Distribution Of Intermediate Filaments Research Articles

Long-term remission of Hailey-Hailey disease by Er:YAG ablative laser therapy.

Hailey-Hailey disease (HHD) is a rare genetic therapy-resistant blistering disease with great disease burden. Treatment is currently focused on symptomatic relief. Er:YAG ablative laser therapy is a therapeutic modality with promising results, though evidence is currently scarce. To analyse the effect of Er:YAG ablative laser therapy on clinical remission of erosive plaques in HHD and on patient's quality of life (QoL). In this observational study eight patients were included and 77 erosive plaques were treated once only by Er:YAG laser and assessed for clinical remission. QoL was evaluated by obtaining Skindex-29 and DLQI questionnaires before laser therapy, 6 weeks and 3 years after laser therapy. Skin biopsies were taken to evaluate the depth of laser ablation. The intercellular distance between keratinocytes, the number of desmosomes and intermediate filament distribution were studied by electron microscopy before and after laser therapy and in clinically uninvolved skin and were compared to subjects without HHD. One single Er:YAG laser ablation to mid-dermis resulted in complete remission of 97.4% of HHD plaques (75/77) after median 38 months (range 7-63 months) and significantly improved QoL. Laser therapy restored the number of desmosomes, decreased intercellular distance and diminished perinuclear retraction of keratin filaments to a level comparable to the patient's clinically uninvolved skin. After laser ablation, the skin showed significantly fewer ultrastructural aberrations compared to the patient's clinically uninvolved skin and rather resembled the skin of healthy control individuals. One single Er:YAG laser treatment resulted in long-term remission of HHD and significantly improved QoL. Our findings support a greater role for ablative laser surgery in the management of this recalcitrant disorder.

Comparative Study of Cytokeratin Immunostaining of Parotid Gland Parenchyma in Normal, Diabetic, and Excretory Duct Ligation of Mongrel Dogs.

The present study aimed to give a glimpse of the normal distribution of intermediate filaments within the parotid gland parenchyma of mongrel dogs and to reveal the pathological changes that may occur as a result of the effects of diabetes mellitus or atrophy of the gland caused by the ligation of the excretory duct to discover whether there is a similarity in these pathological behaviors. Twelve healthy mongrel dogs were used in the experiment and were divided into three groups: group I (the control group), group II (dogs with alloxan-induced diabetes), and group III (dogs with the right-side duct-ligated parotid gland). The dogs were sacrificed 45 days after the parotid excretory duct were tied. The right parotid gland of all groups was dissected and prepared for histological and immunohistochemical expression of cytokeratin 17 assay. Histological findings confirmed that the parotid gland parenchyma of the diabetic group had glandular atrophy characterized by the loss of gland structure, degenerated acini, and dilatation of the duct system. Moreover, there is a predominance of the fibrous component with the presence of fat cells within the gland compartments. On the contrary, the excretory duct-ligated group undergoes severe glandular atrophy of the previous character with the presence of duct-like structure as well as extravasation and vasodilatation. Immunohistochemical expression of cytokeratin 17 in control parotid using an immunoperoxidase technique showed that cytokeratin expression varies from negative to mild in all ducts and some serous acinar cells. The gland parenchyma of the diabetic group showed mild to strong cytokeratin expression of duct cells more concentrated in the apical part with moderate to strong expression of diffuse type in some serous acini. The intensity of cytokeratin 17 in gland compartments of the excretory duct-ligated group revealed a variation in expression that ranged from negative to strong diffuse staining throughout the gland. The severity and prevalence of cytokeratin 17 in our results are predictive of the pathological influence of both diabetes mellitus and duct ligation on the cytokeratin intracellular filaments of the salivary gland parenchyma in a different way that interferes with saliva production and/or secretion leading to xerostomia.

Antimicrobial peptide nisin induces spherical distribution of macropinocytosis-like cytokeratin 5 and cytokeratin 17 following immediate derangement of the cell membrane.

