The attachment complex of brachiolaria larvae of the sea star <Emphasis Type="Italic">Asterias rubens</Emphasis> (Echinodermata): an ultrastructural and immunocytochemical study

The attachment complex of brachiolaria larvae of the asteroid Asterias rubens comprises three brachiolar arms and an adhesive disc located on the preoral lobe. The former are used in temporary attachment and sensory testing of the substratum, whereas the latter is used for permanent fixation to the substratum at the onset of metamorphosis. Brachiolar arms are hollow structures consisting of an extensible stem tipped by a crown of dome-like ciliated papillae. The papilla epidermis is composed of secretory cells (type A, B and C cells), non-secretory ciliated cells, neurosecretory-like cells and support cells. Type A and B secretory cells fill a large part of the papilla epidermis and are always closely associated. They presumably form a duo-gland adhesive system in which type A and B cells are respectively adhesive and de-adhesive in function. The adhesive disc is an epidermal structure mainly composed of secretory cells and support cells. Secretory cells produce the cement, which anchor the metamorphic larva to the substratum until the podia are developed. The relatedness between the composition of the adhesive material in the brachiolaria attachment complex and in the podia of adults was investigated by immunocytochemistry using antibodies raised against podial adhesive secretions of A. rubens. Type A secretory cells were the only immunolabelled cells indicating that their temporary adhesive shares common epitopes with the one of podia. The attachment pattern displayed by the individuals of A. rubens during the perimetamorphic period—temporary, permanent, temporary—is unique among marine non-vertebrate Metazoa.     

Improvement in organophosphorus hydrolase activity of cell surface-engineered yeast strain using Flo1p anchor system

Organophosphorus hydrolase (OPH) hydrolyzes organophosphorus esters. We constructed the yeast-displayed OPH using Flo1p anchor system. In this system, the N-terminal region of the protein was fused to Flo1p and the fusion protein was displayed on the cell surface. Hydrolytic reactions with paraoxon were carried out during 24 h of incubation of OPH-displaying cells at 30°C. p-Nitrophenol produced in the reaction mixture was detected by HPLC. The strain with highest activity showed 8-fold greater OPH activity compared with cells engineered using glycosylphosphatidylinositol anchor system, and showed 20-fold greater activity than Escherichia coli using the ice nucleation protein anchor system. These results indicate that Flo1p anchor system is suitable for display of OPH in the cell surface-expression systems.     

Larval adhesive organs and metamorphosis in ascidians

Summary The larva of Distaplia occidentalis bears three cup-shaped adhesive papillae, each with a prominent axial protrusion. At the onset of metamorphosis these organs rapidly evert through fenestrations in the cuticular layers of tunic exposing hyaline caps of adhesive. Additional adhesive material is secreted from collocytes during eversion. The stickiness of the papillae facilitates attachment to a variety of substrates.Each papilla is composed of more than 900 cells; six different types were identified. The wall of the cup contains about 260 myoepithelial cells with long attenuated processes. These extend from the rim of the cup to the base in the parietal (inner) layer. The apices of the myoepithelial cells are held in place by 11 pairs of specialized anchor cells bearing long bulbous microvilli. When the myoepithelial cells contract they force the axial protrusion forward and transform the papilla into a hyperboloidal configuration. The papilla is innervated by small motor fibers, but sensory fibers were not detected. The adhesive papillae of Distaplia are discussed in relationship to nine other recognizable types of papillae in the ascidians.     

