Fibrillar Meshwork Research Articles

Nephron induction--the epithelial mesenchymal interface revisited.

While more and more humoral factors are being implicated in nephrogenesis, there is no detailed knowledge of the morphological structures at the interface of the nephron inducer and the surrounding mesenchyme. Hence we examined this area in the cortex of neonatal rabbit kidneys by scanning and transmission electron microscopy. Our interest was focused on the basal aspect of the collecting duct ampulla and the surrounding competent mesenchyme where morphogenic signals are exchanged during nephron induction. Close contact between these two tissues is assumed during nephrogenesis to allow direct cellular contact or diffusion of soluble factors across a short distance. However, our data show the presence a wide cleft around the collecting duct ampulla spatially separating the inducer and the competent mesenchyme during nephron induction. This cleft is filled with a characteristic fibrillar mesh-work.

Existence of a dense reticular meshwork surrounding the nephron inducer in neonatal rabbit kidney.

While more and more humoral factors involved in nephrogenesis are being discovered, there is no detailed knowledge of the morphological structures at the interface of the nephron inducer and the surrounding mesenchyme. For that reason we examined this area in the cortex of neonatal rabbit kidneys by scanning electron-microscopical and transmission electron-microscopical techniques. Our interest was focused on the basal aspect of the collecting duct ampulla and the surrounding competent mesenchyme, where morphogenic signals are to be exchanged during nephron induction. Close contact between these two tissues involved in nephrogenesis is assumed to allow direct cellular contact or diffusion of soluble factors across a short distance. Our data, however, show the presence of a dense fibrillar meshwork around the collecting duct ampulla, spatially separating the inducer and the competent mesenchyme during nephron induction.

Degeneration and transdifferentiation of human lens epithelial cells in nuclear and anterior polar cataracts

Purpose To study the possible mechanisms of cataractogenesis by evaluating the characteristics of cataractous lens epithelial cells (LECs) in different types of human cataract. Setting Kangnam St. Mary’s Hospital, The Catholic University of Korea, Seoul, Korea. Methods Lens epithelial cells attached to the anterior capsules in eyes with nuclear or anterior subcapsular were analyzed for morphological characteristics by electron microscopy and for cellular characteristics by immunohistochemistry. Results Human LECs beneath the anterior capsule were degenerated in nuclear cataracts and were transdifferentiated in anterior polar cataracts. In senile nuclear cataractous lenses, LECs beneath the anterior capsule showed degenerative changes in morphology. In nuclear cataracts, LECs were immunohistochemically positive for cytokeratin and vimentin, while those in anterior polar cataracts were positive for vimentin only. The LECs of anterior subcapsular cataracts were transdifferentiated into spindle-shaped fibroblast-like cells without cellular junctions and embedded within a fibrillar meshwork mass. The extracellular matrixes in the anterior capsule of anterior subcapsular cataracts were immunohistochemically positive for fibronectin, laminin, collagen type I, and collagen type IV. Conclusions Lens epithelial cells in different types of cataracts have distinct cellular characteristics and may possess a bipotential nature with the ability to transdifferentiate into mesenchymal cells. This may be an underlying mechanism for the development of cataract and capsule opacification.

Variation in the Structure of Intertracueary Pit Membranes in Abies Sacualinensis, as Observed by Field-Emission Scanning Electron Microscopy

An examination was made of the fine structure of bordered pit membranes in the radial walls between tracheids in the outer sapwood of Abies sachalinensis to improve our understanding of the so-called extended torus, the minute holes in the torus and the imperforate zone near the periphery of the pit membranes, Field-emission scanning electron microscopy revealed that a so-called extended torus was present in many bordered pit membranes. We examined the frequency occurrence of and variations in the extended torus within a single annual ring. The frequency tended to increase from the earlywood to the latewood within a single annual ring. In the tori of many bordered pit membranes, we detected minute holes, and the number and location of such minute holes in a single torus varied among individual pit membranes. The appearance of each minute hole also varied. An imperforate zone was observed near the periphery of the pit membrane. In this imperforate zone, we found amorphous materials, and fine fibrils were visible that were an extension of the fibrillar meshwork of the margo.