The anti-aging effects of Lactococcus lactis are extensively investigated. Nisin is an antimicrobial peptide produced by L. lactis subsp. lactis. We previously reported that 24-hour nisin treatment disturbs the intermediate filament distribution in human keratinocytes. Additionally, we showed that the ring-like distribution of the intermediate filament proteins, cytokeratin (CK) 5 and CK17 is a marker of nisin action. However, two questions remained unanswered: 1) What do the CK5 and CK17 ring-like distributions indicate? 2) Is nisin ineffective under the experimental conditions wherein CK5 and CK17 do not exhibit a ring-like distribution? Super resolution microscopy revealed that nisin treatment altered CK5 and CK17 distribution, making them spherical rather than ring-like, along with actin incorporation. This spherical distribution was not induced by the suppression of endocytosis. The possibility of a macropinocytosis-like phenomenon was indicated, because the spherical distribution was >1 µm in diameter and the spherical distribution was suppressed by macropinocytosis inhibiting conditions, such as the inclusion of an actin polymerization inhibitor and cell migration. Even when the spherical distribution of CK5 and CK17 was not induced, nisin induced derangement of the cell membrane. Nisin treatment for 30 minutes deranged the regular arrangement of the lipid layer (flip-flop); the transmembrane structure of the CK5–desmosome or CK17–desmosome protein complex was disturbed. To the best of our knowledge, this is the first study to report that CK5 and CK17 in a spherical distribution could be involved in a macropinosome-like structure, under certain conditions of nisin action in keratinocytes.

Electron microscopy study of the central retinal fovea in Pied flycatcher: evidence of a mechanism of light energy transmission through the retina

We present unique ultrastructural data on avian retinal cells. Presently and earlier (Zueva et al., 2016) we explored distribution of intermediate filaments (IFs) in retinal cells of the Pied flycatcher (Ficedula hypoleuca, Passeriformes, Aves) in the central foveolar zone. This retinal zone only contains single and double cone photoreceptors. Previously we found that continuous IFs span Müller cells (MC) lengthwise from the retinal inner limiting membrane (ILM) layer up to the outer limiting membrane (OLM) layer. Here we describe long cylindrical bundles of IFs (IFBs) inside the cone inner segments (CIS) adjoining the cone plasma membrane, with these IFBs following along the cone lengthwise, and surrounding the cone at equal spacing one from the other. Double cones form a combined unit, wherein they are separated by their respective plasma membranes. Double cones thus have a common external ring of IFBs, surrounding both cone components. In the layer of cilia, the IFBs that continue into the cone outer segment (COS) follow on to the cone apical tip along the direction of incident light, with single IFs separating from the IFB, touching, and sometimes passing in-between the light-sensitive lamellae of the COS. These new data support our previous hypothesis on the quantum mechanism of light energy propagation through the vertebrate retina (Zueva et al., 2016, 2019).

Foveolar Müller Cells of the Pied Flycatcher: Morphology and Distribution of Intermediate Filaments Regarding Cell Transparency.

Specialized intermediate filaments (IFs) have critical importance for the clearness and uncommon transparency of vertebrate lens fiber cells, although the physical mechanisms involved are poorly understood. Recently, an unusual low-scattering light transport was also described in retinal Müller cells. Exploring the function of IFs in Müller cells, we have studied the morphology and distribution pattern of IFs and other cytoskeletal filaments inside the Müller cell main processes in the foveolar part of the avian (pied flycatcher) retina. We found that some IFs surrounded by globular nanoparticles (that we suggest are crystallines) are present in almost every part of the Müller cells that span the retina, including the microvilli. Unlike IFs implicated in the mechanical architecture of the cell, these IFs are not connected to any specific cellular membranes. Instead, they are organized into bundles, passing inside the cell from the endfeet to the photoreceptor, following the geometry of the processes, and repeatedly circumventing numerous obstacles. We believe that the presently reported data effectively confirm that the model of nanooptical channels built of the IFs may provide a viable explanation of Müller cell transparency.

