Myosin VIIa, the product of the Usher 1B syndrome gene, is concentrated in the connecting cilia of photoreceptor cells

Usher syndrome is the most common form of combined deafness and blindness. The gene that is defective in Usher syndrome 1B (USH1B) encodes for an unconventional myosin, myosin VIIa. To understand the cellular function of myosin VIIa and why defects in it lead to USH1B, it is essential to determine the precise cellular and subcellular localization of the protein. We investigated the distribution of myosin VIIa in human and rodent photoreceptor cells and retinal pigment epithelium (RPE), primarily by immunoelectron microscopy, using antibodies generated against two different domains of the protein. In both human and rodent retinae, myosin VIIa was detected in the apical processes of the RPE and in the cilium of rod and cone photoreceptor cells. Immunogold label was most concentrated in the connecting cilium. Here, myosin VIIa appeared to be distributed outside the ring of doublet microtubules near the ciliary plasma membrane. These observations indicate that a major role of myosin VIIa in the retina is in the photoreceptor cilium, perhaps in such a function as trafficking newly synthesized phototransductive membrane or maintaining the diffusion barrier between the inner and outer segments. Our results support the notion that defective ciliary function is the underlying cellular abnormality that leads to cellular degeneration in Usher syndrome.     

Tumour cell kinetics as prognostic factor in surgically-treated-non-small cell lung cancer

The gene encoding myosin VIIA is responsible for the mouse shaker-1 phenotype, which consists of deafness and balance deficiency related to cochlear and vestibular neuroepithelial defects. In humans, a defective myosin VIIA gene is responsible for Usher syndrome type IB, which associates congenital deafness, vestibular dysfunction and retinitis pigmentosa. In an attempt to progress in the understanding of the function(s) of myosin VIIA, we studied the expression of the myosin VIIA gene during mouse embryonic development. Embryos from day 9 (E9) to E18 were analyzed by in situ hybridization and immunohistofluorescence. The myosin VIIA mRNA and protein were consistently detected in the same embryonic tissues throughout development. Myosin VIIA was first observed in the otic vesicle at E9, and later in a variety of tissues. The olfactory epithelium and the liver express it as early as E10. In the retinal pigment epithelium, choroid plexus, adrenal gland and tongue, expression begins at E12 and in the testis and the adenohypophysis at E13. In the small intestine, kidney and hair follicles of the vibrissae, expression of myosin VIIA starts only at E15. Myosin VIIA expression was observed only in epithelial cell types, most of which possess microvilli or cilia. Interestingly, myosin VIIA expression seems to be concomitant with the appearance of these structures in the epithelial cells, suggesting a role for this myosin in their morphogenesis. The cellular location of myosin VIIA within sensory hair cells and olfactory receptor neurons also argues for a role of this protein in the synaptic vesicle trafficking.     

Morphology and cytology of the nasal cavity and vomeronasal organ in juvenile and adult blind mole rats (Spalax ehrenbergi)

The blind mole rat (Spalax ehrenbergi) is a fossorial solitary rodent which exhibits extensive intraspecific aggression and uses scent markings to deter contraspecific invaders. Mole rats of different ages were captured near Tel Aviv, Israel, and sacrificed by an overdose of Xylazine hydrochloride. Olfactory epithelium sites from the nasal cavity (NC) and the vomeronasal organ (VNO) were dissected and fixed for light and electron microscopy. The mole rat's olfactory epithelium of the NC consists of several cell types, of which two types are supporting cells that comprise both microvilli and cilia but differ in staining and the presence of rough endoplasmic reticulum. The third type has no cilia. Secretory goblet cells were frequent among supporting cells of adults alone. Two types of receptor cells protrude into the NC with olfactory knobs at their apical region; one type has up to 177.6 +/- 9.4 cilia per knob plus microvilli, while the other type has only microvilli. The third type of sensory cell has no knob and contains microvilli only. The basal epithelium layer consists of short-bodied cells with round nuclei. The VNO of the mole rat is situated beneath the nasal septum, consisting of supporting, sensory, and basal cell types, with many cilia at the apical portion. At its anterior part, the VNO is connected to the NC by narrow canals. The abundance of cilia and microvilli in the mole rat olfactory cells provides the first anatomical evidence for their olfactory acuity. Such acuity is important in mole rats, compensating for their loss of vision and enabling them to detect and avoid rivals prior to potential aggressive encounters as well as to select food plants during foraging.     