Formula omitted]A repeated element in the human lamin B2 gene covers most of an intron and reiterates the exon/intron junction

Nuclear lamins are intermediate filament-type proteins forming a fibrillar meshwork that underlies the inner nuclear membrane. We have previously reported the identification of the human lamin B2 gene that maps to the subtelomeric band p13.3 of chromosome 19 in close proximity of a human DNA replication origin. Here we report the identification within the human lamin B2 gene of a novel repeated element ( v ariable n umber of t andem r epeats: VNTR) that appears to have a very recent origin, being absent in the genome of mouse and primates such as cercopitheques, lemurs and macaques. The VNTR is adjacent to exon 8 of the lamin B2 gene which, albeit encoding the nuclear localization signal of the protein, is highly divergent both at amino acid and nucleotide level among species. Moreover the VNTR, characterized by a repeated unit of about 100 bp, covers most of intron 8 of the gene and reiterates both the last 7 bp of the upstream exon and the exon/intron junction. RT–PCR experiments carried out on HeLa cell RNA suggest that none of the downstream junctions is used during the processing of the lamin B2 pre-mRNA.

Expression of fibulin-2 by fibroblasts and deposition with fibronectin into a fibrillar matrix.

The extracellular matrix protein fibulin-2 was shown to be a typical product of cultured human and mouse fibroblasts by several immunological assays. It is secreted and deposited in cells and tissues as a disulfide-bonded oligomer identical in size to the previously described recombinant fibulin-2. Most of the fibroblast fibulin-2 is deposited into a dense fibrillar meshwork which requires treatment with EDTA and/or 6 M urea for solubilization. Fibulin-2 and fibronectin are synthesized at equivalent levels and both colocalize in the fibrils as shown by immunofluorescence. Metabolic labelling and pulse-chase studies demonstrated fibulin-2 oligomers in detergent extracts of cells and their rapid translocation to extracellular EDTA-sensitive assembly forms. Unlike for fibronectin and fibulin-1 only a little fibulin-2 was found in the cell culture medium. Immunogold staining of confluent human fibroblasts showed localization of fibulin-2 to a fine meshwork or bundles of amorphous microfibrils in the matrix. This also demonstrated a distinct colocalization of fibulin-2 and fibronectin at the electron microscope level, indicating that the interaction between these two protein shown in in vitro assays may also exist in situ. No distinct colocalization of both proteins could, however, be observed with cross-striated fibrils of collagen I and collagen VI microfibrils.

Immunocytochemical localization of Tamm-Horsfall protein in the kidneys of normal and nephrolithic rats.

Studies using in vitro systems have indicated that Tamm-Horsfall protein (THP) can interact with calcium oxalate (CaOx) crystals during kidney stone formation. However, information regarding the nature of its participation in this process remains controversial and unclear. In order to better understand the putative interaction of THP and crystals in vivo, we compared the localization of THP in normal rats and in chronic and semi-acute rat models of nephrolithiasis. In these rats, CaOx crystal deposits were induced in the kidneys by administering ethylene glycol (EG) in drinking water. The formation of CaOx mono- and dihydrate aggregates in the urine was confirmed by scanning electron microscopy. Immunohistochemical localization, as well as protein A-gold labeling at the ultrastructural level, demonstrated that in addition to its normal distribution, THP specifically associated with the renal crystal deposits. The THP-containing, organic matrix-like material consisted of a fine, fibrillar meshwork surrounding individual crystals and their aggregates. In addition, THP also appeared in the papilla, where it is normally absent, concurrent with the appearance of crystal deposits in the kidneys. These observations indicate that in nephrolithic rats the normal localization of THP is altered. Such an alteration may indicate an important physiological event related to crystal aggregation and kidney stone formation.

Mechanical Basis of Cell Morphogenesis and Volume Control

Osmotic differences between the cytoplasm and the surrounding fluid belong to the most important factors in shaping an animal tissue cell. This implies an elastic and contractile fibrillar system attached to the cell membrane which counteracts ionic imbalances and determines cell morphology. Therefore cell volume and cell shape are functionally coupled. Many cell types, epithelial cells in particular, are able to control their volume: ie, swelling (or shrinking) induced by varying the osmolarity of the surrounding fluid is compensated and the previous volume is achieved during a time interval of 10-40 min. This compensation reaction includes the opening of ion channels, activation of the Na/K/2C1 co-transport mechanism, and activities of the cytoskeleton. The changes in ion fluxes have been investigated for various cell types. The main question which is still under debate refers to the signal involved in telling a cell to have reached its appropriate volume. One of the most probable factors is the tension in the cell membrane. By action of tension-sensitive ion channels (K+, Ca++-channels) ion fluxes could be modified. Mechanical forces acting on the cell membrane always affect the cortical cytoplasm - membrane complex: In many cell types a fibrillar meshwork is closely connected to the membrane, thus determining its mechanical properties. Destruction of this complex by drugs like cytochalasins alters the mechanics of the cortex and destroys the ability of the cells for volume regulation. If mechanical forces in the cortex are involved in volume control, the loss of this ability would be a reasonable consequence of cytochalasin treatment. Measurement of the forces at the surface of cells during swelling and during the compensation phase is a direct approach to the problem of volume regulation. Scanning acoustic microscopy (SAM) is an elegant method to determine elastic properties on a subcellular level. Measuring the reflection of sound at the surface of a cell gives a good indication of local elasticity (and thus “tension at the surface”). Using the cell line HaCat which was derived from human epidermis, elasticity changes have been followed by SAM during hypotonicity-induced swelling and the subsequent compensatory volume decrease. On the basis of the present results the regulatory volume decrease (RVD) may be achieved by an increase of forces in the cortex by actomyosin-based contractions in addition to altered ion fluxes. Volume increase first results in a destruction of the actin fibrillar system which is immediately restored by stimulated actin polymerisation which strengthens the complex of the plasma membran with the cortical fibrillar layer. The results support the view that tension in the cortex may participate in RVD and be a signal for volume regulation.

