Are extracellular matrix surface network components involved in signalling and protective function?

Endosperm is an interesting model for in vitro experiments, because of its unique origin, development and ploidy level. Here we used Actinidia deliciosa endosperm-derived callus to investigate morphology, histology and chemistry of extracellular matrix (ECM) structures in morphogenically stable tissue from long-term culture. SEM and TEM analysis showed that ECM is a heterogenous layer which consists of amorphous, dark-staining material, osmiophilic granules and reticulated fibres outside the outer callus cell wall. This structure may serve as a structural marker of morphogenic competence in endosperm-derived callus, because of its presence on the surface of callus forming morphogenic domains and its disappearance during organ growth. Based on immunolabelling, histochemistry, solvent and enzyme treatments, we suggest that pectins and lipids are components of the ECM layer. These results might indicate protective, water retention and/or cell communication functions for this ECM layer.Key words: Actinidia deliciosa, morphogenesis, scanning electron microscopy, transmission electron microscopyIn several plants cultured in vitro, SEM analysis revealed that induction of morphogenesis is linked to the appearance of a fibrillar network referred to as the extracellular matrix surface network (ECMSN).¹ Šamaj et al.,^2,3 reported, that the ECMSN plays an important morphoregulatory role during somatic embryogenesis and organogenesis, implying an active role in plant morphogenesis. According to Bobák et al.,⁴ the chemical composition and structural arrangement of the ECMSN on the cell surface indicate that it may play a fundamental role in cell-to-cell recognition and interaction, cell division and differentiation, and also in generation and maintenance of some traits in plant cell populations. There is not a large body of data confirming the occurrence of ECM during in vitro organogenesis.^5,6 Most data concern the formation, structure and chemical composition and function of ECM in somatic and androgenic embryogenesis (reviewed in refs. ^6–10). In previous histological and SEM analysis of morphogenic endosperm-derived callus of A. deliciosa, we described the presence of a membranous layer covering the callus surface, which we termed extracellular matrix (ECM).¹¹ The present experiments employed SEM, TEM and histochemical analysis to study the structure and composition of material coating the callus surface.SEM revealed the presence of heterogeneous material covering the callus surface. A smooth membranous layer coated some parts of the callus surface (Fig. 1A), while other regions were covered by fibrillar structures and granules of mucilage-like secretion forming a network at site of cell-cell adhesion. Similar structures were observed during in vitro induction of somatic embryogenesis, androgenesis and organogenesis in different species (reviewed in refs. ^7–13).Open in a separate windowFigure 1SEM images of kiwifruit endosperm-derived callus after callogenesis induction, long-term culture and organogenesis. (A) Layer composed of membranous, partially damaged (arrows) structure enveloping callus surface after long-term culture. (B–D) Callus induction after 3 weeks of culture. Remnants of seed coat (sc) and cells of non-morphogenic callus (c) are observed (B). However, some part of the callus surface is covered with fibrillar (arrowheads) and membranous extracellular matrix (ecm) with holes (arrows) (C and D). (E and F) Rhizogenesis after 6 weeks of culture. Root with root cap (rc) and root hairs (rh) is emerging from callus domain (d). Partly damaged (arrows) membranous material covering cells of root tip (F). Bars represent 50 µm for (D); 100 µm for (C and F); 200 µm for (A and B); and 500 µm for (E).TEM showed a layer composed of amorphous material with fibrillar and spherical components covering morphogenic cell clusters. The cell wall of non-embryogenic and senescent cells was also covered with ECM, but it differed in appearance: the layer was granular, with amorphous deposits. According to Rumyansteva et al.,¹⁴ loss of embryogenic competence was preceded by modification of the ECM structure from fibrillar to glue-like.Callus induction was observed after 2–3 weeks of culture (Fig. 1B). Membraneous and fibrillar structures were visible on the very small callus surface by SEM (Fig. 1C and D), though the first signs of organogenesis were noted 4–6 weeks later. TEM observations of the first steps of callogenesis also showed fine, amorphous ECM on the cell surface (data not shown). This might suggest that the extracellular matrix described above probably has a protective rather than signalling function.SEM of root tips appearing during culture showed that root cap cells were enveloped by a specific, partially damaged layer (Fig. 1E and F) similar in appearance to membraneous ECM on the callus surface. It is commonly known, that terminal part of the root tip is composed of pectins and performs a protective function, but signalling molecules were also detected on the root tip. In maize roots in vivo, Abeysekera and McCully¹⁵ found ECM occurring as a pellicle covering young meristematic epidermal cells, which contained arabinogalactan proteins.Solvent treatment and histochemical staining with Sudan Black B suggested that some lipophilic substances are part of the ECM in kiwifruit. Damage to the membranous layer observed by SEM after solvent treatment might be the results of removal of lipid components from the ECM. Moreover, TEM of the surface layer revealed globular deposits similar in electron density and appearance to lipid bodies. Apart from Actinidia, lipophilic components of the ECM have been reported only in Triticum.¹⁰ The probable presence of lipids in the examined ECM may be connected with protective and signalling function, especially in oxygen stress conditions.¹⁶ Pectinase digestion caused only partial disappearance of the ECM, exposing granular remnants on the cell surface and filaments forming thick fibrils at sites of cell adhesion, visible by SEM. Cellulose-binding fluorescent dye ruled out cellulose polymers as an element of ECM structure. Additionally, we identified a callose component in morphogenic callus. Deposition of polysaccharides around embryogenic cells has been suggested by Šamaj et al.,³ as an early structural marker for these cells.During these experiments we detected JIM5-labelled low-methylesterified pectins in intercellular spaces and on the surface layer. A comparison of our results with those obtained in Triticum⁶ and Zea³ supports Šamaj et al.,³ assumption that regulation of pectin localization within the ECM differs between monocotyledonous and dicotyledonous.Our results suggest the possible role of the extracellular material during kiwifruit regeneration. Pectins serve as a reservoir of signalling molecules and for water retention. Consequently, ECM may be involved in integration and recognition of morphogenic cells within multicellular callus domains, and a protective function cannot be excluded.     

