The tectorial membrane of the lizard ear: types of structure

This study is concerned with the forms of the tectorial membrane in the lizard ear and its manner of attachment to the ciliary tufts of the hair cells. These structures and their variations were observed in 20 species representing eight families of lizards. Three forms of tectorial membrane were found, a continuous form that extends throughout the length of the auditory papilla, an abbreviated form that reaches the papilla only in one region, and a dendritic form that is particularly narrow at first and then branches extensively to supply all the hair cells. Occasionally the lower edge of the tectorial membrane makes direct connections with the hair tufts. More often there are special connecting structures between the membrane and the hair tufts. Seven types of these structures were identified, as follows: (1) simple fibers, (2) open network, (3) heavy network, (4) fiber plate, (5) finger processes, (6) sallets, and (7) remote connections. These types of tectorial connections are described and illustrated.     

Polymyxin-B-binding sites in the cochlea as demonstrated by polymyxin-B/gold labeling

A polymyxin-B/bovine-serum-albumin/gold complex was used as a probe to detect the binding sites of polymyxin B on thin sections of cochlea embedded in Spurr's resin. The binding sites were found to be mainly located on the stereocilia, the cuticular plate of hair cells, the head plate of Deiters' cells, the tonofilaments in pillar cells and Deiters' cells, fibrous structures in the spiral limbus, the tectorial membrane and the basilar membrane and neural elements such as nerve endings, fibers, and the myelin sheath. The mitochondria, plasma membrane, and chromatin of the nuclei of the cells observed also exhibited binding. Our results suggest that phospholipids, glycoconjugates, cytoskeletal proteins and nucleic acids are responsible for this binding activity.     

Tectorial structures on the inner ear sensory epithelia of Proteus anguinus (Amphibia,caudata)

The tectorial structures of the inner ear of the proteid salamander Proteus anguinus were studied with transmission and scanning electron microscopy in order to analyze the ultrastructure of the otoconial membranes and otoconial masses of the maculae and the tectorial membrane of the papilla amphibiorum. Both otoconial and tectorial membranes consist of two parts: (1) a compact part and (2) a fibrillar part that joins the membrane with the sensory epithelium. Masses of otoconia occupy the lumina above these membranes. There are two types of calcium carbonate crystals in the otoconial masses within the inner ear of Proteus anguinus. The relatively small otoconial mass of the utricular macula occupies an area no greater than the diameter of the sensory epithelium, and it is composed of calcite crystals. On the other hand, the enormous otoconial masses of the saccular macula and the lagenar macula are composed of aragonite crystals. In the sacculus and lagena, globular structures 2–9 m?m in diameter were discovered on the lower surfaces of the otoconial masses above the sensory epithelia. These globules show a progression from smooth-surfaced, small globules to large globules with spongelike, rough surfaces. It is hypothesized that these globules are precursors of the aragonite crystals and that calcite crystals develop similarly in the utriculus. The presence of globular precursors in adult animals suggests that the formation of new crystals in the otoconial membranes of the sacculus and lagena of Proteus is a continuous, ongoing process.     

Polymyxin-B-binding sites in the cochlea as demonstrated by polymyxin-B/gold labeling

Summary A polymyxin-B/bovine-serum-albumin/gold complex was used as a probe to detect the binding sites of polymyxin B on thin sections of cochlea embedded in Spurr's resin. The binding sites were found to be mainly located on the stereocilia, the cuticular plate of hair cells, the head plate of Deiters' cells, the tonofilaments in pillar cells and Deiters' cells, fibrous structures in the spiral limbus, the tectorial membrane and the basilar membrane and neural elements such as nerve endings, fibers, and the myelin sheath. The mitochondria, plasma mimbrane, and chromatin of the nuclei of the cells observed also exhibited binding. Our results suggest that phospholipids, glycoconjugates, cytoskeletal proteins and nucleic acids are responsible for this binding activity.     