Quaternary and secondary structural imaging of a human hair by a VSFG-detected IR super-resolution microscope

IR super-resolution images of cross sections of a human black hair were measured by using a home-made vibrational sum-frequency generation (VSFG) detected IR microscope in the 6–9μm region with a sub-micrometer spatial resolution. For the amide III band, the sample gave clear strong signals at the cortex area. This enabled us to measure the distribution of intermediate filaments, which have an α-helix based quaternary structure of keratin proteins in the hair. On the other hand, the VSFG signal disappeared completely when the amide I band was monitored by the same polarization of incident light. From the polarization dependence of VSFG, it is concluded that the α-helix of keratin proteins are well aligned along the axial direction in human hair.

Cell adhesion in zebrafish myogenesis: Distribution of intermediate filaments, microfilaments, intracellular adhesion structures and extracellular matrix

To overcome the limitations of in vitro studies, we have been studying myogenesis in situ in zebrafish embryos, at a sub-cellular level. While in previous works we focused on myofibrillogenesis and some aspects of adhesion structures, here we describe in more detail cell adhesion structures and interactions among cytoskeletal components, membrane and extracellular matrix during zebrafish muscle development. We studied the intermediate filaments, and we describe the full range of desmin distribution in zebrafish development, from perinuclear to striated, until its deposition around the intersomite septa of older somites. This adhesion structure, positive for desmin and actin, has not been previously observed in myogenesis in vitro. We also show that actin is initially located in the intersomite septum region whereas it is confined to the myofibrils later on. While actin localization changes during development, the adhesion complex proteins vinculin, paxillin, talin, dystrophin, laminin and fibronectin always appear exclusively at the intersomite septa, and appear to be co-distributed, even though the extracellular proteins accumulates before the intracellular ones. Contrary to the adhesion proteins, that are continuously distributed, desmin and sarcomeric actin form triangular aggregates among the septa and the cytoskeleton. We studied the cytoskeletal linker plectin as well, and we show that it has a distribution similar to desmin and not to actin. We conclude that the in situ adhesion structures differ from their in vitro counterparts, and that the actual zebrafish embryo myogenesis is quite different than that which occurs in in vitro systems.

Characterization of Cytoskeletal Proteins in Follicular Structures of Cows with Cystic Ovarian Disease

The distribution of intermediate filaments (vimentin, cytokeratins, desmin) and microfilaments (alpha-smooth muscle actin and muscle specific actin) was studied immunohistochemically in bovine ovaries, with and without cystic ovarian disease. The immunohistochemically stained area (IHCSA), was quantified by image analysis, to evaluate the expression of these cytoskeletal proteins in the follicular wall of healthy antral, atretic, and cystic follicles. The granulosa cell layer of cystic follicles and atretic follicles had a significantly larger IHCSA for vimentin than did healthy antral follicles. Cytokeratins reacted lightly in the granulosa cells of antral follicles of normal ovaries, whereas granulosa cells of atretic and cystic follicles showed significantly higher IHCSA values. Immunohistochemical localization of desmin, muscle specific actin, and alpha-smooth muscle actin was restricted to the theca externa. This study supports earlier suggestions that strongly positive reactions with vimentin and cytokeratin antibodies observed in the granulosa cells of cystic follicles are due to the reorganization that occurs in the follicle during the process of cystic development, and are associated with changes in the expression of cytoskeletal proteins that are essential to proper cellular functioning.

Changes in cell shape and desmin intermediate filament distribution are associated with down-regulation of desmin expression in C2C12 myoblasts grown in the absence of extracellular Ca2+