Developmental SEM observations on an extracellular matrix in embryogenic calli ofDrosera rotundifolia andZea mays

Summary Primary embryogenic callus ofDrosera rotundifolia and long-term cultured embryogenic callus ofZea mays possess a conspicuous extracellular matrix (ECM) around and between embryogenic cells. The structural arrangement of ECM depends on the developmental stage of the embryogenic cells. Single embryoid cells were covered with, and connected by net-like material. However, surface cells of young globular embryoids were covered with a coherent layer of ECM which forms bridges with net-like material between the cells which was gradually reduced to coarse strands. When protodermis was formed on the surface of globular embryoids, the ECM disappeared completely. The ECM network was never observed on the surface of heart- and torpedo-shaped embryoids. Safranine (especially 0.1%) stabilized the structure of ECM. Digestion with pronase E and proteinase K indicated that the ECM contains proteinaceous components. Similar developmental patterns of ECM were observed in dicotyledonous and monocotyledonous examples. The ECM represents a stable morphological structure even during long-term embryogenic culture in maize.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - Dicamba 3,6-dichloro-o-anisic acid - ECM extracellular matrix - KIN kinetin - SEM scanning electron microscopy - TEM transmission electron microscopy     

Extracellular Matrix Surface Network During Plant Regeneration in Wheat Anther Culture

Androgenic plant regeneration from wheat anther callus was accompanied by the formation of a conspicuous extracellular matrix surface network (ECMSN) around the induced callus cells and young embryo-like structures. Microscopic observations at the onset of regeneration revealed the presence of two distinct types of cells on the callus surface: large, loosely attached parenchymatous cells and small tightly packed meristematic cells arranged in multicellular clusters. Parenchyma cells of the callus had smooth surface, while on the surface and between the cells of multicellular clusters numerous fine fibrils of ECMSN were observed. The structural arrangement of the ECMSN changed during culture. On the surface of globular embryo-like structures, before protoderm formation, the ECMSN was the most abundant and arranged as a compact layer of secretion with wide strands visible at the cell junctions. Further development of globular embryos was disturbed, giving rise to branched structures outlined by continuous epidermis. The development of such regenerants was accompanied by gradual degradation of the extracellular network and finally its complete disappearance. Digestion with protease did not destroy the network. Treatment of the calluses with chloroform and washing with ether–methanol led to partial destruction of the network, while digestion with pectinase removed the network completely and resulted in the collapse of surface embryo cells.     

