Fine structure of bovine lens epithelial cells in vitro in relation to modifications induced by a retinal extract (EDGF)

Cultured bovine lens epithelial cells are polygonal in shape. In confluent and multilayer cultures they exhibit elaborate arrays of 6 nm filaments, bundles of intermediate-sized filaments, and a fibrous meshwork of subcellular and intercellular material. Cells grown in the presence of a retinal extract (RE) have a higher growth rate, and are smaller and more regular in shape. In them the 6 nm filaments are mostly aligned in sheets, the intermediate-sized filaments form a fine network, and the cells are closely apposed to the plastic substratum. Some homogeneous material is formed intercellularly in older cultures. Cellular elongation, induced in the former cultures by the addition of RE, is accompanied by an alignment of cytoskeletal elements, including microtubules, parallel to the long axis. Other structural features are similar in all cell types. The response to RE is discussed in terms of shape modulations associated with the restricted expression of structural characteristics acquired in vitro.     

Culture and characterization of dental follicle cells from rat molars

Summary Because the dental follicle is necessary for the eruption of teeth of limited eruption, it was the objective of this study to determine if the cells of the follicle could be cultured in vitro. To achieve this, dental follicles and associated enamel organs were dissected from the first and second mandibular molars of 6–7-day-old rats (secretory stage of amelogenesis), and then cultured in a medium that promotes fibroblast growth — the predominant cell type of the dental follicle. The cultured cells grew to confluency and were kept through 3 passages before experimentation. The cultured cells were fibroblastic in shape, elongate with processes, and transmission electron microscopy revealed that they contained an abundant rough endoplasmic reticulum, but did not form desmosomes. Immunofluorescent staining for anti-vimentin showed that all the cells stained and electron-microscopic immunogold labeling indicated that the antibody was associated with intermediate filaments. As revealed by SDS-polyacrylamide gel electrophoresis and Western blotting, the cultured cells synthesized and secreted the extracellular matrix molecules fibronectin and procollagens. Subsequent immunofluorescence staining of permeabilized and non-permeabilized cells confirmed the presence of fibronectin and type I collagen both intra- and extracellularly. Thus, based on all the above characteristics, the cultured cells appeared to be fibroblasts derived from the dental follicle, although a few of the fibroblasts may be derived from undifferentiated mesenchymal cells interposed between the alveolar bone and follicle. Experiments now can be conducted to determine how these cultured cells respond directly to growth factors that alter the rates of tooth eruption.     

Ultrastructural studies of Austramphilina elongata (Cestoda,Amphilinidea)

Summary The fine structure of larval Austramphilina elongata is described using serial semithin and ultrathin sections. Densely packed germ cells with many ribosomes and mitochondria and with large Golgi complexes fill the middle third of the body. Some necrotic nuclei were observed near the anterior end. The neodermis consists of a subepidermal syncytium connected to pericarya in the parenchyma by means of cytoplasmic processes containing peripheral microtubules; electron-dense ovoid bodies condense in these processes. Myoblasts are connected to muscle fibres by means of cytoplasmic connections rich in mitochondria. Twelve (exceptionally eleven) type I gland cells containing large secretory granules and extensive granular endoplasmic reticulum are located in the dorso-posterior part of the body; they open through 12 (or 11) discrete ducts into an anterior invagination of the tegument which is covered by epidermis and not connected to the outside. Ten type II gland cells containing elongate secretory granules with regularly arranged longitudinal microtubules are located ventral to the type I cell bodies; they open on a ventral papilla a short distance behind the anterior end. Ten type III gland cells containing irregularly round-oval secretory granules with coiled microtubules are located anterior and ventral to the type I gland cells; they open through five discrete ventro-anterior openings on each side of the body. Ducts of all gland cells have mitochondria and microtubules. The spermatozoon has a basic pattern of two axonemes, each with a single central filament, a mitochondrion (mitochondria), and a row of surface microtubules interrupted by the axonemes. In the tips of epidermal cilia, doublet 1 and doublets adjacent to it lose their microtubules B first and close in on the central pair of filaments in a spiral fashion, enclosing an electron-dense rod. Presence of a neodermis and ultrastructure of the spermatozoon support the validity of the taxa Neodermata Ehlers and Trepaxonemata Ehlers and are strong evidence against a phylogenetic relationship of the cestodarians — cestodes with the Acoelomorpha; this is also indicated by the ultrastructure of sense receptors and epidermal ciliary rootlets.     