Papillary cystadenoma of the epididymis. An ultrastructural and immunohistochemical study

Papillary cystadenoma of the epididymis is a rare neoplasm that is sometimes associated with von Hippel-Lindau's syndrome. Electron microscopic study of the present case revealed that neoplastic cells contained abundant glycogen granules and large lipid droplets, but a few organelles. On the apical surface there were numerous microvilli and a few single cilia, but no ciliated cells. Subepithelial basal lamina was noted, but it was occasionally disrupted. Furthermore, microvilli sprang from the circumference of the small tumor-cell nest and became associated with matrix components (microvillus-matrix associations). On immunohistochemical study, neoplastic cells showed epithelial characteristics, but positive reactivity for S-100 protein. These findings resembled those of the epithelial cells of the efferent ductules of the epididymis. In the stroma, prominent vasculature was characteristic and fenestrated-type capillaries were found in the peripheral portion of the tumor. Papillary cystadenoma of the epididymis may originate from non-ciliated epithelial cells of the efferent ductules.     

Shaker-1 mutations reveal roles for myosin VIIA in both development and function of cochlear hair cells

The mouse shaker-1 locus, Myo7a, encodes myosin VIIA and mutations in the orthologous gene in humans cause Usher syndrome type 1B or non-syndromic deafness. Myo7a is expressed very early in sensory hair cell development in the inner ear. We describe the effects of three mutations on cochlear hair cell development and function. In the Myo7a816SB and Myo7a6J mutants, stereocilia grow and form rows of graded heights as normal, but the bundles become progressively more disorganised. Most of these mutants show no gross electrophysiological responses, but some did show evidence of hair cell depolarisation despite the disorganisation of their bundles. In contrast, the original shaker-1 mutants, Myo7ash1, had normal early development of stereocilia bundles, but still showed abnormal cochlear responses. These findings suggest that myosin VIIA is required for normal stereocilia bundle organisation and has a role in the function of cochlear hair cells.     

Identification of epithelial cells and fibroblasts in hypophysis intermediate lobe cultures by scanning electron microscopy

Cell cultures of the rat pituitary intermediate lobe grown for eight days were studied in the scanning electron microscope. The epithelial cells and fibroblasts could be differentiated by the characteristic structures of the cell surface and the cell association features, The epithelial cells were characterized by blebs and rugae, microvilli, and occasionally by some cilia. The surface of the fibroblasts was smooth or bore microvilli. Scanning electron microscopy may provide special information for the characterization of endocrine cell cultures.     

Localization of a G protein Gi2 in the cilia of rat ependyma, oviduct and trachea

In previous studies, the localization of a pertussis toxin-sensitive G protein was demonstrated in ependymal cilia, but the identification of the subtype of G protein was inconsistent. To clarify this issue, we studied the localization of Goalpha, Gi1alpha, Gi3alpha and Gi2alpha in the ciliated ependymal cells and in the cilia of some other tissues of rats using specific antibodies. The cilia of the ependymal cells that line the ventricular cavity of the brain were intensely immunoreactive for Gi2alpha, but not for Goalpha, Gi1alpha or Gi3alpha. Immunoblot analysis demonstrated higher levels of Gi2alpha in the ependymal cilia-rich pellet than in the motor area of the parietal cortex. At the ultrastructural level, the immunoreactivity specific for Gi2alpha was found predominantly in the cilia, but rarely in the microvilli or the basal bodies of ependymal cells. In cross-sections, the immunoreactivity specific for Gi2alpha was observed only in cell membranes, in particular, in the inner electron-dense leaflet of the trilaminar structure. In addition to that in the ependymal cilia, such specific localization of Gi2alpha was observed in the motile cilia in other tissues, including the oviduct and trachea. By contrast, the stereocilia in the ductus deferens were not immunopositive for Gi2alpha. These findings suggest that Gi2 might play an important role in the signal transduction in ciliary membrane-associated function(s) of the ependymal cells, oviduct and trachea.     

Biochemical analysis of fibroblasts from patients with cytochrome c oxidase-associated Leigh syndrome

Cultured skin fibroblasts from four patients with Leigh syndrome and cytochrome c oxidase deficiency were studied. Mitochondrial DNA (mtDNA) analysis excluded large-scale deletions and known point mutations associated with Leigh syndrome. The COX activities were reduced to 18-44% of healthy probands, when measured in the presence of laurylmaltoside. COX activity from patients was shown to be more temperature sensitive than COX activity from control cells. In order to determine the subunit composition of COX immunoblotting studies were performed using mono- and polyclonal antibodies to distinct subunits. A monoclonal antibody to subunit IV crossreacted with two unknown proteins of higher apparent molecular weight in mitochondria from three patients, but not in mitochondria from control and the fourth patient. Quantification of immunoreactivity revealed a decrease of subunits II/III and IV parallel to the determined enzyme activity. In contrast, a variable amount of subunit VIIa (and/or VIIb) was found in mitochondria from different patients. The results indicate a defective COX holoenzyme complex in patients with Leigh syndrome and suggest different molecular origins of the defect.     