Extracellular matrix of plant callus tissue visualized by ESEM and SEM

Actinidia deliciosa endosperm-derived callus culture is stable over a long period of culture. This system was used to investigate the ultrastructure of extracellular matrix occurring in morphogenic tissue. Specimens were prepared by different biological techniques (chemical fixation, liquid nitrogen fixation, glycerol substitution, critical-point drying, lyophilization) and observed by scanning electron microscopy (SEM). Fresh and wet samples were analyzed with the use of environmental scanning electron microscopy (ESEM). Extracellular matrix was observed on the surface of cell clusters as a membranous layer or reticulated network, shrunken or wrinkled, depending on the procedure. Generally, shrunken membranous layers with a globular appearance and fibrils were noted after critical-point drying and liquid nitrogen fixation. Smoother surface layers without visible fibrils and showing porosity were typically seen by environmental scanning electron microscopy. Preservation with glycerol substitution caused wrinkled appearance of examined layer. Analysis of fresh samples yielded images closer to their natural state than did critical-point drying or fixation in liquid nitrogen, but it seems best to compare the results of different visualization methods. This is the first report of ESEM observations of plant extracellular matrix and comparison with SEM images from fixed material.     

Three-dimensional organization of the extracellular matrix secreted by cultured rat smooth muscle cells

Summary Specific interactions between cells and the extracellular matrix (ECM) in which they are embedded play a vital role in tissue organization. In recent years, many of the individual components of the extracellular matrix have been isolated and their molecular structures elucidated, but the detailed topography of most extracellular matrices, as they are deposited by cells, is still largely unknown. In this study, the insoluble extracellular matrix produced by cultured rat vascular smooth muscle cells has been characterized morphologically using high-resolution electron microscopy of rotary platinum replicas. These cells grew as flat sheets in culture, secreting their matrix laterally and basally. The matrix was composed of a cross-linked fibrillar meshwork. Some fine fibers (10 to 15 nm in diameter) were naked, but most of the filamentous mesh was covered with coarse granular material. Limited digestion with trypsin or pancreatic elastase removed most of this coating, indicating that the granules were glycoproteins and proteoglycans. Another subset of matrix fibrils (20 to 40 nm in diameter) was identified as type I collagen by direct comparison with purified bovine skin collagen. In addition to exposing the underlying filamentous substructure of the matrix, protease treatment also revealed large, straight fiber bundles and globules of amorphous material suspended in the filamentous web. This novel view of a complex matrix promises to provide spatial information that will be useful in future studies of cell interactions with the ECM. These studies were supported in part by NIH Biomedical Research Support grant S07-RR-05684.     

Distribution of pectin and arabinogalactan protein epitopes during organogenesis from androgenic callus of wheat

The distribution of several arabinogalactan protein and pectic epitopes were studied during organogenesis in androgenic callus of wheat. In cell wall of mature and degenerating parenchyma cells, the arabinogalactan epitopes JIM4, JIM14, JIM16 or LM2 were expressed differently according to the cells location. LM2 was observed also in meristematic cells of regenerated shoot buds and leaves. Anti-pectin JIM7 labelled the wall of meristematic cells but fluorescence was strongest in outer walls of surface cells of callus and shoot buds coated by extracellular matrix surface network (ECMSN). During leaves growth the ECMSN disappeared, and JIM7 fluorescence decreased. JIM5 epitope was abundant in the cell walls lining the intercellular spaces of callus parenchyma and in tricellular junctions within regenerated buds and leaves.     

中华蜜蜂咽下腺的电镜观察

王浆是由工蜂的咽下腺所分泌,咽下腺位于工蜂头腔内两侧,每侧腺体都有一条长而粗的分泌管,在分泌管的两侧平行排列有许多椭圆形小叶,每个小叶内有十多个细胞,每个细胞有一根胞外管与分泌管直接相通,细胞内有一条胞内管又与胞外管相连。王浆就是通过细胞内分泌块分泌而成,由胞内管把王浆输送到胞外管,最后由分泌管到咽喉部,在此处饲喂王浆给蜂王、幼蜂和雄蜂。通过咽下腺的超微结构观察,不仅了解到咽下腺各种细胞器的形态特征,同时也了解到输送王浆的途径。     

Extracellular matrix surface network of embryogenic units of friable maize callus contains arabinogalactan-proteins recognized by monoclonal antibody JIM4

