp Crawfish,Procambarus clarkii (Crustacea: Decapoda)1

Because heavy branchial infestations are thought to interfere with respiration, we examined the attachment of three stalked ciliates commonly found in the branchial chambers of Louisiana crawfish. Attachments by Cothurnia sp., Epistylis sp. and Acineta sp. differ in their fine structure. Stalks of the peritrichous ciliates. Cothurnia and Epistylis, contain striated tubules that differ in their arrangement, diameter, and in the periodicity of their striations. In both species the striated tubules branch within the basal disk and attach to a pad of adhesive material secreted by the organism during initial attachment to the gill surface. The stalk of the suctorian Acineta is composed of a striated honeycomb-like matrix. Within the basal disc the matrix is disorganized; however, striated elements anchor the stalk to a pad of adhesive material. Attachment sites also differ in the amount of secretory material deposited. Cothurnia forms a multi-layered, granular pad; Epistylis forms an indistinct, microfibrillar layer, and Acineta deposits a thick mucoid pad. None of the ciliates appear to damage the gill epicuticle nor is there an obvious host response. Harmful effects are probably limited to a decrease in respiratory surface area and disruption of normal water flow patterns. This may impair respiration sufficiently to increase the susceptibility of crawfish to low dissolved oxygen concentrations encountered periodically in commercial crawfish ponds.     

The Fine Structure of the Colonial Kinetoplastid Flagellate Cephalothamnium cyclopum Stein

Cell structure, cell adhesion, and stalk formation have been examined by electron microscopy in the colonial flagellate, Cephalothamnium cyclopum. Each cell is obconical or spindle-shaped, pointed posteriorly and truncated anteriorly. The cell membrane is underlain by epiplasm 0.1 μm thick in the posterior region, but bands of microtubules support the anterior region which is differentiated into a flagellar pocket, oral apparatus and contractile vacuole. Each of 2 flagella, joined a short way above their bases by an interflagellar connective, has a paraxial rod and mastigonemes. One flagellum is free and is important in food gathering while the other is recurrent and lies in a shallow groove on the ventral cell surface but projects posteriorly into the stalk. The basal bodies of these flagella are bipartite structures connected by a pair of striated rootlets with accessory microtubular fibers. The oral apparatus consists of a funnel-shaped buccal cavity and cytostome. It is supported by helical and longitudinal microtubules and also has nearby striated and microtubular fibers. Possible roles of associated oral vesicles in relation to ingestion are discussed. A reticulate mitochondrion houses a massive kinetoplast which has a fibrillar substructure resembling that of dinoflagellate chromosomes. Adjacent flagellates adhere by laminate extensions of their posterior regions and attach by their recurrent flagella to a communally secreted stalk composed of finely fibrillar material. This study indicates that Cephalothamnium belongs in the order Kinetoplastida, and has many features in common with members of the family Bodonidae.     

The Fine Structure of the Scopula-Stalk Region of Vorticella convallaria

ABSTRACT. We examined by SEM and TEM the stalk-scopular junction, the stalk, and stalk formation in Vorticella convallaria Linnaeus, 1767. The stalk sheath is anchored to the walls of the scopular lip and to the scopular cilia by thin fibrils. Experimental extraction of these fibrils weakens this junction enough to separate the stalk from the cell body. Telotrochs escape from the stalk by means of violent contractions of the cell body, accelerated beating of the trochal band cilia, and twisting of the cell body against the stalk. The edges of the scopular lip spread over the scopular cilia after escape and, in some cases, fuse to enclose the entire, aboral scopular surface in a cupola-like structure. The sessile cells contain fewer and smaller scopular granules than telotrochs. The presence of disintegrating scopular granules in the stalk matrix of some sessile cells suggests that they contain material which is secreted over a period of time to form the stalk. Eruptive formation of the initial adhesive pad and quick elongation of the distal part of the stalk suggests a rapid exocytosis of the larger, more numerous granules of the telotroch. The stalk sheath is formed of fibrils making up complete and incomplete compartments peripherally arranged along the major stalk axis.     

