Fine Structural Observations on the Turbellarians Stenostomum sp. and Microstomum lineare with Special Reference to the Extracellular Matrix and Connective Tissue Systems

Employing transmission electron microscopy, observations were made on epidermis, muscle cells and connective tissue systems, with special emphasis on extracellular matrix components (ECM), in two rather primitive turbellarians: Stenostomum sp. (Catenulida) and Microstomum lineare (Macrostomida). In Stenostomum the only ECM components found are basal laminae, predominantly situated subepidermally. In Microstomum ECM is well developed and connective tissue filaments abundant in conspicuous extracellular spaces. It is uncertain whether basal laminae exist. The finding of basal lamina structures as the only ECM component present in Stenostomum makes it now possible to establish a complete ECM and connective tissue hierarchy in turbellarians, ranging from a purely cellular type with no ECM present to systems dominated by ECM and very similar to loose connective tissue in vertebrates. Comparative aspects of ECM and connective tissue systems in turbellarians are discussed in addition to the difficulties and ambiguities regarding definition and nomenclature of basal matrices as basal laminae and subepidermal membranes.     

Leaf glands act as nectaries in Diplopterys pubipetala (Malpighiaceae)

Leaf glands of Diplopterys pubipetala were studied with light and electron microscopy. Aspects of their secretion, visitors and phenology were also recorded. Glands occur along the margin, at the apex and at the base of the leaf blade. All the glands begin secretion when the leaf is still very young, and secretion continues during leaf expansion. The highest proportion of young leaves coincides with the beginning of flowering. The glucose‐rich secretion is collected by Camponotus ants, which patrol the newly formed vegetative and reproductive branches. All the glands are sessile, partially set into the mesophyll, and present uniseriate epidermis subtended by nonvascularised parenchyma. The glands at the apex and base are larger and also consist of vascularised subjacent parenchyma. The cytoplasm of epidermal and parenchyma cells has abundant mitochondria, polymorphic plastids filled with oil droplets and a few starch grains. Golgi bodies and endoplasmic reticulum are more abundant in the epidermal cells. The parenchyma cells of the subjacent region contain chloroplasts and large vacuoles. Plasmodesmata connect all the nectary cells. The zinc iodide–osmium tetroxide (ZIO) method revealed differences in the population of organelles between epidermal cells, as well as between epidermal cells and parenchyma cells. Ultrastructural results indicate that leaf glands of D. pubipetala can be classified as mixed secretory glands. However, the secretion released by these glands is basically hydrophilic and composed primarily of sugars, hence these glands function as nectaries.     

Hepatic stellate cells: unique characteristics in cell biology and phenotype

Hepatic stellate cells (HSCs), a mesenchymal cell type in hepatic parenchyma, have unique features with respect to their cellular origin, morphology, and function. Normal, quiescent HSCs function as major vitamin A-storing cells containing over 80% of total vitamin A in the body to maintain vitamin A homeostasis. HSCs are located between parenchymal cell plates and sinusoidal endothelial cells, and extend well-developed, long processes surrounding sinusoids in vivo as pericytes. However, HSCs are known to be 'activated' or 'transdifferentiated' to myofibroblast-like phenotype lacking cytoplasmic lipid droplets and long processes in pathological conditions such as liver fibrosis and cirrhosis, as well as merely during cell culture after isolation. HSCs are the predominant cell type producing extracellular matrix (ECM) components as well as ECM degrading metalloproteases in hepatic parenchyma, indicating that they play a pivotal role in ECM remodeling in both normal and pathological conditions. Recent findings have suggested that HSCs have a neural crest origin from their gene expression pattern similar to neural cell type and/or smooth muscle cells and myofibroblasts. The morphology and function of HSCs are regulated by ECM components as well as by cytokines and growth factors in vivo and in vitro. Liver regeneration after partial hepatectomy might be an invaluable model to clarify the HSC function in elaborate organization of liver tissue by cell-cell and cell-ECM interaction and by growth factor and cytokine regulation.     