Embryogenic units of friable maize callus are formed as globular or oblong packets of tightly associated meristematic cells. These units are surrounded by conspicuous cell walls visible in light microscopy after staining with basic fuchsin. Transmission electron microscopy revealed that embryogenic cells are rich in endoplasmic reticulum, polysomes and small protein bodies, and that the outermost layer of their cell walls is composed of fibrillar material. Electron microscopy has also shown that this material covers the surface of embryogenic cells as a distinct layer which we denote as extracellular matrix surface network (ECMSN). Employing histochemical staining with β-glucosyl Yariv phenylglycoside, we localized arabinogalactan-proteins (AGPs) to the outer cell walls of embryogenic units including ECMSN. The most prominent staining was found in cell-cell junction domains. Large non-embryogenic callus cells were not stained with this AGP-specific dye. Immunofluorescence and silver-enhanced immunogold labelling using monoclonal antibody JIM4 has shown that the ECMSN of embryogenic cells is equipped with JIM4 epitope, while non-embryogenic callus cells are devoid of this epitope. We propose that some specific AGPs of the ECMSN might be relevant for cell-cell adhesion and recognition of embryogenic cells during early embryogenic stages, and that the JIM4 antibody can serve as an early marker of embryogenic competence in maize callus culture. Received: 13 March 1998 / Revision received: 6 June 1998 / Accepted: 1 July 1998     

Adhesion between cells and extracellular matrix with special reference to hepatic stellate cell adhesion to three-dimensional collagen fibers

Hepatic stellate cells are located in the perisinusoidal space (space of Disse), and extend their dendritic, thin membranous processes and fine fibrillar processes into this space. The stellate cells coexist with a three-dimensional extracellular matrix (ECM) in the perisinusoidal space. In turn the three-dimensional structure of the ECM regulates the proliferation, morphology, and functions of the stellate cell. In this review, the morphology of sites of adhesion between hepatic stellate cells and extracellular matrix is described. Hepatic stellate cells cultured in polystyrene dishes spread well, whereas the cells cultured on or in type I collagen gel become slender and elongate their long cellular processes which adhere directly to the collagen fibers. Cells in type I collagen gel form a large number of adhesive structures, each adhesive area forming a face but not a point. Adhesion molecules, integrins, for the ECM are localized on the cell surface. Elongation of the cellular processes occurs via integrin-binding to type I collagen fibers. The signal transduction mechanism, including protein and phosphatidylinositol phosphorylation, is critical to induce and sustain the cellular processes. Information on the three-dimensional structures of ECM is transmitted via three-dimensional adhesive structures containing the integrins.     

Human disease-associated extracellular matrix orthologs ECM3 and QBRICK regulate primary mesenchymal cell migration in sea urchin embryos

Sea urchin embryos have been one of model organisms to investigate cellular behaviors because of their simple cell composition and transparent body. They also give us an opportunity to investigate molecular functions of human proteins of interest that are conserved in sea urchin. Here we report that human disease-associated extracellular matrix orthologues ECM3 and QBRICK are necessary for mesenchymal cell migration during sea urchin embryogenesis. Immunofluorescence has visualized the colocalization of QBRICK and ECM3 on both apical and basal surface of ectoderm. On the basal surface, QBRICK and ECM3 constitute together a mesh-like fibrillar structure along the blastocoel wall. When the expression of ECM3 was knocked down by antisense-morpholino oligonucleotides, the ECM3-QBRICK fibrillar structure completely disappeared. When QBRICK was knocked down, the ECM3 was still present, but the basally localized fibers became fragmented. The ingression and migration of primary mesenchymal cells were not critically affected, but their migration at later stages was severely affected in both knock-down embryos. As a consequence of impaired primary mesenchymal cell migration, improper spicule formation was observed. These results indicate that ECM3 and QBRICK are components of extracellular matrix, which play important role in primary mesenchymal cell migration, and that sea urchin is a useful experimental animal model to investigate human disease-associated extracellular matrix proteins.     

Fine Structure of Established Callus Cultures of Taraxacum officinale Weber

The nodular, brown callus cultures of Taraxacum officinale growslowly on a modified White's medium. A section of nodule revealsa meristematic layer bounded both internally and externallyby parenchymatous tissue. No other types of tissue occur withinthe callus. The cells of the inner parenchyma are often compressedand senescing, whereas in the outer tissue localized de-differentiationapparently contributes to the development of new nodules duringcallus growth. Fine-structural observations of both meristematic and parenchymatoustissues show the normal complement of higher plant cell organellesexcept for the apparent absence of cytoplasmic microtubules.The rough endoplasmic reticulum cisternae are often alignedparallel to the cell wall or in whorls and may show pores, thusresembling annulate lamellae. Numerous lipid bodies, up to 7µm wide, occur and these are sometimes invested by arraysof apparently membranous material. The mitochondria are frequentlyhighly branched and often show a scalariform arrangement ofcristae. The plastids show few internal membranes despite cultureof the callus under continuous illumination. Lomasomes are very common in all cells and in the parenchymatissue membranous wall bodies also occur. The latter bodiesare much larger than lomasomes and consist of wall overgrowthsin which vesicular, myelin-like or isolated membranous elementsare enmeshed in fibrillar material. It is suggested that membranouswall bodies may originate from the amalgamation and subsequentproliferation of several adjacent lomasomes. Taraxacum officinale Weber, dandelion callus cultures, fine structure     