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro : A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.     

Fine structure of the corpus Luteum of the raccoon during pregnancy

Summary Ultrastructure of the granulosa lutein cells of the raccoon from throughout pregnancy has been described. The lutein cells often from epithelial cords which are separated by the connective tissues, capillaries and lymphatics. Based on the arrangements and modifications of the cytoplasmic organelles and inclusions, three types of lutein cells have been recognized. The type I lutein cells predominantly contain tubular, agranular endoplasmic reticulum, juxtanuclear Golgi complexes, a few round to rod-shaped mitochondria, some free ribosomes, and occasional lipid droplets. Occasionally the tubular cristae of mitochondria and tubular smooth endoplasmic reticulum appear contiguous. The type II cells contain abundant lace-like and/or stacked fenestrated endoplasmic reticulum cisternae that frequently form membranous whorls, some tubular, agranular endoplasmic reticulum, mitochondria, and lipid droplets. Mitochondria are usually small, but unusual large ones also occur. The small, rod-to round-shaped mitochondria usually have tubular cristae; but the large, oval, elongate, and cup shaped mitochondria possess tubular, lamellar, plate like, and whorl-like cristae. The plasma membranes of the cells are complexly elaborated and folded, especially when apposing each other. In favorable sections, strands of fenestrated cisternae appose the folds of the plasma membranes. In general, the amount of cytoplasmic organelles and inclusions vary greatly in the cells. The type III cells predominantly contain lipid droplets and sparse cytoplasmic organelles. The type I and II cells are found throughout pregnancy, but the type III cells are observed from mid gestation to term. The cytological features of type I and II cells suggest that they probably secrete most of the steroids, whereas the type III cells primarily store lipids.This research was supported by UPSHS grant AM-11376 and NIH contract 69-2136.     

Relationship between actions of transforming growth factor (TGF)-β and cell surface expression of its receptors in clonal osteoblastic cells

Various osteoblastic cell lines were examined for the relationship between the presence of cell-surface transforming growth factor (TGF)-β receptors and the synthesis of matrix proteins with their responsiveness to TGF-β. Treatment with TGF-β1 inhibited proliferation and stimulated proteoglycan and fibronectin synthesis in MC3T3-E1 and MG 63 cells. The major proteoglycans synthesized by these cells were decorin and biglycan, and TGF-β1 markedly stimulated the synthesis of decorin in MC3T3-E1 and of biglycan in MG 63 cells. SaOS 2 and UMR 106 cells synthesized barely detectable amounts of decorin or biglycan, and TGF-β1 did not stimulate the synthesis of these proteoglycans. In SaOS 2 cells, however, TGF-β1 enhanced fibronectin synthesis. TGF-β1 did not show any of these effects in UMR 106 cells. Receptor cross-linking studies revealed that only MC3T3-E1 and MG 63 cells had both types I and II signal-transducing receptors for TGF-β in addition to betaglycan. SaOS 2 cells possessed type I but no type II receptor on the cell surface. In contrast, SaOS 2 as well as MC3T3-E1 and MG 63 cells expressed type II receptor mRNA by Northern blot analysis, and cell lysates contained type II receptor by Western blot analysis. Thus, it appears that type II receptor present in SaOS 2 cells is not able to bind TGF-β1 under these conditions. UMR 106 cells with no response to TGF-β1 had neither of the signal-transducing receptors by any of the analyses. These observations using clonal osteoblastic cell lines demonstrate that the ability of osteoblastic cells to synthesize bone matrix proteoglycans is associated with the responsiveness of these cells to TGF-β1, that the responsiveness of osteoblastic cells to TGF-β1 in cell proliferation and proteoglycan synthesis correlates with the presence of both types I and II receptors, and that the effect of TGF-β1 on fibronectin synthesis can develop with little binding of TGF-β1 to type II receptor if type I receptor is present. It is suggested that the combination of cell-surface receptors for TGF-β determines the responsiveness of osteoblastic cells to TGF-β and that changes in cell-surface TGF-β receptors may play a role in the regulation of matrix protein synthesis and bone formation in osteoblasts. © 1995 Wiley-Liss, Inc.     