Response of xylem parenchyma by suberization in some hardwoods after mechanical injury

Summary Wound responses of xylem parenchyma by suberization were investigated in some hardwoods by light and electron microscopy. Suberized ray and axial parenchyma cells form a distinct boundary around the wound in all investigated species. Vessels and fibres within and close behind the suberized area appeared more or less occluded; vessels in Fagus, Quercus, and Populus contained suberized tyloses, those in Betula and Tilia contained amorphous and fibrillar deposits. A common mechanism for suberin deposition in the parenchyma cells became evident. Cisternae of the endoplasmic reticulum were apparently involved in suberization. Suberin compounds are extruded by cytoplasmic vesicles, which fused with the plasma membrane, in order to release their content. The suberin layer exhibited the typical lamellated structure; cytoplasmic continuity between suberized cells by plasmodesmata was maintained through the suberin layer. Fagus revealed the most intense suberized area as compared with the other species. Within the reaction zone of Fagus and Quercus, some individual ray and axial parenchyma cells exhibited a subdivision into 2 or 3 compartments prior to suberization. Subdivision was achieved by the formation of a primary wall-like layer. Subsequently, the compartments became individually suberized. Wounding during winter did not induce suberization. Also, samples wounded and kept under water during the vegetation period showed no response. The role of suberization in the effectivity of wound-associated compartmentalization is discussed.     

Extracellular matrix in some microturbellarians

Electron microscopy was used to study the ECM (extracellular matrix) in the epidermis, brain, and parenchyma of the catenulid turbellarians Stenostomum leucops leucops (Dugès) and Stenostomum leucops aquariorum Luther and of the proseriates Archiloa unipunctata (Fabricius) and Promonotus schultzei Meixner. Specimens were fixed with a tannic-acid fixation that enhances staining of extracellular proteins (TARI method) or a conventional electron microscopical method. Differences between species were found in the amount of matrix and extracellular fibrils. In S. leucops leucops the ECM was very sparse, consisting of only a thin electron-dense sheath beneath the epidermis and surrounding neuropil. These sheaths were not found in S. leucops aquariorum which had only rarely encountered extracellular spaces. In P. schultzei and A. unipunctata ECM was more abundant. A. unipunctata had a distinct sheath surrounding the neuropil and a fibrillar subepidermal membrane; the same membranes in P. schultzei appeared only as patches of ECM. In A. unipunctata hemidesmosome-like structures attached the epidermal cells to the underlying membrane, and from these attachments, fibrils projected to the underlying muscle layer. The TARI method revealed exocytotic activity in the brain. The morphology of ECM in these and other flatworms indicate that it could be an internal medium for transport and exchange of substances among cells. Variability in the structure of ECM among species probably does not reflect evolutionary relationships.     

Ultrastructural changes in cells of pea root cortical explants culturedIn vitro

Summary Root cortical explants from seedlings ofPisum sativum L., cv. Little Marvel were cultured on a sterile nutrient medium in the presence of auxins or auxins and cytokinin. Explants were fixed (and subsequently processed for electron microscopic observation) at the outset and after 30, 60, and 72 hours of culture under the two hormonal conditions. In the presence of auxin alone, the cell walls of the cortical parenchyma showed distinctive structural changes involving the deposition of a new, diffusely fibrillar primary wall. A considerable increase of rough ER in the adjacent cytoplasm was associated with the new wall synthesis. These wall changes are interpreted as auxin-induced and prelude to cell enlargement and later cell separation. No dramatic changes occurred in other cytoplasmic organelles or in the nucleus. In the presence of cytokinin and auxin, the striking cytological events observed included marked nuclear changes and greater cytoplasmic density due to increased organelles associated with the onset of DNA synthesis, mitosis and cytokinesis. New cell walls formed from the developed phragmoplasts, cleaving the original parenchyma cells into smaller cellular compartments with no accompanying cell enlargement. No marked changes in the original primary cell walls were observed in cytokinin-auxin-treated explants. By 72 hours some cells already had completed two successive cell divisions. No ultrastructural evidence was obtained suggesting that these cells were committed to their known fate of differentiating into mature tracheary elements in the subsequent 2–4 days. At 72 hours each explant represented a population of actively dividing, still considerably vacuolated meristematic cells.     