Analysis of the BadA stalk from Bartonella henselae reveals domain-specific and domain-overlapping functions in the host cell infection process

Human pathogenic Bartonella henselae cause cat scratch disease and vasculoproliferative disorders. An important pathogenicity factor of B. henselae is the trimeric autotransporter adhesin Bartonella adhesin A (BadA) which is modularly constructed and consists of a head, a long and repetitive neck‐stalk module with 22 repetitive neck/stalk repeats and a membrane anchor. The BadA head is crucial for bacterial adherence to host cells, binding to several extracellular matrix proteins and for the induction of vascular endothelial growth factor (VEGF) secretion. Here, we analysed the biological role of the BadA stalk in the infection process in greater detail. For this purpose, BadA head‐bearing and headless deletion mutants with different lengths (containing one or four neck/stalk repeats in the neck‐stalk module) were produced and functionally analysed for their ability to bind to fibronectin, collagen and endothelial cells and to induce VEGF secretion. Whereas a head‐bearing short version (one neck/stalk element) of BadA lacks exclusively fibronectin binding, a substantially truncated headless BadA mutant was deficient for all of these biological functions. The expression of a longer headless BadA mutant (four neck/stalk repeats) restored fibronectin and collagen binding, adherence to host cells and the induction of VEGF secretion. Our data suggest that (i) the stalk of BadA is exclusively responsible for fibronectin binding and that (ii) both the head and stalk of BadA mediate adherence to collagen and host cells and the induction of VEGF secretion. This indicates overlapping functions of the BadA head and stalk.     

The Stalk of the Suctorian Tokophrya infusionum: Histochemistry, Biochemistry, and Physiology

Tokophrya infusionum, a sessile suctorian with an external stalk and adhesive disc, has in its life cycle a ciliated, swimming embryo which metamorphoses into the adult form. The addition of NiCl₂ to the medium induced metamorphosis immediately; however, other salts had no effect. Incomplete metamorphosis, without stalk formation, occurred if the organism began metamorphosis before its anterior (stalk-forming) end touched a substrate. The stalk was studied by histochemical and biochemical technics to determine its composition. The stalk stained with mercury-bromphenol blue, and Alcian blue under a variety of conditions, but not with PAS. These results suggest that the stalk contains protein and sulfate groups, possibly in the form of a sulfated protein-polysaccharide. The stalk was insoluble in several common laboratory reagents, but did dissolve in hot 6 N HCl, 2 N NaOH, and papain. It was evident from amino acid analysis of the stalk and of the whole organism that 15% of the total protein is located in the stalk.     

Calculation of free energy barriers to the fusion of small vesicles

The fusion of small vesicles, either with a planar bilayer or with one another, is studied using a microscopic model in which the bilayers are composed of hexagonal- and lamellar-forming amphiphiles. The free energy of the system is obtained within the self-consistent field approximation. We find that the free energy barrier to form the initial stalk is hardly affected by the radius of the vesicle, but that the barrier to expand the hemifusion diaphragm and form a fusion pore decreases rapidly as the radius decreases. As a consequence, once the initial barrier to stalk formation is overcome, one which we estimate at 13 k_BT for biological membranes, fusion involving small vesicles should proceed with little or no further input of energy.     

Origin and function of the stalk-cell vacuole in Dictyostelium

Large vacuoles are characteristic of plant and fungal cells, and their origin has long attracted interest. The cellular slime mould provides a unique opportunity to study the de novo formation of vacuoles because, in its life cycle, a subset of the highly motile animal-like cells (prestalk cells) rapidly develops a single large vacuole and cellulosic cell wall to become plant-like cells (stalk cells). Here we describe the origin and process of vacuole formation using live-imaging of Dictyostelium cells expressing GFP-tagged ammonium transporter A (AmtA-GFP), which was found to reside on the membrane of stalk-cell vacuoles. We show that stalk-cell vacuoles originate from acidic vesicles and autophagosomes, which fuse to form autolysosomes. Their repeated fusion and expansion accompanied by concomitant cell wall formation enable the stalk cells to rapidly develop turgor pressure necessary to make the rigid stalk to hold the spores aloft. Contractile vacuoles, which are rich in H⁺-ATPase as in plant vacuoles, remained separate from these vacuoles. We further argue that AmtA may play an important role in the control of stalk-cell differentiation by modulating the pH of autolysosomes.     