Culture on basement membrane does not reverse the phenotype of lens derived mesenchyme-like cells

Definitive epithelia suspended within type I collagen gel give rise to individual, freely migrating cells that express the mesenchymal phenotype. They become elongate in shape, invade collagenous matrices and develop abundant RER. We investigated whether mesenchyme-like cells that derive from lens epithelia retain the mesenchymal phenotype or revert to epithelial phenotype when cultured on basement membrane (BM). Mesenchyme-like cells placed on top of BM gel or lens capsule BM retain the elongate, bipolar morphology of mesenchymal cells. They migrate individually along and into the BM matrix. Mesenchyme-like cells on or in BM ultrastructurally resemble true mesenchymal cells. They extend pseudopodia and filopodia, exhibit a circumferential actin cortex, and contain well developed RER. Mesenchymal products, such as type I collagen, continue to be expressed. We conclude that the phenotype of mesenchyme-like cells derived from definitive epithelia is stable even in or on matrix known to promote the epithelial genetic program. Their behavior, thus, is similar to that of true (secondary) mesenchymal cells in the embryo.     

The epidermal intermediate filament proteins of tunicates are distant keratins; a polymerisation-competent hetero coiled coil of the Styela D protein and Xenopus keratin 8

Two novel cytoplasmic intermediate filament (IF) proteins (C and D) from the tunicate (urochordate) Styela are characterised as putative keratin orthologs. The coexpression of C and D in all epidermal cells and the obligatory heteropolymeric IF assembly of the recombinant proteins argue for keratin orthologs, but the sequences do not directly reveal which protein behaves as a keratin I or II ortholog. This problem is solved by the finding that keratin 8, a type II keratin from man or Xenopus, forms chimeric IF when mixed with Styela D. Mutant proteins of Styela D and keratin 8 with a single cysteine in equivalent positions show that these chimeric IF are, like vertebrate keratin filaments, based on the hetero coiled coil. We propose that Styela D retains, in spite of its strong sequence drift, important molecular features of type I keratins. By inference Styela C reflects a type II ortholog. We discuss that type I to III IF proteins are expressed along the chordate branch of metazoa.     

Different types of cell-to-cell connections mediated by nanotubular structures

Communication between cells is crucial for proper functioning of multicellular organisms. The recently discovered membranous tubes, named tunneling nanotubes, that directly bridge neighboring cells may offer a very specific and effective way of intercellular communication. Our experiments on RT4 and T24 urothelial cell lines show that nanotubes that bridge neighboring cells can be divided into two types. The nanotubes of type I are shorter and more dynamic than those of type II, and they contain actin filaments. They are formed when cells explore their surroundings to make contact with another cell. The nanotubes of type II are longer and more stable than type I, and they have cytokeratin filaments. They are formed when two already connected cells start to move apart. On the nanotubes of both types, small vesicles were found as an integral part of the nanotubes (that is, dilatations of the nanotubes). The dilatations of type II nanotubes do not move along the nanotubes, whereas the nanotubes of type I frequently have dilatations (gondolas) that move along the nanotubes in both directions. A possible model of formation and mechanical stability of nanotubes that bridge two neighboring cells is discussed.     