Comparative ultrastructure of adhesive systems in the turbellaria

Summary Glandular adhesive organs and other structures by which turbellarians attach themselves temporarily to surfaces have been studied by electron microscopy. Adhesive organs in representatives of the turbellarian orders Haplopharyngida, Macrostomida, Polycladida, Rhabdocoela, Proseriata, and Tricladida are composed of three cell types: two gland cell types and a modified epidermal cell type through which the necks of the glands project. One of the gland types is characterized by its dense membrane-bound, 0.2–0.7Μm diameter, secretion granules and is called here theviscid gland on the basis of evidence that it secretes the adhesive substance by which the organs attach to surfaces. The other gland type is characterized by smaller (0.1Μm), less-dense, membrane-bound secretion granules and is called thereleasing gland on the basis of evidence that it secretes a substance by which adhering organs are released from surfaces. The modified epidermal cell is called theanchor cell; it has a well-developed cell web and bears microvilli with fibrous cores surrounding the tips of the gland necks in a collar-like fashion. Adhesive organs that have the two gland types, viscid and releasing glands, are referred to here asduo-gland adhesive organs. Other turbellarians, including orders Nemertodermatida, Acoela, and Lecithoepitheliata, have adhesive glands or other adhesive structures with a morphology unlike that of duo-gland adhesive organs. Ultrastructural characters of the adhesive organs show that the Macrostomida and Haplopharyngida are related, and that the Polycladida, Rhabdocoela, Proseriata, and Tricladida are related, and that these two groups of orders share a common ancestor. The Nemertodermatida, Acoela, and Lecithoepitheliata, with morphologically different adhesive systems, are apparently derived separately from these orders.     

Der Ankerapparat vonSida crystallina (Crustacea,Cladocera)

Zusammenfassung Die in eine Grundsubstanz eingebetteten Klebanker und Ankerfäden, mit deren Hilfe sichSida crystallina am Substrat befestigt, liegen auf der Epicuticula. Die Fäden sind mit der Epicuticula über Trennvorrichtungen verbunden. Die Sekretion der Ankerstrukturen durch die Epidermiszellen erfolgt zunächst in oberflächenparallelen Schichten. Später wird das Sekretionsfeld mit den Fadenenden nach hinten verschoben. Es folgt die Bildung der Trennstellen und schließlich die Sekretion der Cuticula. Nach der Häutung klappt die Ankerleiste in ihre funktionelle Stellung und die Procuticula skierotisiert.

---

The anchor apparatus ofSida crystallina (Crustacea, Cladocera)II. Ultrastructure and formation

Summary Sida crystallina attaches itself to the substratum by adhesive anchors. Anchors and anchor threads are embedded in a matrix and situated on the epicuticle. The anchor threads are connected to the epicuticle by special junctions at which they can be separated. The new structures of the anchor organs, which are secreted by epidermal cells, are firstly laid down in layers parallel to the surface. Later the area of secretion becomes shifted backward. Then the junctions are formed and finely the secretion of the cuticle follows. After molting the anchor ledge turns out in its functional position and sclerotisation of the procuticle takes place.

     

Larval morphology of the Nemertean Carcinonemertes epialti (Nemertea: Hoplonemertea)

The morphology of the newly hatched larva of Carcinonemertes epialti Coe has been examined by light and electron microscopy. The newly hatched larva is covered with cilia and measures about 110 μm in length. Four types of epidermal cells are recognizable: (1) Multiciliated cells, (2) vacuolated cells, (3) mucous cells, and (4) “knob cells”. The knob cells protrude from the posterior end of the larva and contain granules and bundles of microfilaments. The gut is incomplete and is located ventral to the bipartite proboscis. A bilobed brain and two subepidermal ocelli are found in the anterior end of the larva. The anterior and posterior cirri are composed of long, tightly appressed cilia that arise from an invagination of the epidermis at each end of the larva. The anterior cirrus is surrounded by two types of glandular cells. It is proposed that the knob cells have a role in larval attachment, combining the functions of the adhesive cells and anchor cells described in the duo-gland system of turbellarians. The cirri are believed to be larval sensory structures that function in substrate selection. Histological and ultrastructural observations suggest that the larvae of Carcinonemertes are relatively long lived and develop into juveniles without a drastic metamorphosis.     