The ultrastructure of ependymoma: personal experience and the review of the literature

I report here the ultrastructure of 29 ependymal tumors. The ultrastructural pattern was florid and characteristic with a picture dominated by the presence of microlumina, cilia with basal bodies (blepharoplast), microvilli and long, interdigitating intercellular junctions of the zonulae adherentes (adhesive plaque junctions) type. Tumor cells themselves were not particularly peculiar but they formed typical patterns of rosettes (so called mini- or ultrastructural rosettes) cell gatherings around small, electron-lucent lumina which are filled with numerous microvilli. Empty microlumina were rare. The apical and lateral portions of the cells surrounding microlumina were sealed by intercellular junctions which are long, tortuous and clearly different from the zonulae occludentes (tight junctions) of epithelial tumors. Clusters of apparently "redundant" junctions were occasionally visible comprising segments of different lengths. Ependymoma cells contained myriads of 10 nm intermediate filaments (glial filaments), occasionally forming thick bundles, virtually identical to those encountered in astrocytic tumors and forming an ultrastructural correlate for the GFAP immunostaining. The glycogen granules were often remarkably numerous. Numerous cilia, with a typical 9+1 pattern or with a distorted pattern were frequently observed in longitudinal or cross-sections.     

Maternal urinary free beta-subunit of human chorionic gonadotrophin: creatinine ratios and fetal chromosomal abnormalities in the second trimester of pregnancy

We observed a 56-year-old woman with Kartagener's syndrome and severe seropositive rheumatoid arthritis. This is the third case of such association in the world literature and a second one being diagnosed in our Department. The patient was also as the previous one a carrier of HLA DR1 and B27 antigens. An electromicroscopic study showed normal bronchial cilia in contrast to classical course of the disease. A number of immunological disturbances were observed, especially defective granulocyte function. We suggest that the severe course of rheumatoid arthritis may be related to the chronic stimulation of immune system by microbes continuously present in the patients airways.     

Structure and regeneration of the eyes of strombid gastropods

The tips of the eyestalks of three species of strombid gastropods were amputated and the structure of the fully developed eye in investigated. The retina contains at least two types of cell: sensory cells bearing long tufts of microvilli with a central cytoplasmic core, and pigment cells with short microvilli. New eyes became visible at the tips of the eyestalk stump 5-16 days after amputation. When the regenerated eyes first appear, they consist of hollow balls of cells with a pigment lined cavity; two types of retinal cells are already distinguishable but their microvilli and cilia are small and sparse. The microvillous tufts and sensory cell contents develop quickly and about 14 days after their first appearance, the eye is a fully formed but miniature organ.     

Electron paramagnetic resonance evidence of the generation of superoxide (O2.-) and hydroxyl (.OH) radicals by irradiation of a new photodynamic therapy photosensitizer, Victoria Blue BO

Fetal and postnatal ontogenesis of the rat cochlea, from the 16th gestational day (16DG) until 3 months post partum, were studied using scanning electron microscopy with emphasis on the stereocilia during the earliest stages of development. The epithelium of the cochlear duct in 16DG rat consisted of plygonal cells topped with numerous microvilli and one central kinocilium, which form the so-called K?lliker's organ. Inner hair cells (IHCs) appeared at 18DG in the basal cochlea. They were characterized by tufts of cilia of the same height and with a kinocilium. The first outer hair cells (OHCs) can be seen at 20DG. The earliest stages of ciliary differentiation, at 18DG for IHCs and 20DG for OHCs, were similar on both types of cells and were characterized by the presence of round bundles of cilia arising from the surrounding microvilli. A three-dimensional V-shaped organization for OHCs and the linear arrangement for IHCs appeared by the end of the first postnatal week, accompanied by the disappearance of transient cilia on the modiolar side of the hair cell and the kinocilium on the external side. The apical pole of OHCs reached adult-like morphology before that of IHCs. Various links between stereocilia were detected already at birth. Morphometric analysis showed that auditory cells from the base of the cochlea reached adult size by the end of the first postnatal week while those from the apex increased their size later. A review of the literature including comparative observations across species on the ontogenesis of the stereocilia shows that hair cells of the stato-acoustic system may present the same early ontogenesis.     