Migratory phenotypes of HSC-3 squamous carcinoma cell line induced by EGF and PMA: relevance to migration of loosening of adhesion and vinculin-associated focal contacts with prominent filopodia

Cell migration is involved in carcinoma cell invasion and wound healing. We examined motogenic cytokines that potentiated migration of human HSC-3 carcinoma cells. To assess migratory activity, modified Boyden chambers were used. Among a variety of potential motogenic cytokines, epidermal growth factor (EGF) enhanced migration of HSC-3 cells both on collagen and fibronectin. Phorbol myristate acetate (PMA) also enhanced migration. Inhibitors of protein kinase C completely inhibited PMA-induced migration, but only partly inhibited EGF-induced migration. Protein kinase A was also involved in the EGF-induced signaling pathway for migration. Although the signaling pathways were independent, and the cell shape on collagen was different from that on fibronectin, migratory cells stimulated by EGF or PMA showed common morphology on different ligands. The cells were polygonal or round in shape and the loss of long cytoplasmic extensions was noted. Migratory HSC-3 cells stimulated by EGF or PMA became less adhesive to collagen and fibronectin. Since both EGF- and PMA-stimulated migration did not require de novo protein synthesis, the signaling pathways possibly lead to assembly and disassembly of an actin cytoskeleton. Immunofluorescence for vinculin was concentrated into focal contacts in EGF- and PMA-stimulated HSC-3 cells, whereas the fluorescence signal was hardly detected in non-stimulated cells. Talin and beta1 integrin were immunolocalized at focal contacts in non-stimulated cells, and it remained unchanged in stimulated cells. Numerous filopodia visualized with actin immunofluorescence were formed around stimulated HSC-3 cells, whereas filopodia were short and sparse around elongated cytoplasms in non-stimulated cells. Thus, shortening of cytoplasmic extensions with numerous filopodia, loosening of adhesion, and vinculin-associated focal contacts were regarded as migratory phenotypes.     

Three-dimensional architecture of the higher plant nucleolonema disclosed on serial ultrathin sections

Summary— The three-dimensional architecture of the nucleolonema of Vicia faba has been studied by applying a silver impregnation technique to serial ultrathin sections. This technique disclosed lateral and transverse segments of the nucleolonema which were heavily impregnated with silver. The lateral profiles of the nucleolonema segments were classified into three main categories; a segment made up of one to several rod-like filaments (type I); a ladder-like segment consisting of two parallel and of transverse filaments (type II); and a last type constructed from two parallel filaments (type III). Tracing of the lateral segments through serial sections has indicated that type I first appears, then either type II or III and finally type I reappears at the corresponding sites on sections. Types II and III remained constant in width, about 1.0 μm, along their longitudinal axes whereas the width of type I was significantly smaller than that of the two former. The lateral filaments of both types II and III showed heterogeneity in width on account of the presence of knobs intermittently distributed along them. The thickness of these knobs was about 0.35 μm. Combining the observations on serial ultrathin sections and the morphometrical data it is very probable that the elementary structure of the nucleolonema is a 0.35-μm thick filament that tightly coils up into a solenoid structure with a thickness of approximately 1.0 μm. This model can explain the appearance of open- and closed-argyrophilic rings in serial sections since transverse segments of the solenoid are expected to show the argyrophilic rings. The elementary filament of the nucleolonema solenoid was sometimes loosened. Judging from our cytochemical data at the electron microscope level, some argyrophilic proteins appear to reside in the axial space of the solenoid but both DNA and RNA were not detectable in this space.     

Switch in Fas-activated death signaling pathway as result of keratin 8/18-intermediate filament loss