Desmin is the main intermediate filament (IF) protein of muscle cells. In skeletal muscle, desmin IFs form a scaffold that interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton and cytoplasmic organelles. The interaction between desmin and the sarcolemma is mediated by a number of membrane proteins, many of which are Ca2+-sensitive. In the present study, we analyzed the effects of the Ca2+ chelator EGTA (1.75 mM) on the expression and distribution of desmin in C2C12 myoblasts grown in culture. We used indirect immunofluorescence microscopy and reverse transcription polymerase chain reaction (RT-PCR) to analyze desmin distribution and expression in C2C12 cells grown in the presence or absence of EGTA. Control C2C12 myoblasts showed a well-spread morphology after a few hours in culture and became bipolar when grown for 24 h in the presence of EGTA. Control C2C12 cells showed a dense network of desmin from the perinuclear region to the cell periphery, whereas EGTA-treated cells showed desmin aggregates in the cytoplasm. RT-PCR analysis revealed a down-regulation of desmin expression in EGTA-treated C2C12 cells compared to untreated cells. The present results suggest that extracellular Ca2+ availability plays a role in the regulation of desmin expression and in the spatial distribution of desmin IFs in myoblasts, and is involved in the generation and maintenance of myoblast cell shape.

Extracellular Matrix Components and Matrix Degrading Enzymes in the Feline Placenta during Gestation

In the endotheliochorial placenta of the cat, the maternal surface epithelium and parts of the connective tissue have to be removed to bring the fetal blood vessels in close contact to the maternal capillaries. The composition of the extracellular matrix (ECM) in the feline uterus is not known and it is still not clear if and which parts of the maternal ECM persist during gestation in the placental labyrinth. We demonstrated various extracellular matrix components (collagen types I, III, IV, and laminin) and matrix metalloproteinases (MMP-1, -2, -13) using immunohistochemistry and studied the distribution of intermediate filaments (vimentin, cytokeratin) and alpha-smooth muscle actin (SMA) in the placental girdle on specimens of different stages of gestation. Collagen types I and III were mainly present in the fetal chorionic lamellae whereas diminished in the maternal placental labyrinth part. Collagen IV and laminin were expressed in fetal basement membranes and mesenchyme. Maternal endothelial cells and stromal cells showed a positive immunoreaction for anti-collagen type IV and laminin. MMP-2 was identified in the maternal stroma, including decidual cells. Endothelia of maternal blood vessels within the labyrinth contained MMP-1, -2 and -13, probably associated with angiogenesis. In the trophoblast MMP-1 and -13 were demonstrated. Maternal stem vessels were accompanied by a thick layer of syncytiotrophoblast. Around these vessels, collagen type I and SMA were present in a periendothelial region between the endothelium and the trophoblast. These findings indicate that a strictly regulated balance between ECM deposition and ECM degradation in the feline placental labyrinth is necessary for proper placental development and function.

Role of Phosphorylation in Ethanol-Induced Aggregation of Keratin Intermediate Filaments

Keratins are members of a diverse group of tissue-specific cytoskeletal components known as intermediate filaments. Regulation of the structure and intracellular distribution of intermediate filaments is known to be related to the phosphorylation state of their structural subunits. It also is known that disruption of the keratin filaments of hepatocytes in response to chronic ethanol ingestion is characteristic of alcoholic liver disease. To characterize the mechanism of ethanol-induced keratin filament reorganization and dephosphorylation, cells were grown in culture with and without ethanol, and then were treated at the end of the incubation period for 1 hr with either 8-bromo-adenosine 3':5'-cyclic monophosphate (8Br), water-soluble forskolin (ws-forskolin), H-89 diHCL, or okadaic acid. Morphology of the cells was examined by immunofluorescence microscopy, and keratin phosphorylation levels were determined by analysis of 32p labeling. We found that treatment of hepatoma cells with 300 mM ethanol results in disruption and aggregation of the keratin network in the vicinity of the nucleus as well as a hypophosphorylation of keratin subunits from ethanol-treated cells compared with non-ethanol-treated controls. 8Br and ws-forskolin treatment of ethanol groups restored keratin phosphorylation to control levels and reversed the ethanol-induced aggregation of keratin filaments. When H-89, an inhibitor of A-kinase, was added to control cells, keratin filament disorganization and dephosphorylation was observed. H-89 produced only a slight additional decrease in keratin phosphorylation in ethanol-treated cells, with no change in keratin distribution. Okadaic acid treatment of control cells produced hyperphosphorylation and filament network disruption, whereas in ethanol groups a reversal of the ethanol-mediated hypophosphorylation was observed but without reversal of the keratin filament aggregation. These results suggest that site-specific phosphorylation of keratin filaments is important in maintaining their integrity and that activation of the A-kinase system can antagonize the effects of ethanol, whereas its inhibition results in filament dephosphorylation and reorganization, mimicking effects of ethanol treatment.