The anterior-like cells in Dictyostelium are required for the elevation of the spores during culmination

 Shortly after initiation of Dictyostelium fruiting body formation, prespore cells begin to differentiate into non-motile spores. Although these cells lose their ability to move, they are, nevertheless, elevated to the tip of the stalk. Removal of the amoeboid anterior-like cells, located above the differentiating spores in the developing fruiting body, prevents further spore elevation although the stalk continues to elongate. Furthermore, replacement of the anterior-like cells with anterior-like cells from another fruiting body largely restores the ability to lift the spores to the top of the stalk. However, if amoeboid prestalk cells are used to replace the anterior-like cells, there is no restoration of spore elevation. Finally, when a droplet of mineral oil replaces differentiating spores, it is treated as are the spores: the mineral oil is elevated in the presence of anterior-like cells and becomes arrested on the stalk in the absence of anterior-like cells. Because a similar droplet of mineral oil is totally ignored by slug tissue, it appears that there is a dramatic transformation in the treatment of non-motile matter at this point in Dictyostelium development. Received: 26 January 1998 / Accepted: 27 May 1998     

HISTOCHEMICAL STUDIES OF THE EXTRACELLULAR CARBOHYDRATE OF COLACIUM MUCRONATUM (EUGLENOPHYCEAE)1

The stalk of Colacium mucronatum Bourr. & Chad. is composed primarily of a cylindrical shaft with a lightly staining inner core and diffuse peripheral cortex. The shaft and cortex arise from a ring-shaped region around the canal opening whereas the core appears continuous with the canal which may be associated with initial cell attachment. All parts of the stalk, as well as the lining and contents of the reservoir, canal and flagellum exhibit stain reactions associated with neutral or mildly acidic carbohydrate with widely spaced anionic groups in low concentration.     

Extracellular DNA facilitates the formation of functional amyloids in Staphylococcus aureus biofilms

Persistent staphylococcal infections often involve surface‐associated communities called biofilms. Staphylococcus aureus biofilm development is mediated by the co‐ordinated production of the biofilm matrix, which can be composed of polysaccharides, extracellular DNA (eDNA) and proteins including amyloid fibers. The nature of the interactions between matrix components, and how these interactions contribute to the formation of matrix, remain unclear. Here we show that the presence of eDNA in S. aureus biofilms promotes the formation of amyloid fibers. Conditions or mutants that do not generate eDNA result in lack of amyloids during biofilm growth despite the amyloidogeneic subunits, phenol soluble modulin peptides, being produced. In vitro studies revealed that the presence of DNA promotes amyloid formation by PSM peptides. Thus, this work exposes a previously unacknowledged interaction between biofilm matrix components that furthers our understanding of functional amyloid formation and S. aureus biofilm biology.     

Loss of the beta-catenin homologue aardvark causes ectopic stalk formation in Dictyostelium

Aardvark (Aar) is a Dictyostelium beta-catenin homologue with both cytoskeletal and signal transduction roles during development. Here, we show that loss of aar causes a novel phenotype where multiple stalks appear during late development. Ectopic stalks are preceded by misexpression of the stalk marker ST-lacZ in the surrounding tissue. This process does not involve the kinase GSK-3. Mixing experiments show that ectopic ST-lacZ expression and stalk formation are cell non-autonomous. The protein-cellulose matrix surrounding the stalk of aar mutant fruiting bodies is defective, and damage to the stalk of wild-type fruiting bodies leads to ectopic ST-lacZ expression. We postulate that poor synthesis of the stalk tube matrix allows diffusion of a stalk cell-inducing factor into the surrounding tissue.     

THE DEVELOPMENT OF CELLULAR STALKS IN BACTERIA

Extensive stalk elongation in Caulobacter and Asticcacaulis can be obtained in a defined medium by limiting the concentration of phosphate. Caulobacter cells which were initiating stalk formation were labeled with tritiated glucose. After removal of exogenous tritiated material, the cells were subjected to phosphate limitation while stalk elongation occurred. The location of tritiated material in the elongated stalks as detected by radioautographic techniques allowed identification of the site of stalk development. The labeling pattern obtained was consistent with the hypothesis that the materials of the stalk are synthesized at the juncture of the stalk with the cell. Complementary labeling experiments with Caulobacter and Asticcacaulis confirmed this result. In spheroplasts of C. crescentus prepared by treatment with lysozyme, the stalks lost their normal rigid outline after several minutes of exposure to the enzyme, indicating that the rigid layer of the cell wall attacked by lysozyme is present in the stalk. In spheroplasts of growing cells induced with penicillin, the stalks did not appear to be affected, indicating that the stalk wall is a relatively inert, nongrowing structure. The morphogenetic implications of these findings are discussed.     