Electron microscopic study of intercalated duct cells in the chicken pancreatic islet and effects of tolbutamide administration

Intercalated duct cells are present in the alpha and beta islets of chicken. The intercalated duct cells adhere to each other via intercellular junctional complexes at the apical side, projecting many microvilli and a few cilia into the lumen. They also extend slender cytoplasmic processes between the islet endocrine cells. These intercalated cells appear to have a stellate form, and to wrap their cytoplasm around endocrine cells. Administration of tolbutamide led to increased electron density in the cytoplasm of intercalated duct cells. Lysosomes are present in these cells in various numbers and sizes and tend to increase with time after administration of tolbutamide. These observations suggested that the intercalated ducts not only pass through the islet, but also play a role in supplying islet cells.     

Three-dimensional structure of the rat pancreatic duct in normal and inflammated pancreas

We observed the corrosion casts of the Wistar rats' pancreatic ducts with scanning electron microscopy (SEM), and their conventionally fixed pancreatic tissue with SEM and transmission electron microscopy (TEM). These findings revealed the following facts about the three-dimensional structure of pancreatic duct. (1) The interlobular and intralobular ducts branch like a tree, and the intercalated ducts wind and fork into two branches, although parts of the intercalated ducts anastomose with each other. The intercellular secretory canaliculi extend from the central lumina, which run straight through the center of the acini, finally approaching close to the basement membranes of acini. (2) The lumina of pancreatic ducts (i.e., the interlobular up to the intercalated ducts) are cylindric and have smooth surfaces. The luminal surface of each epithelial cell, however, is decorated by numerous microvilli and a single cilium. The length of the latter tends to be short in proportion to the diameter of pancreatic duct. Moreover the epithelial cell surfaces, which border each central lumen, have various densities of microvilli. (3) The intraductal cilium core is provided with nine microtubules, which is different from the number of microtubules encountered within the cilium core of uterine tube or bronchial epithelium. The number of microtubules in the cross-sectioned intraductal cilia decreases toward the distal portion of cilia. SEM and TEM observations on WBN/Kob rats' pancreatic ducts suggest that increased pancreatic ductal pressure causes the helical shape of the pancreatic ductal lumen. Such a helical form might also be caused by the protrusion of epithelial cell boundaries into their lumen and the hypertrophy and hyperplasia of epithelial cells, thus leading to the formation of numerous depressions equipped with elongated cilia.     

Histamine sustains fMLP-stimulated leukocyte adhesion in rat mesenteric venules in vivo

Hereditary non-syndromic profound deafness affects about 1 in 2000 children prior to language acquisition. In 80% of the cases, the mode of transmission is autosomal recessive. The number of genes involved in these recessive forms of isolated deafness (DFNB genes) has been estimated to between 30 and 100. So far, ten DFNB genes have been mapped to human chromosomes, one of which has been isolated. By linkage analysis of a single family whose members were affected with profound deafness, some of them presenting with vestibular dysfunction, DFNB2 has been mapped to chromosome 11q13 (ref. 3). The gene responsible for a form of Usher syndrome type I, USH1B, has been assigned to the same chromosomal region. Usher syndrome associates profound congenital deafness and vestibular dysfunction with retinitis pigmentosa. In the homologous murine region are located the shaker-1 mutations responsible for deafness and vestibular dysfunction. It has been demonstrated that the murine shaker-1 and human USH1B phenotypes result from mutations in the gene encoding myosin-VIIA. Based on mapping data as well as on the similarities between the phenotypes of DFNB2-affected patients and shaker-1 mouse mutants, we have proposed that a defective myosin-VIIA may also be responsible for DFNB2 (ref. 1). Sequence analysis of each of the coding exons of the myosin-VIIA gene (MYO7A) was thus undertaken in the DFNB2-affected family. In the last nucleotide of exon 15, a G to A transition was detected, a type of mutation that is known to decrease the efficiency of splicing. Accordingly, this result shows that different mutations in MYO7A result in either an isolated or a syndromic form of deafness.     