Plectin is concentrated at intercellular junctions and at the nuclear surface in morphologically differentiated rat Sertoli cells.

Intermediate filaments in Sertoli cells have a well-defined pattern of distribution. They form a basally situated perinuclear network from which filaments extend peripherally to adhesion plaques at the plasma membrane and to sites of codistribution with other major elements of the cytoskeleton, particularly with microtubules. Although the general pattern of intermediate filament distribution is known, the molecular components involved with linking the filaments to organelles and attachment plaques in these cells have not been identified. One candidate for such a linking element is plectin. In this study we test for the presence of, and determine the distribution of, plectin in Sertoli cells of the rat testis. Fixed frozen sections and fixed epithelial fragments of rat testis were probed for plectin and vimentin using antibodies. Tissue was evaluated using standard fluorescence microscopy and confocal microscopy. Plectin in Sertoli cells was concentrated in a narrow zone surrounding the nucleus, and at focal sites, presumably desmosome-like plaques, at interfaces with adjacent cells. Plectin was also concentrated at sites where intermediate filament bundles project into specialized actin-filament containing plaques at sites of attachment to elongate spermatids. Plectin in Sertoli cells is concentrated at the nuclear surface and in junction plaques associated with the plasma membrane. The pattern of distribution is consistent with plectin being involved with linking intermediate filaments centrally (basally) to the nucleus and peripherally to intercellular attachment sites.

Spatial distribution of cytoskeleton intermediate filaments during fetal rat hepatocyte differentiation.

The construction of the liver parenchyma throughout fetal development depends on the elaboration of intercellular contacts between epithelial cells and between epithelial and mesenchymal cells. During this time, the spatial distribution of cytokeratins in hepatocytes shows a striking evolution as demonstrated by confocal microscopy and image analysis. In the early stages of fetal rat development, the liver is mainly a hematopoietic organ and hepatocytes represent fewer than 40% of all liver cells. At this time, cytokeratin filaments are scarce and are randomly distributed inside the cytoplasm. A coexpression of desmin and cytokeratin is found in some cells. Intercellular contacts between epithelial and mesenchymal cells are more numerous than between epithelial cells. Later in development, hepatocytes are arranged in a "muralium duplex" architecture (two-cell-thick sheets). Contacts between hepatocytes become more numerous and bile canaliculi become well developed. The density of cytokeratin filaments increases and appears to be very high near the bile canaliculi. In adult liver, hepatocytes are arranged in a "muralium simplex" architecture. Cytokeratin filaments show a symmetrical distribution in relation to the nuclear region. The highest density of filaments is found near the cytoplasmic membrane. Variations of the spatial distribution of intermediate filaments throughout hepatocyte differentiation were investigated in a pilot study using computerized image analysis. We found significant differences between the filament networks in fetal and adult hepatocytes.

Changes in the distribution of intermediate filaments in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development.