Analysis of extracellular matrix proteins produced by cultured cells

The extracellular matrix (ECM) is a highly organized multimolecular structure essential for the vital functions of any organism. Although much of the data of extracellular matrix components has been accumulated, the isolation of an entire set of these proteins remains a complex procedure due to the high content of fibrillar proteins and proteoglycans, which form multidomain, netlike structures. In the study presented, we developed a method for isolating ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membranes. Subsequent treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibers significantly improved the fractioning of ECM proteins. The extraction of remaining proteins from the surface of the culture plate was preformed by a buffer developed based on Laemmli probe buffer. Using this method, we isolated ECM proteins synthesized by cultured cells, and the extracted proteins were suitable for future analysis by SDS PAGE and two-dimentional electrophoresis, as well as for identifying individual proteins by mass spectrometry. This study may allow us to compare assortments of ECM proteins isolated from different sources, and elucidate impact of various proteins on structure and property of extracellular matrix of investigated cells.     

Interaction of rhizobacteria with leafy spurge (Euphorbia esula L.) callus tissue cells

The interaction of two rhizobacterial isolates,Pseudomonas fluorescens isolate LS 102 andFlavobacterium balustinum isolate LS 105, with leafy spurge cells at the cellular level was studied using scanning and electron microscopy. Leafy spurge callus tissue inoculated with either isolate showed considerable changes compared to non-inoculated tissue. The attachment of rhizobacteria to cell surfaces was associated with the elaboration of fibrillar material which may anchor bacteria to surfaces and contribute to mediation of the phytotoxic effect caused by rhizobacteria. At the ultracellular level, inoculated callus tissue showed numerous cell alterations including esiculation and convolution of the plasmalemma, cell wall degradation and disorganization of the cytoplasm, similar to those detected in the whole plant. It is concluded that callus tissue may provide an excellent working model to investigate the mode and/or mechanism of action of potential biocontrol agents on their host plants.     

Cranial meninges of goldfish: age-related changes in morphology of meningeal cells and accumulation of surfactant-like multilamellar bodies

In the optic tectum of goldfish, the outer, middle and inner layers of the endomeninx were evident in animals ranging in age from 1 month to several years. The outer layer in young animals consisted of closely overlapping cells with intertwined processes, whereas in the older animals it contained large extracellular spaces. The intermediate layer cells were always arranged in a single continuous layer, but in young animals they overlapped extensively with one another toward their edges whereas in the oldest animals they became extremely flat and non-overlapping. The inner layer included an outer tier of cells with their bases adhering to the intermediate layer, and an inner tier of cells detached from both the intermediate layer and the basal lamina overlying the brain parenchyma. Inner layer cells contained many large vacuoles that were in continuity with the extracellular space. With age, the extracellular space and the vacuolar system expanded, and the inner layer evolved into a meshwork of attenuated cytoplasmic processes embedded in the granular extracellular matrix. Another age-related feature was the accumulation adjacent to the basal lamina of uniform disc-shaped membranous structures, resembling multilamellar bodies of lung surfactant. These disc bodies were apparently generated by the coalescence of vesicles formed at the surface of the inner layer cells, possibly as a by-product of protein secretion by these cells.     