Fas-induced apoptosis is initiated through the recruitment of FADD and procaspase 8 to form the death-inducing signaling complex (DISC). In some cells (type I cells) the initiator caspase 8 directly activates effector caspases such as procaspase 3, whereas in others (type II cells) the death signal is amplified through mitochondria. In epithelial cells, Fas-induced hierarchic caspase activation is also linked with DEDD, a member of the DED family that binds to keratin (K) intermediate filaments (IFs). Hepatocytes are type II cells and their IFs are made exclusively of K8/K18. We have shown previously that K8-null mouse hepatocytes, lacking K8/K18 IFs, are more sensitive than their wild-type counterparts to Fas-induced apoptosis. Here, by examining the cell-death kinetics and death-signaling ordering, we found that K8-null hepatocytes exhibited prominent DISC formation, higher procaspase 8 activation and direct procaspase 3 activation as reported for type I cells; however they experienced a reduced Bid cleavage and a stronger procaspase 9 activation. In addition, the K8/K18 loss altered the DEDD ubiquitination status and nuclear/cytoplasmic distribution. Together, the results suggest that the K8/K18 loss induces a switch in Fas-induced death signaling, likely through a DEDD involvement. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression of the intermediate-filament-associated protein synemin in chicken lens cells.

Synemin, a 230-kilodalton polypeptide component of avian muscle and erythrocyte intermediate filaments, is also found in association with the vimentin filaments of lens tissue. In chicken lens cells, synemin is bound to the core vimentin polymer with the same 180-nm periodicity that it exhibits in erythrocytes. Its solubility properties are characteristic of those of intermediate filaments in general and similar to those of synemin in muscle cells and erythrocytes. Synemin appears at an early stage of lens development and undergoes a dramatic accumulation as the epithelial cells elongate and differentiate into fiber cells. In contrast to synemin in cultured skeletal muscle, lens synemin is not confined to postmitotic, terminally differentiating cells but is present in proliferative cells as well. It is lost from the fibers near the center of the lens, as are many other cellular structures including intermediate filaments. These findings provide new information about the occurrence and expression of avian synemin and new insight regarding its presumptive role as a modulator of intermediate-filament function.     

Intermediate filaments in the developing type II cell of fetal monkey lung

Anticytokeratin monoclonal antibody was used to study epithelial cell development in fetal monkey lungs taken from animals of different ages. It is well established that the overall maturity of fetal lung depends greatly on the maturation of type II epithelial cells in the alveolus. In this study, we have correlated the cytokeratin phenotype of mammalian epithelial cells with pneumocyte maturation. We show that differentiation and maturation of the type II cell is related to intermediate filament expression. Twenty-four fetal monkeys (Macaca nemestrina) were delivered by cesarean section at a gestational age of 135-140 days (term = 168 days) and divided into two groups. One group of animals was sacrificed during the first 3 hr of life, and the other group was maintained in incubators for 92-120 hr. Anticytokeratin monoclonal antibody recognizes only alveolar type I and type II epithelial cells. In the first 3 hr of life, the cytokeratin was localized only at the alveolar surface and at the cytoplasmic periphery of the type II cells of these premature animals. However, at the age of 92-120 hr, the epithelia in the lungs reacted more intensely than they did during the first 3 hr. Electron microscopy revealed and confirmed that the type II cells were matured and abundant intermediate filaments appeared in the cytoplasm. The filaments appeared to form either aggregates or parallel filament bundles and few were closely associated with the lamellar bodies. In the immature type II cells at 0-3 hr of life, few intermediate filaments could be localized in the cytoplasm, and no parallel filament bundle was observed, though many appeared in the 92-120 hr lungs. This suggests that the intermediate filaments have a functional significance in the development and maturation of the type II cell. The location and stability of keratin filaments in type II cells may confer the structural strength necessary for cells covering a free surface in the alveoli during lung maturation.     

Actin in peripheral rat lung: S1 labeling and structural changes induced by cytochalasin

Actin was identified with S1-labeling in type I and II cells, pericytes, and capillary endothelial cells in the peripheral lung of the adult rat. In type II cells abundant actin was present in the apex of cells, in microvilli, and in close association with lamellar bodies near the cell surface. Lamellar bodies secreting their content into the lumen of alveoli were always surrounded by a thick layer of actin-like material. In specimens treated with cytochalasin D the surface of type I, type II and contractile interstitial cells became irregular. In type II cells, lamellar bodies were no longer surrounded by actin-like material and no exocytic profiles of lamellar bodies were encountered. In type II cells actin filaments may be involved in moving lamellar bodies through the cytoplasm and/or in their secretion into alveoli. These observations also suggest that intact actin filaments may be required for maintenance of cell shape in various lung cells and that cells containing actin may be capable of limited contraction.     