The role of caveolin-1 in pulmonary matrix remodeling and mechanical properties

Caveolin-1 (cav1) is a 22-kDa membrane protein essential to the formation of small invaginations in the plasma membrane, called caveolae. The cav1 gene is expressed primarily in adherent cells such as endothelial and smooth muscle cells and fibroblasts. Caveolae contain a variety of signaling receptors, and cav1 notably downregulates transforming growth factor (TGF)-beta signal transduction. In pulmonary pathologies such as interstitial fibrosis or emphysema, altered mechanical properties of the lungs are often associated with abnormal ECM deposition. In this study, we examined the physiological functions and the deposition of ECM in cav1(-/-) mice at various ages (1-12 mo). Cav1(-/-) mice lack caveolae and by 3 mo of age have significant reduced lung compliance and increased elastance and airway resistance. Pulmonary extravasation of fluid, as part of the cav1(-/-) mouse phenotype, probably contributed to the alteration of compliance, which was compounded by a progressive increase in deposition of collagen fibrils in airways and parenchyma. We also found that the increased elastance was caused by abundant elastic fiber deposition primarily around airways in cav1(-/-) mice at least 3 mo old. These observed changes in the ECM composition probably also contribute to the increased airway resistance. The higher deposition of collagen and elastic fibers was associated with increased tropoelastin and col1alpha2 and col3alpha1 gene expression in lung tissues, which correlated tightly with increased TGF-beta/Smad signal transduction. Our study illustrates that perturbation of cav1 function may contribute to several pulmonary pathologies as the result of the important role played by cav1, as part of the TGF-beta signaling pathway, in the regulation of the pulmonary ECM.     

Ultrastructure and histochemistry of the supportive structures associated with the radula of the slug,Limax maximus

The odontophore and connective tissue-filled portion of the radular sac (called the “collostyle”) of the slug, Limax maximus, were examined by light and electron microscopy. While both of these structures grossly resemble vertebrate cartilage, neither is composed of a type of tissue with the microscopic appearance and histochemical properties of cartilage. The roughly U-shaped odontophore possesses a thin capsule composed of connective tissue. The parenchyma of the odontophore consists of modified muscle cells which are organized into irregular groups by incomplete trabeculae composed of conventional muscle cells. The odontophoral cells are variable in size; they contain glycogen-filled “cores” as well as bundles of peripherally located filaments resembling myofilaments; and they are innervated like muscle cells. The nuclei of the cells are located eccentrically in the glycogen-filled portions of the cells and typically contain prominent nucleoli. The nuclei are surrounded by multiple small Golgi complexes and pleomorphic dense bodies resembling lysosomes. The extracellular matrix of the odontophore is very sparse and contains glycogen and fibrillar material but no histochemically demonstrable acidic mucosubstances. The collostyle consists of a gelatinous type of tissue somewhat like vertebrate mucoid connective tissue. The abundant extracellular matrix contains cross banded filaments, a flocculent material disposed in wavy indefinite strands, and small electron-dense particles. The matrix contains histochemically demonstrable neutral and weakly acidic mucosubstances. The cell population of the collostyle includes solitary muscle cells and fibrocytes containing large quantities of glycogen.     

Extracellular matrix and matrix receptors in blood-brain barrier formation and stroke

The blood-brain barrier (BBB) is formed primarily to protect the brain microenvironment from the influx of plasma components, which may disturb neuronal functions. The BBB is a functional unit that consists mainly of specialized endothelial cells (ECs) lining the cerebral blood vessels, astrocytes, and pericytes. The BBB is a dynamic structure that is altered in neurologic diseases, such as stroke. ECs and astrocytes secrete extracellular matrix (ECM) proteins to generate and maintain the basement membranes (BMs). ECM receptors, such as integrins and dystroglycan, are also expressed at the brain microvasculature and mediate the connections between cellular and matrix components in physiology and disease. ECM proteins and receptors elicit diverse molecular signals that allow cell adaptation to environmental changes and regulate growth and cell motility. The composition of the ECM is altered upon BBB disruption and directly affects the progression of neurologic disease. The purpose of this review is to discuss the dynamic changes of ECM composition and integrin receptor expression that control BBB functions in physiology and pathology.     