Electron-microscopic localization of type II,IX, and V collagen in the organ of Corti of the gerbil

Summary The presence of types II, IX and V collagen was probed in the organ of Corti of the adult gerbil cochlea by use of immunocytochemistry at the light- and electron-microscopic levels. Type II collagen is found in the connective tissues of the osseous spiral lamina and spiral limbus. In the region of the sensory hair cells it is present in the tectorial membrane and antibodies bind to the thick unbranched radial fibers. Type IX collagen co-localizes with type II collagen in the tectorial membrane, where antibodies bind to the thick unbranched radial fibers. Type V collagen is present in the connective tissue of the spiral limbus, the osseous spiral lamina, the eighth nerve, and the tectorial membrane. In the tectorial membrane, the staining with antibodies to type V collagen is more diffuse than that seen for types II and IX collagen and antibodies to type V bind to the thin, highly branched fibers in which the thick fibers are embedded. The results indicate that collagens characteristic of cartilage are localized in the organ of Corti. Within the tectorial membrane, types II and IX collagen form heterotypic thick fibers embedded in a reticular network of type V collagen fibers. These collagens form a highly structured matrix which contributes to the rigidity of the tectorial membrane and allow it to withstand the physical stresses associated with transmission of the stimuli necessary for sensory transduction.     

Scanning electron microscope studies of the papilla basilaris of some turtles and snakes

The papillae basilares of three species of turtles and four species of snakes were studied by SEM. The papillae of turtle are relatively large among reptiles and are characterized by a long, horizontal middle section resting on wide basilar membrane. Both terminal ends of the papilla extend onto the surrounding limbus in the form of a forked or "T" -shaped end or as a curved, "hook"- like processes. Details vary with the species. In the three species of turtles studied, there were between 1,100 and 1,400 hair cells on a papilla. The tectorial membrane covering the horizontal portion of the papilla is heavy in appearance and tightly attached to the kinocilial bulbs. The terminal ends of the papilla are covered by a thin gelatinous material. In addition, mat-like tectorial network covers the supporting cells and extends from the microvilli of the supporting cells to the overlying tectorial membrane. All hair cells are unidirectionally and abneurally oriented. The supporting cell surfaces form a large part of the papilla and, thus, hair cell density is low. The papillae of the two boid snake species studied are moderately long among snakes and contain a moderate number of hair cells (574 in Epicrates and 710-780 in Constrictor). Papillar form is elongate, avoid, or canoe-shaped. The tectorial membrane may be either highly fenestrated or moderately dense and covers all but a few of the terminal hair cells. A tectorial-like mat covers all but a few of the terminal hair cells. Most hair cells are unidirectionally and abneurally oriented. A few terminal cells in boids may show reverse orientation. Hair cell density is similar to that of turtles.     

A COMPLETE RECONSTRUCTION OF THE NEURAL RETINA OF CHICK EMBRYO GRAFTED ONTO THE CHORIO-ALLANTOIC MEMBRANE

When dissociated neural retinal cells of 6-to 10-day-old chick embryos were grafted as a pellet onto the chorio-allantoic membrane and allowed to develop, complete retinal structures were reconstructed. Especially when the retinal cells of 6-day-old embryos were used, well orientated retinal structures, which possesed three nuclear layers and two plexiform layers, were formed. The fundamental steps in this complete reconstruction were as follows; rosette formation, formation of a fibrillar lumen, differentiation of receptor and ganglion cells, fusion of the fibrillar lumen, fusion of the receptor lumen and finally the formation of a three-layered neural retina. Reconstruction by the retinal cells of older embryos was less complete. This stagedependent difference in the capacity for reconstruction was due to a difference in the ability to form well developed rosettes at an early phase of the process of reconstruction.     

Loss of mammal-specific tectorial membrane component carcinoembryonic antigen cell adhesion molecule 16 (CEACAM16) leads to hearing impairment at low and high frequencies

The vertebrate-restricted carcinoembryonic antigen gene family evolves extremely rapidly. Among their widely expressed members, the mammal-specific, secreted CEACAM16 is exceptionally well conserved and specifically expressed in the inner ear. To elucidate a potential auditory function, we inactivated murine Ceacam16 by homologous recombination. In young Ceacam16(-/-) mice the hearing threshold for frequencies below 10 kHz and above 22 kHz was raised. This hearing impairment progressed with age. A similar phenotype is observed in hearing-impaired members of Family 1070 with non-syndromic autosomal dominant hearing loss (DFNA4) who carry a missense mutation in CEACAM16. CEACAM16 was found in interdental and Deiters cells and was deposited in the tectorial membrane of the cochlea between postnatal days 12 and 15, when hearing starts in mice. In cochlear sections of Ceacam16(-/-) mice tectorial membranes were significantly more often stretched out as compared with wild-type mice where they were mostly contracted and detached from the outer hair cells. Homotypic cell sorting observed after ectopic cell surface expression of the carboxyl-terminal immunoglobulin variable-like N2 domain of CEACAM16 indicated that CEACAM16 can interact in trans. Furthermore, Western blot analyses of CEACAM16 under reducing and non-reducing conditions demonstrated oligomerization via unpaired cysteines. Taken together, CEACAM16 can probably form higher order structures with other tectorial membrane proteins such as α-tectorin and β-tectorin and influences the physical properties of the tectorial membrane. Evolution of CEACAM16 might have been an important step for the specialization of the mammalian cochlea, allowing hearing over an extended frequency range.     