Intermediate filaments (IFs) are components of the cytoskeleton. In mammalian Sertoli cell, IFs are formed by vimentin. Previous studies have shown some characteristics of its distribution in Sertoli cells, however, very little is known of its distributional changes during the seminiferous epithelium cycle and during postnatal development. Immunohistochemical and electron microscopic methods were used to determine the distribution of vimentin-type IFs in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development. The distribution of IFs in adult rat Sertoli cell showed distinct cyclic changes during the seminiferous epithelium cycle. At stages I-VI, bundles of IFs extend from the perinuclear region to the supranuclear and apical regions of the Sertoli cell. These apical extensions became shorter at stage VII, and at stages VIII-X IFs were observed only in the perinuclear region. Short apical extensions reappeared at stages XI-XII; and at stages XIII-XIV, they extended again into the apical region. During this cycle, IFs were always closely associated with the heads of elongate spermatids. IFs were also shown to be in close apposition to some specialized structures on the cell membrane, such as the ectoplasmic specialization between adjacent Sertoli cells. During postnatal (p.n.) development, IFs were mainly observed at the basal nuclear region on p.n. day 7. The IFs in the supranuclear or apical regions first appeared at p.n. day 14 and gradually increased during the development. The perinuclear IFs network was fully established by p.n. day 28 and the adult distribution pattern of the IFs was established by p.n. day 42. Vimentin-type IFs in rat Sertoli cells are a delicate endocellular network, which is centered in the perinuclear region and extends to the apical region of the cell. During the seminiferous epithelium cycle, the distribution of IFs changes in a stage-dependent manner and is closely related to the location of the heads of elongate spermatids. During postnatal development, IFs gradually increase in numbers and the main distribution area is transferred from the basal nuclear to the perinuclear and supranuclear regions.

The pattern of desmin filaments in myocardial disarray

“Myofiber disarray” defines a nonparallel arrangement of cardiac myocytes. The presence of a sufficient quantity of myocardial fibers showing this change is considered to be a specific histological feature of hypertrophic cardiomyopathy (HCM). However, small zones of myofiber disarray are found in both cardiac hypertrophy and other pathological conditions. Recently, we demonstrated an altered pattern of desmin intermediate filaments in disarrayed myofibers from specimens of HCM. To test the hypothesis that desmin alterations might be specific for cardiomyopathy, we performed an immunohistochemical study on myocardial surgical samples from 11 patients with HCM and from 12 patients with tetralogy of Fallot (toF) on 14 endomyocardial biopsy specimens (EMBs) from transplant recipients with myofiber disarray surrounding areas of scarring (previous biopsy site) and on speciments of four autoptic hearts with severe acquired left ventricular hypertrophy. Disarrayed myofibers from all specimens of HCM showed the following abnormalities in the pattern of desmin intermediate filament distribution (1) decrease or loss of labeling of intercalated discs and Z bands, (2) longitudinal arrangement of desmin intermediate filaments, and (3) intense, granular staining of several myocytes. This spectrum of desmin alterations was never observed in disarrayed myofibers in specimens of toF or acquired myocardial hypertrophy or in EMBs. Altered distribution of desmin intermediate filaments seems to be specific to myofiber disarray in HCM and it may play a role in the altered myocyte arrangement in HCM.

Cytoskeletal Organization of the Vestibular Sensory Epithelia

The cytoskeletal organization of the guinea pig vestibular sensory epithelium was investigated by employing the saponin perfusion method and scanning electron microscopy. The skeletal framework of a a cell was found to consist of actin, intermediate filaments and microtubules. The membrane-bound organelles such as nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, etc. were also well documented. This made it possible to investigate the three-dimensional organization of cytoskeletons as well as their complex interaction with various membrane-bound organelles. The intermediate filaments were demonstrated not only in the supporting cells but also in the sensory cells. They are usually seen surrounding the nucleus and extending through the cytoplasm which connects the nuclear membrane to the plasma membrane, cuticular plate, reticular lamina or other cyto-organelles. The intermediate filaments are also closely related to the desmosomes. These findings suggest that the intermediate filaments provide mechanical support to the cell and its nucleus. In the sensory cells, microtubules are found mainly in the supranuclear portion of the cells, running parallel to the main axis of the cell body, which is directly connected to the cuticular plate. These findings suggest that the microtubules provide the cell with mechanical support and may be closely related to the sensory cell transduction system. Inside the supporting cells, the microtubules are closely related to the secretory granules, Golgi apparatus and intermediate filaments, which supports the idea that the microtubules may control the distribution of intermediate filaments, and also play an important role for transport of the secretory granules.