Novel characteristics of a myosin isolated from mammalian retinal pigment epithelial and endothelial cells

We have isolated a novel, high Mr protein from human retinal pigment epithelial cells and endothelial cells by affinity chromatography on Sepharose 4B. Two polypeptides are present on SDS-gels of the 8 M urea eluent with apparent molecular mass of approximately 210 and 47 kDa. In the absence of dithiothreitol, the two polypeptides migrate as one protein band with an apparent molecular mass of approximately 550 kDa. "Piglet," as this molecule is tentatively named, is present in retinal pigment epithelial and endothelial cells of several species, but could not be detected in the nonepithelial cells we examined. Immunofluorescent localization using an antibody to the 210-kDa polypeptide revealed a filamentous network in the cytoplasm of cultured cells. This antibody was used to identify a cDNA for piglet in a bovine aortic endothelial cell expression library. Sequence data indicate a high degree of identity with non-muscle myosin II heavy chain. We subsequently found that piglet had an actin-activated ATPase activity, colocalized with actin in cells, and reacted on Western blots with a pan-non-muscle myosin II heavy chain antiserum. The protein was also recognized by antibodies specific for myosin heavy chain isoform A, but did not react with anti-isoform B antibodies. Although piglet has several features in common with known forms of non-muscle myosin II, the distinctly unconventional features it displays suggest that it is a novel myosin.     

Immotile cilia syndrome in pigs. A model for human disease

This paper describes the ultrastructural alterations observed in the tracheal epithelium of six sibling swine suffering from porcine immotile cilia syndrome (PICS) compared with those in human immotile cilia syndrome (HICS). As in some human cases, the tracheal epithelium of these pigs was lined by cilia-lacking cells. A variety of dynein defects in other pigs suffering from PICS have been previously observed. The spectrum of defects affords evidence that the PICS is genetically heterogeneous. Available data suggests that there are many similarities between HICS and PICS. Therefore, it is proposed that PICS may prove to be a useful animal model for the human disease.     

An evaluation of the effectiveness of osteopathic treatment on symptoms associated with myalgic encephalomyelitis. A preliminary report

To improve the study of epithelial function in rat ductuli efferentes (efferent ductules) and initial segment epididymis, we developed a primary cell culture system with modification of the Klinefelter method (1992). The cultured efferent ductal epithelium was grown to confluence and the cells maintained many of the organelles characteristic of these cells in vivo, including dense-staining granules, indented nuclei and apical cilia. Ciliary beat was observed for up to 10 days in culture, Cultured initial segment epithelial cells were elongated and characterized by apical branched microvilli. Electron microscopy revealed intact cell junctions, and endocytotic apparatus and lysosomal granules. Ultrastructurally, the initial segment epithelium contained a well developed Golgi apparatus. For both epithelia, cell characteristics were also confirmed by indirect immunofluorescent staining for cytokeratins 8, 18. Endocytotic activity was detected by the uptake of cationic ferritin at the apical surface and within vesicles. Estrogen receptor and clusterin mRNAs were expressed in the cultured epithelia and no difference was found in their expressions when cultured with or without 10(-9)M 17-beta estradiol. Indirect immunofluorescent staining for clusterin further indicated that this protein was present in the cultures. In conclusion, these in vitro methods will be useful for the investigation of epithelial function in the head of the epididymis.     

Apical endocytosis of lectins by the absorptive cells of the suckling rat jejunum in vivo

Apical endocytosis in the absorptive cells of the suckling rat jejunum was examined in vivo using the intraluminal injection of a range of different lectin-horseradish peroxidase (HRP) conjugates. Con A-HRP, PNA-HRP, LCA-HRP and RCA120-HRP bound strongly to components of the glycocalyx on the plasma membrane of the microvilli, apical coated pits and the small tubular pits at the base of the microvilli of the absorptive cells. The lectin-conjugates on the plasma membranes were endocytosed from the coated pits and small tubular pits, and then transported into coated vesicles, vesicles and small tubules. Lectin-HRP conjugates were later found within the intercellular space. In contrast, SBA-HRP and DBA-HRP bound weakly to the plasma membranes. The absorptive cells demonstrated little uptake or transepithelial transport. When WGA-HRP was injected into the intestinal lumen in vivo, the jejunum absorptive cells formed a deep and wide apical invagination at the base of the microvilli. WGA-HRP bound strongly to the components of the glycocalyx on the plasma membranes of the microvilli, the deep and wide apical invaginations, the apical coated pits and the small tubular pits. The WGA-HRP conjugate was also found in the coated vesicles, tubules, early endosomes, late endosomes, multivesicular bodies and lysosomes, and within the intercellular space. The lectin-HRP conjugate on the plasma membranes however was almost entirely transported into the early endosomes, late endosomes, multivesicular bodies and lysosomes. Therefore, lectin-HRP conjugates may bind to the glycocalyx component on the apical membrane domains, thus resulting in different membrane formations of apical endocytosis by adsorption to the apical plasma membrane specific glycoconjugates in the absorptive cells of the suckling rat jejunum. In addition, WGA induces deep and wide invaginations in the dynamic (not static) membrane domain of the apical plasma membrane in the suckling rat jejunum in vivo.