Quick‐Freeze, deep‐etch studies of renal basement membranes

The fine structure of the renal (i.e., glomerular, tubular, and capillary) basement membranes was re-evaluated with the aid of a deep-etch replica method. The structure of the laminae rarae interna and externa of the rat glomerular basement membrane (GBM) and laminae lucida of other basement membranes were basically identical in that 6 to 8 nm fibrils were interconnected to form a three-dimensional, polygonal network. By contrast, all of the laminae densa examined were composed of closely packed granules, and a filamentous substructure was identified only in a limited area. These granular components were demonstrated to be an integral component of the lamina densa. From additional observations on the trypsinized bovine GBM, it appeared that the basic structure of renal basement membranes was almost identical, namely, that a three-dimensional fibrillar meshwork existed throughout the individual layers to form a structural framework upon which fine particles were variably attached. In addition, we observed some of the fine structure of the pars fibroreticularis; the laminae densa of the tubular and capillary basement membranes continued to the fibrillar meshwork resembling the structural backbone of the glomerular basement membrane. The network was sometimes directly connected to the extracellular matrix, but more often changed into a rough fibrillar framework and connected to the extracellular matrix.

Composition of Articular Cartilage

The current state of knowledge on the structure and function of the macromolecules of articular cartilage is summarized. This highly hydrated tissue is composed of relatively few cells embedded in an extracellular matrix. The matrix consists of a fibrillar meshwork composed of mainly cross-linked type II collagen molecules, but with small amounts of type XI and IX collagen. The interstices of this meshwork are filled with the proteoglycan aggrecan bound, with link proteins, to chains of hyaluronan to form proteoglycan aggregates. The tissue also contains the small proteoglycans decorin, biglycan, and fibromodulin. The adhesion protein type VI collagen surrounds the chondrocytes and, in the extracellular matrix, binds to hyaluronan and other matrix molecules, probably to organize the matrix into higher ordered structures. The organization of these macromolecular structures with the water of the tissues endows articular cartilage with its characteristic mechanical properties.

Nucleic acid cytochemistry of the nucleus and microtubule-organizing centers in dictyostelium discoideum

We used fluorescence microscopy with DAPI, Hoechst 33258, acridine orange, and ethidium bromide, as well as ultracytochemical regressive staining, the Feulgen-type reaction with osmium-ammine, and the enzyme-gold method to investigate the presence and distribution of DNA and RNA in the nucleoplasm, nucleolus, nucleus-associated body, and spindle pole bodies. We found that the nucleoplasm of interphase nuclei contains mostly DNA dispersed in a fibrillar meshwork, with which some RNA is probably associated as perichromatin granules or fibers. With DNase-gold and RNase-gold the nucleolus, which consists of interspersed fibrillar and granular components, was the most heavily labelled of five cellular compartments analyzed. Accordingly, its fluorescence with acridine orange and ethidium bromide was brightest. In mitotic nuclei the nucleolus was dispersed, filling most of the nuclear volume. Chromosomes were brightly stained by DNA-specific fluorochromes and the osmium-ammine reaction revealed that only the innermost layer of the trilaminar kinetochores contains DNA. Neither the nucleus-associated body of interphase cells nor the spindle pole bodies of mitotic cells contain DNA or RNA.

Is classical consolidation theory applicable to articular cartilage deformation?