The distribution of novel intermediate filament proteins defines subpopulations of myenteric neurons in rat intestine

Background/Aims : Recent studies with neurofilament antibodies as neuronal markers have shown subpopulations of myenteric neurons that do not contain neurofilament proteins. Novel neuronal intermediate filament proteins α-internexin, peripherin, and nestin have been identified. The aim of this study was to examine the distribution of these novel intermediate filaments in comparison with neurofilaments in myenteric plexus neurons. Methods : Using indirect immunofluorescence techniques in whole-mount cryostat sections from neonate and adult rat small intestine and in primary cultures of myenteric neurons, the distribution of neurofilaments, α-internexin, peripherin, and nestin was studied in comparison with the neuronal marker protein gene product (PGP) 9.5 in myenteric neurons. Results : Sixty-five percent of neurons contained neurofilament triplet proteins, α-lnternexin and/or peripherin were found in the neurofilament-negative neurons. PGP 9.5 was present in 80% of the myenteric neurons. Of the neurons that were PGP negative,95% contained peripherin or α-internexin. Nestin was not found in either neonate or adult myenteric neurons but was seen in glial cells in culture. Conclusions : The results suggest that a subpopulation of myenteric neurons lacks neurofilament triplet proteins but contains either peripherin, α-internexin, or both. This selective distribution of intermediate filaments in subpopulations of enteric neurons may support differential roles in these structurally unique neurons.

The determination and intracellular distribution of intermediate filaments of cultured inner ear melanocytes

The determination and intracellular distribution of intermediate filaments of cultured inner ear melanocytes

Distribution of cytoskeletal proteins (neurofilaments, peripherin and MAP-tau) in the cochlea of the human fetus.

We report here an immunohistochemical study of the distribution of intermediate filaments (neurofilament, peripherin) and a microtubule-associated protein, tau, in the human fetal cochlea at 27 weeks of gestation. Neurofilament immunoreactivity (160 and 200 KDa) was localized in afferent and efferent fibers of the cochlear innervation and restricted to a few small spiral ganglion neurons. Peripherin immunoreactivity was specifically distributed in some small ganglion neurons and in their central and peripheral extensions, particularly in fibers reaching the lower part of the outer hair cells. Double immuno-labelling studies with these neurofilaments and peripherin antibodies show that only small neuron cell bodies were stained. Morpholometrical data indicate that immunostained neurons could be related to the Type II neuron population in the spiral ganglion. Tau protein was localized in intraganglionic spiral bundle fibers and in fibers that reach the lower part of hair cells. These observations suggest that neurofilament and peripherin antibodies stain a particular population of human spiral ganglion neurons with Type II characteristics. Moreover, the specificity of peripherin labelling in Type II cells and their processes suggest that peripherin could be used as a probe for the developmental study of this system in the human cochlea. On the other hand, tau antibody appeared as a marker for efferent fibers during development and could give information on the ontogenesis of efferent innervation.

Cytoskeletal organization of the vestibular supporting cells. Saponin perfusion method for observing intracellular structures by scanning electron microscopy.

The cytoskeletal organization of the guinea pig vestibular supporting cells was investigated employing the saponin perfusion method using scanning electron microscopy. The skeletal framework of a cell was composed of actin, intermediate filaments and microtubules. The membrane bound organelles such as nucleus mitochondria, endoplasmic reticulum, Golgi apparatus, etc. were also well documented. This made it possible to investigate the three-dimensional organization of cytoskeletons as well as their complex interaction with various membrane bound organelles. The intermediate filaments were usually seen surrounding the nucleus and extending through the cytoplasm which connected the nuclear membrane to the plasma membrane, reticular lamina or other cytoorganelles. The intermediate filaments also closely related to the desmosomes. These findings suggest that the intermediate filaments provide mechanical support to the cell and its nucleus. The microtubules were closely related to the secretory granules, Golgi apparatus and intermediate filaments, which supports the idea that the microtubules may control the distribution of intermediate filaments, and also play an important role for the transport of the secretory granules.