Electron microscopy of human articular cartilage]

Scanning and transmission microscopy of the articular cartilage was performed in femoral condyles of persons at the age of 30-50 years. It was demonstrated that hyaline cartilage is covered with a protective fibrillar layer consisting of tightly pressed collagenous fibrillae with an underlying layer of fibroblastic cells. In the intracellular substance of the hyaline cartilage fibrillar structures form a complex reticular web with vertical arrangement of the main collagenous fasiculi. In the superficial layer of the hyaline cartilage the collagenous fibrillae and their fasciculi form arcade-like structures. Lacunar chondrocytes have a rough villose surface, cellular secrete is discharged as round granules through cytoplasmic membrane. Ultrastructural changes in chondrocytes are observed simultaneously with their degenerative-dystrophic changes.     

A targeted deletion in alpha-tectorin reveals that the tectorial membrane is required for the gain and timing of cochlear feedback

alpha-tectorin is an extracellular matrix molecule of the inner ear. Mice homozygous for a targeted deletion in a-tectorin have tectorial membranes that are detached from the cochlear epithelium and lack all noncollagenous matrix, but the architecture of the organ of Corti is otherwise normal. The basilar membranes of wild-type and alpha-tectorin mutant mice are tuned, but the alpha-tectorin mutants are 35 dB less sensitive. Basilar membrane responses of wild-type mice exhibit a second resonance, indicating that the tectorial membrane provides an inertial mass against which outer hair cells can exert forces. Cochlear microphonics recorded in alpha-tectorin mutants differ in both phase and symmetry relative to those of wild-type mice. Thus, the tectorial membrane ensures that outer hair cells can effectively respond to basilar membrane motion and that feedback is delivered with the appropriate gain and timing required for amplification.     

Role of Endoplasmic Reticulum in the Formation of Peribacteroid Membranes

The relation between the endoplasmic reticulum and peribacteroid membranes during the development of infected cells of Chinese soybean (Glycine max L. cv. Harvest 11) root nodules by transmission electron microscopy was observed. After the host cells are infected by bacteria, the ultrastructures of the infected cells appear to have many changes, such as that their cytoplasm becomes thicker, the vacuoles decrease in size and organelles rapidly increase in number, among these organelle changes are more obvious than the others. However, changes of endoplasmic reticulum is mostly striking. It is not only increases greatly in number but often swells and forms wider inter-spaces. The swelling of endoplasmic reticulum is especially conspicuous at its ends and often form various vesicles. Sometimes, the front part of the endoplasmic reticulum also forms a gourd-shaped structure, which together with the vesicles usually contain fibrillar material. After they are released from the endoplasmic reticulum to the host cytoplasm, they continuously move towards neighbouring bacteria and close to the peribacteroid membranes. The gourd-shaped structures always locate near but never fuse with the peribacteroid membranes. However, the vesicles can do that and form a kind of papillae, often containing fibrillar material, on the peri bacteroid membranes. These papillae and their fibrillar material gradually disappear whilst the membrane of the vesicle derived from endoplasmic reticulum becomes one part of the peribacteroid membrane by way of fusing with the latter to form a papilla on it.     