Early stalked stages in ontogeny of the living isocrinid sea lily Metacrinus rotundus

The early stalked stages of an isocrinid sea lily, Metacrinus rotundus, were examined up to the early pentacrinoid stage. Larvae induced to settle on bivalve shells and cultured in the laboratory developed into late cystideans. Three‐dimensional (3D) images reconstructed from very early to middle cystideans indicated that 15 radial podia composed of five triplets form synchronously from the crescent‐shaped hydrocoel. The orientation of the hydrocoel indicated that the settled postlarvae lean posteriorly. In very early cystideans, the orals, radials, basals and infrabasals, with five plates each in the crown, about five columnals in the stalk, and five terminal stem plates in the attachment disc, had already formed. In mid‐cystideans, an anal plate appeared in the crown. Late cystideans cultured in the field developed into pentacrinoids about 5 months after settlement. These pentacrinoids shared many crown structures with adult sea lilies. On the other hand, many features of the stalk differed from those in adult isocrinids, while sharing many characteristics with the stalk of feather star pentacrinoids, including disc‐like proximal columnals, high and slender median columnals, synarthrial articulations developmentally derived from the symplexial articulations, limited formation of cirri only in the proximal columnal(s), and an attachment disc. On the basis of these findings, phylogenetic relationships among extant crinoid orders are discussed.     

Genetic control of yield related stalk traits in sugarcane

A major focus of sugarcane variety improvement programs is to increase sugar yield, which can be accomplished by either increasing the sugar content of the cane or by increasing cane yield, as the correlation between these traits is low. We used a cross between an Australian sugarcane variety Q165, and a Saccharum officinarum accession, IJ76-514, to dissect the inheritance of yield-related traits in the complex polyploid sugarcane. A population of 227 individuals was grown in a replicated field trial and evaluated over 3 years for stalk weight, stalk diameter, stalk number, stalk length and total biomass. Over 1,000 AFLP and SSR markers were scored across the population and used to identify quantitative trait loci (QTL). In total, 27 regions were found that were significant at the 5% threshold using permutation tests with at least one trait; individually, they explained from 4 to 10% of the phenotypic variation and up to 46% were consistent across years. With the inclusion of digeneic interactions, from 28 to 60% of the variation was explained for these traits. The 27 genomic regions were located on 22 linkage groups (LGs) in six of the eight homology groups (HGs) indicating that a number of alleles or quantitative trait alleles (QTA) at each QTL contribute to the trait; from one to three alleles had an effect on the traits for each QTL identified. Alleles of a candidate gene, TEOSINTE BRANCHED 1 (TB1), the major gene controlling branching in maize, were mapped in this population using either an SSR or SNP markers. Two alleles showed some association with stalk number, but unlike maize, TB1 is not a major gene controlling branching in sugarcane but only has a minor and variable effect.     

Effects of Cyclic Nucleotides and Nucleoside Triphosphates on Stalk Formation in Caulobacter crescentus

Extensive stalk elongation in Skl mutants of Caulobacter crescentus occurs when they are grown in complete medium. This stalk elongation is less pronounced in synthetic medium with glucose as the sole carbon source than in complex peptone yeast extract medium. Addition of exogenous nucleoside triphosphates (adenosine triphosphate [ATP], guanosine triphosphate [GTP], cytidine triphosphate, and uridine triphosphate) inhibits stalk elongation of the Skl mutants, whereas cyclic guanosine 3',5'-monophosphate (GMP) stimulates stalk elongation in the Skl strains grown in synthetic glucose medium. Cyclic GMP also produces stalk elongation in wild-type C. crescentus and concurrently produces a cell division defect resulting in cellular filament formation. Under conditions tested, cyclic adenosine 3',5'-monophosphate and dibutyryl cyclic adenosine monophosphate did not enhance stalk elongation. Endogenous ATP and GTP levels in the mutants are significantly lower than corresponding nucleotide concentrations of the parent wild-type strains. Control of syntheses resulting in stalk formation in C. crescentus appears to be related to intracellular concentrations of nucleotides, with cyclic GMP as a prominent candidate for an important regulatory role in this aspect of morphogenesis.