Enzymatic dissection of embryonic cell adhesive mechanisms: II. Developmental regulation of an endogenous adhesive system in the chick neural retina

Previous studies have demonstrated the presence of two functionally distinct intercellular adhesive systems operating among embryonic chick neural retina cells. These systems differ in their proteolytic sensitivity, protection by calcium against proteolysis, dependence on calcium for function, and in vitro morphogenetic potential. In this report we demonstrate that functional expression of the calcium-dependent adhesive system of embryonic chick neural retina cells is developmentally regulated between Days 7 and 16 of development, whereas the calcium-independent adhesive system is not. Age-dependent changes are described in terms of the ability to produce adhesive-competent cells bearing the calcium-dependent adhesive system and in terms of the responses of these cells during aggregation to perturbations with various drugs. Enzyme and ion combinations other than calcium and typsin are shown to yield calcium-dependent adhesive-competent cells. We also describe the protective effect of calcium on the histological and ultrastructural organization of trypsinized embryonic neural retina tissue. The possible role of the calcium-dependent adhesive system in retinal development is discussed.     

Insights into the autotransport process of a trimeric autotransporter,Yersinia Adhesin A (YadA)

Trimeric autotransporter adhesins (TAAs) are a subset of a larger protein family called the type V secretion systems. They are localized on the cell surface of Gram‐negative bacteria, function as mediators of attachment to inorganic surfaces and host cells, and thus include important virulence factors. Yersinia adhesin A (YadA) from Yersinia enterocolitica is a prototypical TAA that is used extensively to study the structure and function of the type Vc secretion system. A solid‐state NMR study of the membrane anchor domain of YadA previously revealed a flexible stretch of small residues, termed the ASSA region, that links the membrane anchor to the stalk domain. In this study, we present evidence that single amino acid proline substitutions produce two different conformers of the membrane anchor domain of YadA; one with the N‐termini facing the extracellular surface, and a second with the N‐termini located in the periplasm. We propose that TAAs adopt a hairpin intermediate during secretion, as has been shown before for other subtypes of the type V secretion system. As the YadA transition state intermediate can be isolated from the outer membrane, future structural studies should be possible to further unravel details of the autotransport process.     

Toxoplasma gondii protease TgSUB1 is required for cell surface processing of micronemal adhesive complexes and efficient adhesion of tachyzoites

Host cell invasion by Toxoplasma gondii is critically dependent upon adhesive proteins secreted from the micronemes. Proteolytic trimming of microneme contents occurs rapidly after their secretion onto the parasite surface and is proposed to regulate adhesive complex activation to enhance binding to host cell receptors. However, the proteases responsible and their exact function are still unknown. In this report, we show that T. gondii tachyzoites lacking the microneme subtilisin protease TgSUB1 have a profound defect in surface processing of secreted microneme proteins. Notably parasites lack protease activity responsible for proteolytic trimming of MIC2, MIC4 and M2AP after release onto the parasite surface. Although complementation with full‐length TgSUB1 restores processing, complementation of Δsub1 parasites with TgSUB1 lacking the GPI anchor (Δsub1::ΔGPISUB1) only partially restores microneme protein processing. Loss of TgSUB1 decreases cell attachment and in vitro gliding efficiency leading to lower initial rates of invasion. Δsub1 and Δsub1::ΔGPISUB1 parasites are also less virulent in mice. Thus TgSUB1 is involved in micronemal protein processing and regulation of adhesive properties of macromolecular adhesive complexes involved in host cell invasion.     