The lizard ear: Cordylus, Platysaurus and Gerrhosaurus

In further consideration of the lizard ear, the fine structure of the cochlea has been investigated and related to auditory sensitivity in members of the family Cordylidae. The ear of this group of lizards is unusual in that a tectorial membrane is present only in a modified and seemingly vestigial form, and this membrane makes no connections with the auditory hair cells. These cells are provided instead with a series of sallets, small bodies extending in a single row through the dorsal and middle regions of the cochlea, where they rest upon the tips of the ciliary tufts and evidently bring about a stimulation of the hair cells because of their inertia. At the ventral end of the cochlea this line of sallets ends, and here is a single, relatively enormous structure, the culmen papillae, that serves a similar purpose for a large group of hair cells. Consideration is given to the manner of stimulation of the auditory sense cells in these species in relation to others with the usual arrangements involving connections between the ciliary tufts and a tectorial membrane. Included also is a study of a species of Gerrhosaurus, which some have included in the cordylid family and others have placed in a family of its own. The cochlear structure in this species is similar to that of the cordylids in many respects but differs in the ventral region, where instead of the culmen there is a heavy tectorial plate, similarly covering a large number of hair cells but connected to a tectorial membrane. The functioning of these ears is assessed in terms of the cochlear potentials, and is found to vary with species from better than average to excellent in comparison with other lizards investigated. The structural differentiation also is of fairly high degree, and hence it appears that ears without tectorial connections, or with such connections only in a limited region of the cochlea, can perform in a highly serviceable manner.     

Dual role for the latent transforming growth factor-beta binding protein in storage of latent TGF-beta in the extracellular matrix and as a structural matrix protein

The role of the latent TGF-beta binding protein (LTBP) is unclear. In cultures of fetal rat calvarial cells, which form mineralized bonelike nodules, both LTBP and the TGF-beta 1 precursor localized to large fibrillar structures in the extracellular matrix. The appearance of these fibrillar structures preceded the appearance of type I collagen fibers. Plasmin treatment abolished the fibrillar staining pattern for LTBP and released a complex containing both LTBP and TGF-beta. Antibodies and antisense oligonucleotides against LTBP inhibited the formation of mineralized bonelike nodules in long-term fetal rat calvarial cultures. Immunohistochemistry of fetal and adult rat bone confirmed a fibrillar staining pattern for LTBP in vivo. These findings, together with the known homology of LTBP to the fibrillin family of proteins, suggest a novel function for LTBP, in addition to its role in matrix storage of latent TGF-beta, as a structural matrix protein that may play a role in bone formation.     

Gelsolin immunoreactivity and development of the tectorial membrane in the cochlea of normal and hypothyroid rats

Summary Gelsolin was localized by immunocytochemistry in the developing cochlea of the rat. In normal animals, the protein appeared at 18 th day in utero in cells of the Kölliker's organ, which are involved in the secretion of the tectorial membrane. The Kölliker's organ cells were not immunoreactive after the first postnatal week, which is when they cease their secretory activity. Gelsolin immunoreactivity was similar in thyroid-deficient rats until the second postnatal week but, at this age, Kölliker's organ did not transform and its gelsolin immunoreactivity persisted, together with its secretory activity. As a result, the tectorial membrane was greatly distorted and out of contact with the hair cells, which dramatically impaired the mechanical properties of the organ of Corti. The developing cochlea thus provides an example of the involvement of gelsolin in a secretory process that is of importance in the development of hearing.     

Experimental approaches used to quantify physical parameters at cellular and subcellular levels

From a mechanical point of view, plant and hyphal cells are more complex than their animal counterparts because the variety of structural components determining cellular architecture is broader. In addition to cytoskeletal elements and the plasma membrane, the cell wall and turgor pressure equip plant and hyphal cells with structures analogous to an exoskeleton and a hydroskeleton, respectively. To quantify the physical properties of plant and hyphal cells, researchers have developed a plethora of experimental methods. This review provides an overview of experimental approaches that have been used to measure turgor pressure and to determine the mechanical properties of the plant cell wall at the subcellular level. It is completed by a glimpse into the arsenal of techniques that has been used to characterize the physical properties of cytoskeletal elements. These have mostly been used on animal cells, but we hope they will find their way into plant cell research. Finally, assays and tests to measure the generation of forces by cells and subcellular structures are discussed.     