In this paper, classical consolidation theory has been used to investigate the time-dependent response of articular cartilage to static loading. An experimental technique was developed to measure simultaneously the matrix internal pressure and creep strain under conditions of one-dimensional consolidation. This is the first measurement of the internal stress state of loaded cartilage. It is demonstrated that under static compression the applied load is shared by the components of the matrix (i.e. water, the proteoglycans, and the collagen fibrillar meshwork), during which time a maximum hydrostatic excess pore pressure is developed as initial water exudation occurs. This pressure decays as water is further exuded from the matrix and effective consolidation begins with a progressive transfer of the applied stress from water to the collagen fibrils and proteoglycan gel. Consolidation is completed when the hydrostatic excess pore pressure is reduced to zero and the solid components sustain in full the applied load.

Alterations in rabbit vitreal fine structure following C3F8 injection

This study examines the morphological and histochemical changes in the cortical vitreous of 36 rabbit eyes following C3F8 intravitreal gas injection. Eyes were examined by light microscopy (LM) using a modified cryofixation and cryosectioning technique that prevented the loss of soluble tissue moieties and permitted collagen and proteoglycan histochemistry as well as enzyme digestion with hyaluronidase. LM and scanning electron microscopy (SEM) of cryosectioned normal eyes revealed an elaborate fibrillar matrix extending 100-190 microns from the basal lamina of the retina into the vitreous proper, which seemed to be composed of collagen fibrils intimately associated or wrapped in proteoglycan. Following the full expansion of the C3F8 gas bubble in the vitreous, the cortical fibrillar meshwork was absent from the retinal surface and a dense, collagenous material accumulated in the anterior vitreous, especially between the ciliary processes and over the posterior face of the lens. At 41 days postinjection, the fibrillar matrix was reforming and the vitreal cavity was filled with fluid and numerous fibrillar-mucinous islands. These islands did not form sheets or membranes, nor did they attach to either the posterior or the anterior retinal surface. The cortical fibrillar meshwork had reformed at 61 days' recovery; however, the condensed fibrillar material against the lens and filling the spaces between the ciliary processes had not resorbed. Neither shearing of the cortical gel or fibrillar matrix nor congestion of the anterior vitreous was observed in eyes only partially filled with gas.

Cloning and sequencing of cDNA clones encoding chicken lamins A and B 1 and comparison of the primary structures of vertebrate A- and B-type lamins

Nuclear lamins are intermediate-filament-type proteins forming a fibrillar meshwork underlying the inner nuclear membrane. The existence of multiple isoforms of lamin proteins in vertebrates is believed to reflect functional specializations during cell division and differentiation. Although biochemical criteria may be used to classify many lamin isoforms into A- and B-type subfamilies, the structural features distinguishing the members of these subfamilies remain to be characterized fully. Here, we report the complete primary structures of chicken lamins A and B 1 as they are deduced from cloned cDNAs; in the accompanying paper we present the complete sequence of lamin B 2, a second avian B-type lamin. Comparisons of the chicken lamin sequences with each other and with those of other lamins allow us to establish structural features that are common to members of both subfamilies. Conversely, multiple sequence alignments make it possible to identify a number of structural motifs that clearly differentiate B-type lamins from A-type lamins. With this information at hand, we attempt to correlate different biochemical properties of A- and B-type lamins with the presence or absence of specific sequence motifs.

Immunological detection of spectrin during differentiation and in mature ciliated cells from quail oviduct.

A protein that was immunologically related to the erythrocyte and brain alpha-240-subunit and to the brain beta-235-subunit of spectrin was characterized by immunoblotting and was detected by immunofluorescence in the apical part of ciliated cells from quail oviduct. After immunogold-labeling electron immunocytochemistry, spectrin was detected mainly in a fibrillar meshwork located between the proximal parts of the basal bodies. It was also observed to be in contact with the basal foot of basal bodies, but was not found to be associated with the apical plasma membrane. Cilia and microvilli were unlabeled. In contrast, spectrin was detected in close contact with the lateral plasma membrane of mature ciliated cells as well as in stem epithelial cells in unstimulated oviduct. During ciliogenesis induced by estrogen, spectrin gradually appeared at the apex of the cells as the apical cytoskeleton differentiated.

Structure of the zona pellucida and cumulus oophorus in three species of native Australian rodents

The sperm head of many Australian hydromyine rodents has three curved hooks projecting from its anterior margin; the structure of the hooks has been characterized, but their function is unknown. In this study, we have investigated whether the hooks might have evolved to assist sperm penetration through more formidable egg vestments, particularly the zona pellucida. Cumulus-oocyte complexes were obtained from two species that possess a three-hooked sperm head (Pseudomys australis and P. nanus) and one species that does not (Notomys alexis) and examined by light and electron microscopy. After fixation in the presence of ruthenium red, the zona pellucida was found to consist of a fibrillar meshwork, but there were no interspecific structural differences. A corona radiata was absent, and the cumulus extracellular matrix was composed of filaments and electron-dense granules in each species. Measurements of the zona thickness in freshly ovulated, unfixed oocytes revealed that it was thinnest (7.8 microns) in P. australis, which has a three-hooked sperm head, and thickest (11.4 microns) in N. alexis, the species in which the ventral hooks are absent. Hence, no correlation was found between the thickness of the zona pellucida or the structure of the cumulus-oocyte complex, and the presence of three hooks on the sperm head. We conclude, therefore, that it is unlikely that the evolution of the three-hooked sperm head is an adaptation for penetration of increased barriers around the oocyte.