Recellularization of decellularized human adipose-tissue-derived extracellular matrix sheets with other human cell types

Decellularized human extracellular matrices (ECMs) are an extremely appealing biomaterial for tissue engineering and regenerative medicine. In this study, we decellularized human adipose tissue, fabricated a thin ECM sheet and explored the potential of this human adipose-derived ECM sheet as a substrate to support the formation of tissues other than adipose tissue. Acellular ECM sheets were fabricated from human adipose tissue through successive physical and chemical treatments: homogenization, centrifugation, casting, freeze-drying and sodium dodecyl sulfate treatment. The ECM sheets exhibited good mechanical properties, despite their porous structure. They degraded quickly in the presence of collagenase and the degradation rate increased with the collagenase concentration in phosphate-buffered saline. Five different human cell types, covering a broad range of cells and applications (normal human dermal fibroblasts, human aortic smooth muscle cells, human chondrocytes, human umbilical vein endothelial cells and human adipose-derived stem cells), were seeded onto the ECM sheets. All the human cell types spread well, proliferated and were successfully integrated into the decellularized ECM sheet. Overall, the results suggest that recellularized ECM sheets are a promising substitute for defective or damaged human tissues.     

Diversity of extracellular matrix morphology in vertebrate skeletal muscle

Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle consists of several morphologically distinct layers: an endomysium, perimysium, and epimysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These ECM layers are hypothesized to serve important functional roles within muscle, influencing passive mechanics, providing avenues for force transmission, and influencing dynamic shape changes during contraction. The morphology of the skeletal muscle ECM is well described in mammals and birds; however, ECM morphology in other vertebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skeletal muscle, and whether functional roles attributed to the ECM should be considered in mechanical analyses of non-mammalian and non-avian muscle. To explore the prevalence of a multilayered ECM, we used a cell maceration and scanning electron microscopy technique to visualize the organization of ECM collagen in muscle from six vertebrates: bullfrogs (Lithobates catesbeianus), turkeys (Meleagris gallopavo), alligators (Alligator mississippiensis), cane toads (Rhinella marina), laboratory mice (Mus musculus), and carp (Cyprinus carpio). All muscles studied contained a collagen-reinforced ECM with multiple morphologically distinct layers. An endomysium surrounding muscle fibers was apparent in all samples. A perimysium surrounding groups of muscle fibers was apparent in all but carp epaxial muscle; a muscle anatomically, functionally, and phylogenetically distinct from the others studied. An epimysium was apparent in all samples taken at the muscle periphery. These findings show that a multilayered ECM is a common feature of vertebrate muscle and suggest that a functionally relevant ECM should be considered in mechanical models of vertebrate muscle generally. It remains unclear whether cross-species variations in ECM architecture are the result of phylogenetic, anatomical, or functional differences, but understanding the influence of such variation on muscle mechanics may prove a fruitful area for future research.     

Schwann cell extracellular matrix molecules and their receptors

The major cellular constituents of the mammalian peripheral nervous system are neurons (axons) and Schwann cells. During peripheral nerve development Schwann cells actively deposit extracellular matrix (ECM), comprised of basal lamina sheets that surround individual axon-Schwann cell units and collagen fibrils. These ECM structures are formed from a diverse set of macromolecules, consisting of glyco-proteins, collagens and proteoglycans. To interact with ECM, Schwann cells express a number of integrin and non-integrin cell surface receptors. The expression of many Schwann cell ECM proteins and their receptors is developmentally regulated and, in some cases, dependent on axonal contact. Schwann cell ECM acts as an organizer of peripheral nerve tissue and strongly influences Schwann cell adhesion, growth and differentiation and regulates axonal growth during development and regeneration.     