Ultrastructural study of two glandular systems in the proboscidial glandular epithelium of Riseriellus occultus (Nemertea, Heteronemertea)

 Two different types of glandular system in the proboscidial epithelium of Riseriellus occultus have been investigated by transmission electron microscopy. As expected, most of the epithelial cells are glandular in nature. With regard to differences in the ultrastructure of these gland cells and in the formation and morphology of their secretory granules, we have categorized and described four types of gland cell, indicated as G₁, G₂, G₃, and G₄. Each gland cell has a completely intraepithelial body characterized by a prominent nucleus, developed rough endoplasmic reticulum, Golgi complexes, and numerous secretory granules at different stages of maturation. These four types of gland cell appear associated in pairs forming numerous glandular systems of two types (A, B). These glandular systems are restricted to the ventral surface of the proboscis and are scattered irregularly throughout its length. Each glandular system consists of two gland cells of different types. The gland cell necks in each glandular system extend together to the epithelial surface; they protrude onto this and form a papilla where they open in a common area. The epithelial supportive cells adjacent to the glandular systems have long, stout microvilli which have a core of tonofilaments. These tonofilaments gather into dense bundles which pass vertically through the supportive cells and attach to the extracellular matrix underlaying the cells by hemidesmosomes. Moreover, a single sensory process stands close to each papilla. The ultrastructural morphology of the type A glandular systems suggests that they have an adhesive function operating in a similar way to that of the duo-gland adhesive systems in other invertebrate groups, although they are not homologous with these. The spatial arrangement of the secreted products of the type B glandular systems suggests that these may contribute to increasing the grip of the proboscis on the prey. The secretory granules (=pseudocnids) of the type G₃ gland cells are very likely an autapomorphy of the Anopla, providing a character by which the relationships within the Nemertea can be evaluated. Accepted: 9 October 1997     

Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis

Two alternative cell-surface display systems were developed in Pichia pastoris using the α-agglutinin and Flo1p (FS) anchor systems, respectively. Both the anchor cell wall proteins were obtained originally from Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was displayed functionally on the cell surface of P. pastoris using the anchor proteins α-agglutinin and FS. The activity of CALB displayed on P. pastoris was tenfold higher than that of S. cerevisiae. The hydrolytic and synthetic activities of CALB fused with α-agglutinin and FS anchored on P. pastoris were investigated. The hydrolytic activities of both lipases displayed on yeast cells surface were more than 200 U/g dry cell after 120 h of culture (200 and 270 U/g dry cell, respectively). However, the synthetic activity of CALB fused with α-agglutinin on P. pastoris was threefold higher than that of the FS fusion protein when applied to the synthesis of ethyl caproate. Similarly, the CALB displayed on P. pastoris using α-agglutinin had a higher catalytic efficiency with respect to the synthesis of other short-chain flavor esters than that displayed using the FS anchor. Interestingly, for some short-chain esters, the synthetic activity of displaying CALB fused with α-agglutinin on P. pastoris was even higher than that of the commercial CALB Novozyme 435.     

Cell surface hydrophobicity and the orientation of certain bacteria at interfaces

Summary Individual cells of Flexibacter aurantiacus CW7 and Hyphomicrobium vulgare ZV580 orientate themselves perpendicularly to the interface in air-water, oil-water and solid-water systems. Electrostatic phenomena probably are not involved in this orientation, since no evidence was found of any localized distribution of positively-charged ionogenic groups on the bacterial surface. It is suggested that the orientation results from a relatively hydrophobic portion of each cell being rejected from the aqueous phase of the system. This property also may be related to the formation of rosettes by these bacteria. Electron micrographs of thin sections of cells sorbed to araldite blocks show that the cell proper is not in contact with the solid surface, but is anchored to it by extracellular adhesive material. The extracellular materials may be of a polysaccharide nature.     