Microinjection of lectins, hyaluronidase, and hyaluronate fragments interferes with cleavage delay and mesoderm induction in embryos of Patella vulgata

In embryos of Patella vulgata at the 32-cell stage, one of the four vegetally located macromeres makes contacts with overlying animal micromeres. As a result, this macromere (designated 3D) divides significantly later than the other macromeres and forms the mesodermal stem cell 4d. Shortly before and during this interaction two types of extracellular matrix are present: a basal lamina-like layer on the tips of the micromeres and a loose fibrillar meshwork in the blastocoel. In this paper we examine the role of the matrix in cleavage delay and mesoderm determination. The microinjection of extracellular matrix-binding lectins, or of hyaluronidase, or of decasaccharide fragments of hyaluronate into the blastocoel results in embryos in which either no or two macromeres are delayed in cleavage and are presumably determined as mesodermal stem cells. We suggest that the fibrillar meshwork is needed for macromere elongation toward the micromeres and that the basal lamina-like layer is involved in the determination process itself.

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

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.

Fractionation of desmosomes and comparison of the polypeptide composition of desmosomes prepared from two bovine epithelial tissues

Desmosomes isolated from bovine tongue mucosa or muzzle epidermis appeared identical by ultrastructural analyses but had some differences in their polypeptide compositions as determined by SDS-PAGE. These preparations were extracted in 9 M urea, 10 mM Tris-HCl (pH 9), and 25 mM B-mercaptoethanol and then centrifuged at 240,000g for 30 min. The urea-soluble and insoluble fractions were analyzed by SDS-PAGE. The urea soluble fractions of both tongue and muzzle desmosomes were enriched in polypeptides of 240, 210, 81, and 75 kDa and also polypeptides (40 to 70 kDa) that were keratin-like, as determined by immunoblotting analyses with keratin antisera. The urea insoluble fraction of tongue desmosomes contained glycoproteins of 165, 160, 140, 110, and 100 kDa, while this fraction from muzzle contained glycoproteins of 165, 115, and 105 kDa. Ultrastructural examinations of insoluble pellets obtained from urea extracted tongue and muzzle desmosomes showed that most of the components at the cytoplasmic faces of the desmosomes were removed, while the membrane regions of the desmosomes resisted the treatment. The urea soluble proteins were dialyzed against 10 mM Tris-HCl (pH 7.6), and the resulting preparation was pelleted by centrifugation and examined by electron microscopy. Ultrastructural examination of this material revealed that it had assembled into a fibrillar meshwork, similar to the fibrillar region adjacent to the submembranous plaque of isolated desmosomes. Thus, treatment of isolated desmosomes with 9 M urea allowed the fractionation of membrane-associated desmosomal proteins from cytoplasmic desmosomal proteins. A comparison of these fractions from tongue and muzzle indicated that the polypeptide compositions of the desmosomes varied between tissues, especially with respect to the fractions enriched in either glycoproteins or keratin.

Observations on the substructural organisation of the exine in Fagus sylvatica L. (Fagaceae) and Scorzonera hispanica L. (Compositae: Lactuceae)

Treatment of acetolysed pollen grains with potassium permanganate exposes substructures by removing part of the exine. New surface details are exposed between tectal and infratectal elements. In sectioned pollen grains ectexinous components including columellae and the tectum are eventually reduced to a system of spaces delimited by a layer which is resistant to the treatment. This is referred to as a “boundary layer” and is composed of a granular or fibrillar meshwork. Results from investigations of Fagus sylvatica and Scorzonera hispanica are compared with earlier studies of exine dissolution and a new hypothesis for exine substructure is presented. In this interpretation the boundary layer is conceived as delimiting areas which are receptive to sporopollenin during early development and so define ectexine stratification and ornamentation. Subsequent addition of tapetal or microsporal sporopollenin masks the boundary layer to a greater or lesser extent in different taxa. The ability to reveal the boundary layer in mature pollen may provide ontogenetically significant information concerning the organisation of pollen walls.