The effect of beta,beta'-iminodipropionitrile (IDPN) on cytoskeletal organization in cultured human skin fibroblasts

By impairing the axonal transport of neurofilaments, IDPN induces large, filament-filled swellings in the proximal portion of axons of spinal motor neurones. We have reported that 2,5-hexanedione, another agent producing focal accumulation of neurofilaments, induces aggregation of intermediate filaments of the vimentin type in human skin fibroblasts grown in tissue culture. IDPN was tested in this model to determine if this chemical also affects a general mechanism involved in cytoplasmic transport of the cytomatrix or a process exclusive to axonal flow of neurofilaments. Although aggregates of intermediate filaments were seen in fibroblasts exposed to IDPN, they did not occur in the absence of generalized cytotoxicity and were labelled by antisera against tubulin, actin and fibronectin in addition to vimentin. It is probable the effect of IDPN on the distribution of intermediate filaments was secondary to changes in cell shape occurring during slow detachment from the substratum.     

Disassembly of F-actin filaments in human endothelial cells cultured on type V collagen.

We examined the inhibitory activity of type V collagen on cell attachment and cell growth and the role of stress fibers and beta 1 integrin in cultured human endothelial cells. Human endothelial cells cultured on type V collagen attached temporarily to the substrate and formed stress fibers. However, the cells failed to proliferate and gradually detached from the substrate. After 24 h, the cells on type V collagen lacked discernible stress fibers (F-actin filaments) and exhibited dots in small aggregates of F-actin. In addition, the cells expressed little or no proteins as focal adhesions, including vinculin and beta 1 integrin. In contrast, the cells on fibronectin and type I collagen formed complete F-actin filaments, exhibited sufficient vinculin and beta 1 integrin, and grew logarithmically from 2 days. On the other hand, human smooth muscle cells formed complete F-actin filaments, revealed typical focal adhesions, and started to proliferate rapidly after 24 h on type V collagen as well as on fibronectin and type I collagen. Thus, the disassembly of F-actin filaments was observed as a specific phenomenon in human endothelial cells cultured on type V collagen. Moreover, the F-actin filaments disappeared from endothelial cells treated with cytochalasin D after 24 h and the cells detached from fibronectin and type I collagen with time, a result consistent with the observations on type V collagen. Accordingly, the disassembly of F-actin filaments in focal adhesions may result in the detachment of endothelial cells from type V collagen.     

Light microscopy of glial cells in turtles and birds

Summary Four classes of glial cells can be recognized in the central nervous system of turtles and birds on the basis of nuclear characteristics (methylene blue) and external morphology (Golgi technique). It seems likely that astrocytes and ependymal cells have a similar origin and function, but no evidence has been seen to indicate that transitional forms exist between astrocytes and oligodendrocytes or microgliacytes. Ependymal cells in the tectum and forebrain are covered by lamellate excrescences which are absent on cells in the spinal cord. Protoplasmic astrocytes are restricted to the gray matter. In the turtle they have an elongate shape characteristic of primitive elements, but stellate forms typical of mammals predominate in the bird. Fibrous astrocytes are abundant in the white matter. Endfeet are lacking in the turtle except on cells located near the pia; they are common for all elements in the bird and can sometimes be observed to outline the course of capillaries. Oligodendrocytes are identical to mammalian and amphibian forms. Small, round somata and long, thin processes are typical of types I and II while a tubular reticulum or membranous sheath characterizes type IV. The lack of a well defined somata and absence of transitional forms (type III) are compatible with the possibility that type IV is not a true cell type but corresponds to the inner cytoplasmic tongue of myelin. Microgliacytes are present in gray and white matter; they have a smaller overall size in the turtle and young chicken than in adult birds.Supported by a postdoctoral fellowship from the United States National Institutes of Health, NB 28,013-01Al.