Physiological type I collagen organization induces the formation of a novel class of linear invadosomes

Invadosomes are F-actin structures capable of degrading the matrix through the activation of matrix metalloproteases. As fibrillar type I collagen promotes pro-matrix metalloproteinase 2 activation by membrane type 1 matrix metalloproteinase, we aimed at investigating the functional relationships between collagen I organization and invadosome induction. We found that fibrillar collagen I induced linear F-actin structures, distributed along the fibrils, on endothelial cells, macrophages, fibroblasts, and tumor cells. These structures share features with conventional invadosomes, as they express cortactin and N-WASP and accumulate the scaffold protein Tks5, which proved essential for their formation. On the basis of their ability to degrade extracellular matrix elements and their original architecture, we named these structures "linear invadosomes." Interestingly, podosomes or invadopodia were replaced by linear invadosomes upon contact of the cells with fibrillar collagen I. However, linear invadosomes clearly differ from classical invadosomes, as they do not contain paxillin, vinculin, and β1/β3 integrins. Using knockout mouse embryonic fibroblasts and RGD peptide, we demonstrate that linear invadosome formation and activity are independent of β1 and β3 integrins. Finally, linear invadosomes also formed in a three-dimensional collagen matrix. This study demonstrates that fibrillar collagen I is the physiological inducer of a novel class of invadosomes.     

Fine structure of the mesosome and nucleoid in frozen-etchedBacillus subtilis

Summary An electron microscopical investigation ofBacillus subtilis prepared by freeze-etching revealed the fine structural changes that occur in the cell prior to spore formation. The initiation of growth from lyophilized cells was characterized by the appearance of numerous vesicular structures embedded in and attached to the plasma membrane. As growth continued, the number of vesicular structures decreased and lamellar membrane structures began to appear. Prior to spore formation, a fine, fibrillar material was found in the central portion of the cell and was believed to be the DNA.     

Enhanced synthesis of basement membrane proteins during the differentiation of rat mammary tumour epithelial cells into myoepithelial-like cells in vitro

Rama 25, an epithelial cell line obtained from a dimethylbenzanthracene-induced rat mammary tumour differentiates spontaneously in culture forming elongated myoepithelial-like cells. The elongated cells form multilayered ridge structures from which cultures of elongated cells, relatively uncontaminated by epithelial cells, can be obtained. By using immunofluorescence techniques, both the elongated cells and the cells in ridges, but not undifferentiated Rama 25 cells, have been demonstrated to synthesize three basement membrane proteins, laminin, type IV collagen, and fibronectin. The identity of these basement membrane proteins has been confirmed by immunoprecipitation. These proteins appear to be located in a fibrillar extracellular matrix. We suggest that the ability to synthesize basement membrane proteins by mammary epithelial cells in vitro on plastic is a characteristic of myoepithelial-like cells.     

Water and the cytoskeleton.

The diffusion of intracellular fluid and solutes is mainly limited by the density and the geometry of crossbridges between cytoskeletal polymers mediating the formation of an integrated cytoplasmic scaffold. Evidence for specific relationships between water and cytoskeletal polymers arises from the effect of heavy water on their polymerization process in vitro and on the cytoskeleton of living cells. The hydration of cytoskeletal subunits is modified through polymerization, a mechanism which may be involved in the direct contribution of the cytoskeleton to the osmotic properties of cells together with changes of hydration of polymers within networks. The dynamic properties of the hydration layer of cytoskeletal polymers may reflect the repetitive distribution of the surface charges of subunits within the polymer lattice, thus inducing a local and long range ordering of the diffusion flows of water and solutes inside polymer networks. The interactions between subunits in protofilaments and between protofilaments determine the specific viscoelastic properties of each type of polymer, regulated by associated proteins, and the mechanical properties of the cell through the formation of bundles and gels. Individual polymers are interconnected into dynamic networks through crossbridging by structural associated proteins and molecular motors, the activity of which involves cooperative interactions with the polymer lattice and likely the occurence of coordinated modifications of the hydration layer of the polymer surface. The cytoskeletal polymers are polyelectrolytes which constitute a large intracellular surface of condensed anionic charges and form a buffering structure for the sequestration of cations involved in the regulation of intracellular events. This property allows also the association of cytoplasmic enzymes and multimolecular complexes with the cytoskeleton, facilitating metabolic channelling and the localization of these complexes in specific subdomains of the cytoplasm. The consequences of interactions between membranes and the cytoskeleton in all cellular compartments range from the local immobilization and clustering of lipids and membrane proteins to the regulation of water and ion flows by the association of cytoskeletal subunits or polymers with transmembrane channels. The possibility that the polyelectrolyte properties of the cytoskeletal polymers contribute to the modulation of membrane potentials supports the hypothesis of a direct involvement of the cytoskeleton in intercellular communications.