Immobilization of Lactococcus lactis to cellulosic material by cellulose‐binding domain of Cellvibrio japonicus

Aims: Immobilization of whole cells can be used to accumulate cells in a bioreactor and thus increase the cell density and potentially productivity, also. Cellulose is an excellent matrix for immobilization purposes because it does not require chemical modifications and is commercially available in many different forms at low price. The aim of this study was to construct a Lactococcus lactis strain capable of immobilizing to a cellulosic matrix. Methods and Results: In this study, the Usp45 signal sequence fused with the cellulose‐binding domain (CBD) (112 amino acids) of XylA enzyme from Cellvibrio japonicus was fused with PrtP or AcmA anchors derived from L. lactis. A successful surface display of L. lactis cells expressing these fusion proteins under the P45 promoter was achieved and detected by whole‐cell ELISA. A rapid filter paper assay was developed to study the cellulose‐binding capability of these recombinant strains. As a result, an efficient immobilization to filter paper was demonstrated for the L. lactis cells expressing the CBD‐fusion protein. The highest immobilization (92%) was measured for the strain expressing the CBD in fusion with the 344 amino acid PrtP anchor. Conclusions: The result from the binding tests indicated that a new phenotype for L. lactis with cellulose‐binding capability was achieved with both PrtP (LPXTG type anchor) and AcmA (LysM type anchor) fusions with CBD. Significance and Impact of the Study: We demonstrated that an efficient immobilization of recombinant L. lactis cells to cellulosic matrix is possible. This is a step forward in developing efficient immobilization systems for lactococcal strains for industrial‐scale fermentations.     

Spatial distribution of proteins in the quagga mussel adhesive apparatus

The invasive freshwater mollusc Dreissena bugensis (quagga mussel) sticks to underwater surfaces via a proteinacious ‘anchor’ (byssus), consisting of a series of threads linked to adhesive plaques. This adhesion results in the biofouling of crucial underwater industry infrastructure, yet little is known about the proteins responsible for the adhesion. Here the identification of byssal proteins extracted from freshly secreted byssal material is described. Several new byssal proteins were observed by gel electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize proteins in different regions of the byssus, particularly those localized to the adhesive interface. Byssal plaques and threads contain in common a range of low molecular weight proteins, while several proteins with higher mass were observed only in the plaque. At the adhesive interface, a plaque-specific ~8.1 kDa protein had a relative increase in signal intensity compared to the bulk of the plaque, suggesting it may play a direct role in adhesion.     

Functional morphology of the locomotory podia ofHolothuria forskali (Echinodermata,Holothuroida)

Summary The ventral surface ofHolothuria forskali (Holothuroida, Aspidochirotida) is almost completely covered by small-sized podia that are locomotory. Each podium consists of a stem that allows the podium to lengthen, to flex, and to retract, and this is topped by a disc that allows the podium to adhere to the substratum during locomotion. Podia ofH. forskali do not end in a sucker and their adhesion to the substratum thus relies entirely on the disc epidermal secretions. The disc epidermis is made of five cell types: non-ciliated secretory cells of two different types that contain granules whose content is either mucopolysaccharidic (NCS1 cells) or mucopolysaccharidic and proteinic in nature (NCS2 cells), ciliated secretory cells containing small granules of unknown nature (CS cells), cilitated nonsecretory cells (CNS cells), and support cells. The cilia ofCS cells are subcuticular whereas those ofCNS cells, although also short and rigid, traverse the cuticle and protrude in the outer medium. During locomotion, epidermal cells of the podial disc are presumably involved in an adhesive/de-adhesive process functioning as a duogland adhesive system. Adhesive secretions would be produced byNCS1 andNCS2 cells and de-adhesive secretion byCS cells. All these secretions would be controlled by stimulations of the two types of ciliated cells (receptor cells) which presumably interact with the secretory cells by way of the nerve plexus. The lack of suckers and the coexistence of two adhesive cell types in the disc epidermis give the locomotory podia ofH. forskali a compromise structure which would perhaps explain their ability to move as efficiently along soft and hard substrata.     