Organization of extracellular matrix components during differentiation of adipocytes in long-term culture

Summary Scanning electron microscopy (SEM) observation showed that fully differentiated spherical adipocytes were embraced by a network of collagens and fibroblastic preadipocytes. The properties of both the collagen networks and the preadipocytes allow the adipocytes to be interconnected, forming a fat-cell cluster, which can anchor to the bottom of a culture dish. In this network structure, collagen fibrils and fibrillar bundles were closely arranged and stratified. We found that immunostained collagens appeared to form extracellular network structures, which can be observed by SEM. The extracellular network of fibronectin was the first to develop among the extracellular matrix (ECM) components, though it became degraded with the progress of adipocyte differentiation. The type I collagen network was the last to develop and remained well organized through the late stage of adipocyte differentiation. The extracellular networks of type III, V, and VI collagen developed by the mid-stage and remained in the late stage of adipocyte differentiation. The network structures of type IV collagen and laminin became degraded during the differentiation process and localized at the surface of spherical cells. In addition to these basement membrane components, types III, V, and VI collagens also showed pericellular spherical staining patterns. These results demonstrated that the constitution and distribution of the ECM are altered during adipocyte differentiation, suggesting that the organization of each ECM component into a suitable structure is a requirement for the differentiation and maintenance of unilocular adipocytes.     

Fine Structure of the Oocyst Wall of Eimeria nieschulzi

SYNOPSIS. Oocysts of Eimeria nieschulzi from the laboratory rat, Rattus, norvegicus , were studied by scanning and transmission electron microscopy. Oocysts had a rough outer wall with apparent random depressions. The oocyst wall is composed of 2 layers: an osmiophilic outer layer consisting of a rough external and smooth internal surface, and a relatively thick, electron-lucent inner layer. The outer layer is composed of a dense, coarsely granular matrix. The inner layer consists of homogeneous fine granular material interspersed with coarse osmiophilic granules and contains one closely applied membrane on the outermost surface. Several raised lenticular areas are seen on the coarse outer surface of the inner layer. These layers are 102 (75–128) and 176 (135–204) nm thick, respectively.
The sporocyst wall is thin, consisting of 3 to 4 unit membranes, and measures 27 (18–34) nm thick.     

Intercellular matrix in colonies of Candida.

The histochemistry and fine structure of typical colonies of six species of Candida were studied, using a total of 31 clinical isolates. The colonies consisted of viable and degenerate cells which lay in an intercellular matrix. This matrix was made up of amorphous, granular, and fibrillar components, the relative proportions and total amount of which varied from species to species. The cells of all species were surrounded by a zone of homogeneous amorphus material, which may be a highly cross-linked carbohydrate. This separated intact cells from irregularly distributed granular debris derived from the cytoplasm of degenerate cells. Focal cellular degeneration and associated granular debris were present within the colonies of all species and were most common in the surface layers of cells of colonies of C. albicans and C. tropicalis. The large amounts of intercellular matrix in this region formed a surface coat on colonies of these two species. Intercellular strands of cell wall material, and to a lesser extent other membranous elements from degenerate cells, formed a prominent fibrillar meshwork in the colonies of C. albicans and C. tropicalis, but were less common in those of C. pseudotropicalis and C. guilliermondii and seldom seen in those of C. parapsilosis and C. krusei.     

Colony Organization in the Green Alga Botryococcus braunii (Race B) Is Specified by a Complex Extracellular Matrix

Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous β-1, 4- and/or β-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form “drapes” between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.     

Ultrastructural changes in the oviduct of the laying hen during the laying cycle

The ultrastructural changes occurring in the fully functional oviduct of Isa Brown laying hens were studied during various stages of the laying cycle. Hens were killed at different positions of the egg in the oviduct. The oviduct was lined by ciliated and non-ciliated cells (also referred to as granular cells). The granular cells in the infundibulum contributed to secretion during egg formation, whereas ciliated cells showed little evidence of secretion. Ultrastructural changes were recorded in the granular and glandular cells of the distal infundibulum. In the magnum, the surface ultrastructure revealed glandular openings associated with the ciliated and granular cells. Cyclic changes were recorded in the glandular cells of the magnum. With respect to the three observed types of glands, the structure of gland type A and C cells varied at different egg positions in the oviduct, whereas type B cells represented a different type of gland cell containing amorphous secretory granules. The surface epithelium of the isthmus was also lined by mitochondrial cells. Two types of glandular cell (types 1 and 2) were recorded in the isthmus during the laying cycle. Intracisternal granules were found in type 2 cells of the isthmus. A predominance of glycogen particles occurred in the tubular shell gland. The granular cells in the shell gland contain many vacuoles. During egg formation, these vacuoles regressed following the formation of extensive rough endoplasmic reticulum; the reverse also occurred. The disintegrated material found in the vacuoles may have been derived from the disintegrating granules. The Physiology Teaching Unit, University of New England, provided financial support to K. Chousalkar for this study.     

Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.Abbreviations SEM Scanning electron microscopy - TEM transmission electron microscopy