Cytospectrophotometry of hepatocyte RNA in regenerating and neoplastic liver]

The present work consists in a quantitative cytospectrophotometric investigation of the cytoplasmic hyperbasophilia that characterizes the foci of neoplastic transformation and the tumor cells in rats fed hepatocarcinogens. It reveals that the increase in the dye-binding capacity shown by the cytoplasmic RNA of these cell populations results primarily form a qualitative alteration which raises the affinity for basic dyes by a factor of nearly 2, and also to a change in concentration due to volumetric changes which may again double the staining intensity of these hepatocytes. This phenomenon of hyperbasophilia differs radically from the weak variations in basophilia observed in normal regenerating liver and in hyperplastic liver parenchyma of rats fed the carcinogenic diet in which cases the changes appear to be related mainly to de nova RNA synthesis. Biochemical assays on cellular fractions indicate that the ribosomes are the organelles responsible for the hyperbasophilic properties that hepatocytes acquire in areas of neoplastic transformation.     

Biomechanics of the lung parenchyma: critical roles of collagen and mechanical forces.

The biomechanical properties of connective tissues play fundamental roles in how mechanical interactions of the body with its environment produce physical forces at the cellular level. It is now recognized that mechanical interactions between cells and the extracellular matrix (ECM) have major regulatory effects on cellular physiology and cell-cycle kinetics that can lead to the reorganization and remodeling of the ECM. The connective tissues are composed of cells and the ECM, which includes water and a variety of biological macromolecules. The macromolecules that are most important in determining the mechanical properties of these tissues are collagen, elastin, and proteoglycans. Among these macromolecules, the most abundant and perhaps most critical for structural integrity is collagen. In this review, we examine how mechanical forces affect the physiological functioning of the lung parenchyma, with special emphasis on the role of collagen. First, we overview the composition of the connective tissue of the lung and their complex structural organization. We then describe how mechanical properties of the parenchyma arise from its composition as well as from the architectural organization of the connective tissue. We argue that, because collagen is the most important load-bearing component of the parenchymal connective tissue, it is also critical in determining the homeostasis and cellular responses to injury. Finally, we overview the interactions between the parenchymal collagen network and cellular remodeling and speculate how mechanotransduction might contribute to disease propagation and the development of small- and large-scale heterogeneities with implications to impaired lung function in emphysema.     

Ultrastructure of the subepidermal musculature of Xenoturbella bocki, the adelphotaxon of the Bilateria

 Xenoturbella bocki is the only species of the high-ranked taxon Xenoturbellida. The species lives on marine mud bottoms at a depth of 20–120 m and moves extremely slowly by ciliary gliding. Nevertheless it possesses a well-developed body wall musculature with outer circular muscles, a prominent layer of inner longitudinal muscles and radial muscles that extend from the outer circular myocytes to the musculature surrounding the gastrodermis. The longitudinal myocytes are not compact cells, but form fascicles of fibrils running parallel to each other. Fine cytoplasmic cords connect the fibres of a cell to each other and with its nuclear region. The muscles are embedded within a sometimes expansive extracellular matrix (ECM) that lacks any fibrillar components. All muscle cells display conspicuous and numerous cytoplasmic extensions that are intermingled with each other. Tight coupling between adjacent cell membranes is not found, but zonula adhaerens-like junctions exist. Fibrils belonging to different myocytes, but also fibrils of the same cell, are coupled by such cytoplasmic extensions. Circular, radial and at least the peripheral longitudinal myocytes display cell-matrix connections with the internal lamina, a component of the subepidermal ECM. This internal lamina projects down into the centres of the fascicles with longitudinal muscle fibrils and forms extensive attachment zones with the muscle cells, reminiscent of focal contacts. For the ingestion of food, X. bocki opens the simple mouth pore and protrudes the aciliated gastrodermis. The body wall musculature is responsible for this protrusion and also for the withdrawal of the gastrodermis. In the past, possible phylogenetic kinships with the Acoelomorpha (Plathelminthes) or the Enteropneusta and Holothuroidea were discussed, but, on the basis of all information available, X. bocki is hypothesized to be the sister taxon of the Bilateria. Accepted: 2 April 1997     

Ultrastructural studies on the secretory cavities ofCitrus deliciosa ten. I. Early stages of the gland cells differentiation