Connection of vulval and uterine epithelia in Caenorhabditis elegans

In the Caenorhabditis elegans hermaphrodite, the establishment of the egg-laying system requires the connection of two epithelial tubes: the uterus of the gonad and the vulva in the underlying ectoderm. A specialized uterine cell, the anchor cell (AC), plays a central role in specifying the fates of the uterine and vulval precursor cells via the EGF-Ras-MAP kinase and the Notch/Delta signaling pathways. This central and common inducing source ensures that the two sets of cells are in register and it specifies the cell types that build the T-shaped connection between uterus and vulva. On either side, progeny of the induced cells form lumen structures and undergo stereotyped cell-to-cell fusion, thereby building epithelial tubes. Finally, the anchor cell fuses with a uterine syncytium and thus leaves only a thin cellular process between the lumen of the uterus and the vulva. In the adult, the fertilized eggs exit the lumen of the uterus through the vulva. This relatively simple developmental process serves as a model to study the biology of cells during organogenesis, such as intercellular signaling, cell polarity, invasion of basal laminae and epithelia, cell recognition and cell fusion. The anchor cell is a particularly interesting cell as it coordinates the development of its neighboring cells by using different signaling pathways at different times.     

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.     

Novel surface display system for heterogonous proteins on Lactobacillus plantarum

Aims: To establish a novel cell surface display system that would enable the display of target proteins on Lactobacillus plantarum. Methods and Results: Blast P analysis of the amino acids sequence data revealed that the N‐terminus of the putative muropeptidase MurO from L. plantarum contained two putative lysin motif (LysM) repeat regions, implying that the MurO was involved in bacterial cell wall binding. To investigate the potential of MurO for surface display, green fluorescent protein (GFP) was fused to MurO at its C‐terminus and the resulting fusion protein was expressed in Escherichia coli. After being mixed with L. plantarum cells in vitro, GFP was successfully displayed on the surfaces of L. plantarum cells. Increases in the fluorescence intensities of chemically pretreated L. plantarum cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for MurO. SDS sensitivity assay showed that the GFP fluorescence intensity was reduced after being treated with SDS. To demonstrate the applicability of the MurO‐mediated surface display system, β‐galactosidase from Bifidobacterium bifidium, in place of GFP, was functionally displayed on the surface of L. plantarum cells via MurO. Conclusions: The MurO was a novel anchor protein for constructing a surface display system for L. plantarum. Significance and Impact of Study: The success in surface display of GFP and β‐galactosidase opened up the feasibility of employing the cell wall anchor of MurO for surface display in L. plantarum.     

Unusual adhesive production system in the barnacle Lepas anatifera: An ultrastructural and histochemical investigation

Adhesives that are naturally produced by marine organisms are potential sources of inspiration in the search for medical adhesives. Investigations of barnacle adhesives are at an early stage but it is becoming obvious that barnacles utilize a unique adhesive system compared to other marine organisms. The current study examined the fine structure and chemistry of the glandular system that produces the adhesive of the barnacle Lepas anatifera. All components for the glue originated from large single‐cell glands (70–180 μm). Staining (including immunostaining) showed that L ‐3,4‐dihydroxyphenylalanine and phosphoserine were not present in the glue producing tissues, demonstrating that the molecular adhesion of barnacles differs from all other permanently gluing marine animals studied to date. The glandular tissue and adhesive secretion primarily consisted of slightly acidic proteins but also included some carbohydrate. Adhesive proteins were stored in cytoplasmic granules adjacent to an intracellular drainage canal (ICC); observations implicated both merocrine and apocrine mechanisms in the transport of the secretion from the cell cytoplasm to the ICC. Inside the ICC, the secretion was no longer contained within granules but was a flocculent material which became “clumped” as it traveled through the canal network. Hemocytes were not seen within the adhesive “apparatus” (comprising of the glue producing cells and drainage canals), nor was there any structural mechanism by which additions such as hemocytes could be made to the secretion. The unicellular adhesive gland in barnacles is distinct from multicellular adhesive systems observed in marine animals such as mussels and tubeworms. Because the various components are not physically separated in the apparatus, the barnacle adhesive system appears to utilize completely different and unknown mechanisms for maintaining the liquid state of the glue within the body, as well as unidentified mechanisms for the conversion of extruded glue into hard cement. J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.