Summary Secretory cavities ofCitrus deliciosa seem to originate from a pair of meristematic cells (an epidermal cell and a second one placed under it). These cells undergo successive divisions resulting in the formation of a globular/oval gland situated in the parenchyma, and a conical stalk, which joins the gland with the epidermis. The young gland consists of a central group of polyhedral cells ensheathed by layers of radially flattened cells.During the early differentiation stages of the gland cells a close association of cytoplasmic microtubules with various organelles is observed. Plastids increase progressively in number and size and their matrix locally contains tubular networks accompanied by small oil droplets. In peripheral cytoplasm numerous myelin-like lomasomes have been observed. Peripheral cells of the gland are gradually modified from the inner cells following a different developmental pattern. Their walls become thicker and plastids do not contain tubular complexes, but only a few thylakoids partly surrounding the newly formed starch grains.     

Distribution of extracellular matrix proteins type I collagen, type IV collagen, fibronectin, and laminin in mouse folliculogenesis

The extracellular matrix (ECM) plays a prominent role in ovarian function by participating in processes such as cell migration, proliferation, growth, and development. Although some of these signaling processes have been characterized in the mouse, the relative quantity and distribution of ECM proteins within developing follicles of the ovary have not been characterized. This study uses immunohistochemistry and real-time PCR to characterize the ECM components type I collagen, type IV collagen, fibronectin, and laminin in the mouse ovary according to follicle stage and cellular compartment. Collagen I was present throughout the ovary, with higher concentrations in the ovarian surface epithelium and follicular compartments. Collagen IV was abundant in the theca cell compartment with low-level expression in the stroma and granulosa cells. The distribution of collagen was consistent throughout follicle maturation. Fibronectin staining in the stroma and theca cell compartment increased throughout follicle development, while staining in the granulosa cell compartment decreased. Heavy staining was also observed in the follicular fluid of antral follicles. Laminin was localized primarily to the theca cell compartment, with a defined ring at the exterior of the follicular granulosa cells marking the basement membrane. Low levels of laminin were also apparent in the stroma and granulosa cell compartment. Taken together, the ECM content of the mouse ovary changes during follicular development and reveals a distinct spatial and temporal pattern. This understanding of ECM composition and distribution can be used in the basic studies of ECM function during follicle development, and could aid in the development of in vitro systems for follicle growth.     

A Simple Method for Making Tetrahedra to Illustrate Stereochemicai Principles

Actin and myosin, the major proteins of the contractile complex actomyosin, have now been demonstrated to be important constituents of many eukaryotic cells. As in muscle, their role is primarily that of a contractile system. This system is thought to underly all aspects of cellular motility: locomotion, shape change, mitosis and meiosis, cell division, cytoplasmic streaming, organelle motion, endo-cytosis (pinocytosis, phagocytosis), and exocytosis.

We describe here a simple experimental system to demonstrate quantitatively aspects of motility and its regulation in the slime mould Physarum polycephalum     

The ultrastructure of the morula cells of Eupentacta quinquesemita (Echinodermata: Holothuroidea) and their role in the maintenance of the extracellular matrix

The ultrastructure of the morula cells of Eupentacta quinquesemita and the distribution of these cells in the dermal connective tissue are described. Morula cells are abundant in the dermis and appear to function in the maintenance of the extracellular matrix (ECM) as a source of ground substance material. The synthetic activity of these cells is described in detail. Morula cells are filled with large secretory vesicles containing three electrondense materials which are derived from rough endoplasmic reticulum and Golgi activity. The synthetic product of these cells contains glycosaminoglycans and is secreted into the ECM by degranulation. The ultrastructural and histochemical similarity of the degranulation product to the ECM ground substance suggests that they are comprised of the same material. Morula cells appear to function primarily in connective tissues where ground substance predominates. The cells often contain secretory vesicles at various stages of formation, all of which eventually mature and degranulate. The synthetic pathway of the morula cells appears to result ultimately in the complete disruption and death of the cells. The function of morula cells in the holothuroid ECM is discussed, and the synthetic activity of the cells is compared with that of other secretory cells.